In vitro Studies with Human Colon Cells in Culture

151 INTRODUCTION

Colorectal cancer (CRC) is one of the most frequent causes of death due to cancer in populations of developed countries

who consume ‘Western style diets'.¹⁾ Studies reported that dietary patterns, lifestyle exposure patterns, physical inactivity and obesity increased CRC risks, especially in genetically predisposed populations.²⁾ Additionally, strong predisposing factors are age and, with age, a change in the composition of the gut microflora.^3,4) For instance, in humans older than ∼55 y, the counts of faecal Bifidobacteria, a purportedly beneficial genus, are known to show a marked decrease in comparison to those of younger persons.⁵⁾ With age, also the incidence of CRC and of preneoplastic lesions increases.³⁾ For instance at an age ≥70 every second person consuming a Western-style type

Experimental Approaches to Assess Dietary Fibreꠂ mediated Mechanisms of Chemoprotection:

In vitro Studies with Human Colon Cells in Culture

Beatrice L. Pool-Zobel and Michael Glei Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University, Dornburger Str. 25, D-07743 Jena, Germany

Dietary fibres have been shown to reduce colorectal cancer (CRC) risks, although the epidemiological data is controversial. Obviously, studies are needed to analyse more in depth the role of these important food ingredients in cancer prevention. Dietary fibres reach the colon unaltered, where they are fermented by the gut flora to yield products such as short chain fatty acids. Of these, butyrate inhibits growth of colon cancer cells, a mechanism related to tumour suppression. This review describes an experimental approach on how to characterise different plant foods for their capacity to yield butyrate and how to study the fermentation products for biological activities in human colon cells. Batch culture fermentations with human faces and fibres were performed under anaerobe incubation conditions. The supernatants were added to human colon cells in culture and parameters of chemoprotection (antigenotoxicity, cell growth kinetics, gene expression patterns, activities of enzymes, cells death and content of short chain fatty acids (SCFA)) were measured. A marked increase of SCFA was detected in fermentations from inulin-type fructans and whole wheat. The samples inhibited growth of tumour cells in vitro, partly due to their content of butyrate and propionate. Other growth inhibitory components are unknown and still need to be identified. Samples from fermented isolated wheat arabinoxylans did not inhibit growth of tumour cells, compared to the faeces control, but enhanced the activity of glutathione S-transferases, a cellular detoxifying system. These fermentation samples also reduced the genotoxicity of 4-hydroxy- nonenal, pointing to antigenotoxic effects. Using this approach, dietary fibres may be investigated for potential chemoprotective properties in human colon cells. This will reveal mechanisms of actions that can be assessed in later studies in vivo. These types of studies are needed to elucidate the roles of non-soluble dietary ingredients in cancer initiation, progression and prevention. (Cancer Prev Res 11, 151-161, 2006)

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: Dietary fibre, Probiotics, Prebiotics, Gut flora, Fermentation products, Tumour cell growth

Correspondence to：Prof. Dr. rer. nat. Beatrice L. Pool-Zobel Department of Nutritional Toxicology, Institute for Nutritional Sciences, Biologisch-Pharmazeutische Fakultat, Friedrich-Schiller-University Jena, Dornburger Str. 25, 07743 Jena, Germany

Tel: +49-3641-949670, Fax: +49-3641-949672 E-mail: [email protected]

Received March 6, 2006, Accepted March 17, 2006

of diet is probably an adenoma carrier.⁶⁾ CRC is known to be causally related to this type of high fat diet since it delivers risk factors that cause mutations and initiate cancer or enhance growth by genetic and epigenetic mechanisms.⁷⁾ Diets high in animal fat and meat have been shown to increase the genotoxic activity of faecal water, which reflects the genotoxic burden that occurs in the gut lumen.⁸⁾ Opposed to this, nutrition may also supply products which counteract the causative factors.⁹⁾ Thus it was shown that when providing a diet high in dietary fibre and low in animal fats and meat, the faecal water genotoxicity was reduced.⁸⁾ The mechanisms involved are that some types of dietary fibres can “feed” the gut flora, thereby increasing the population of Bifidobacteria considerably.¹⁰⁾ Bifidobacteria are purportedly beneficial since they may sup- press the growth of harmful bacteria and thus maintain the integrity of a healthy gut flora.¹¹⁾ Bifidobacteria have also been shown to act antigenotoxic in vitro and in vivo, and thus contribute to a lower exposure risk in the gut lumen.^12,13) The question that needs to be answered is to which extent this relates to a reduced risk for sporadic CRC, especially with increasing age.

In epidemiological studies, dietary fibre has^14,15) and has not been shown to lower the risk of CRC.¹⁶⁾ Reasons for the controversies are numerous and possibly include limitations in the epidemiological assessments.¹⁷⁾ Other reasons for not finding beneficial effects by dietary fibre in CRC could be due to an insufficient uptake (＜ 30 g dietary fibre), quality of the fibre (fermentation by the gut flora and production of short chain fatty acids) or the inadequate presence of additional plant ingredients (phytoprotectants with antioxidant activities). ^18-20) Opposed to this, there are numerous reports from experimental studies in mammalian cells in culture, in animal studies and in human intervention trials that have disclosed very distinctive mechanisms by which plant foods (and their individual types of dietary fibres) may have positive impacts on intestinal diseases and disorders, and may also reduce the age-associated deterioration of gut flora composition. Examples are inulin-type fructans, which in numerous studies have shown different types of beneficial health effects related to gut flora composition.^5,19,21) Other dietary fibres, however, which fulfil the criteria of fermentation and which also increase the supply of antioxidants in the gut, such as wheat, could be even more effective.²²⁾ For the benefit of public health and for developing new foods to target predisposed patients, who are at risk for

colorectal cancer, it is therefore of importance to assess how such fibres are functional in the gut. For this, investigation strategies are needed to characterize not only the fibre itself, but rather more its fate in the gut, after fermentation by the gut flora. The fermentation has consequences for the composition of the gut flora and stool and results not only in a changed pattern of the bacterial population but also in the production of new metabolites. The formed products have the potential to interact with cells of the gut mucosa and to elicit a number of different biological activities that need to be elucidated in more detail. It is therefore of high interest to study these compounds and their biological activities in human gut cells and to identify possible chemoprotective activities resulting from ingestion of dietary fibres.

FERMENTATION PRODUCTS WITH CHEMOPROTECTIVE POTENTIALS

Dietary fibres have been described to have chemoprotective activities on account of several putative mechanisms.^23,24) For one, dietary fibre causes stool bulking, increases passage of faeces and scavenges toxic compounds.^25∼27) On the other hand, dietary fibre is fermented by the gut flora, resulting in an increased generation of short chain fatty acids, butyrate, propionate and acetate.²⁸⁾ Of these, butyrate is physiologically relevant to the colonic epithelium in which it serves as a principle energy source.²⁹⁾ Interest in its role as a possible protective agent has arisen from its properties to inhibit proliferation of cells in vitro,³⁰⁾ including colon tumour cell lines.^31,32) Other findings show that it also protects from hydrogen peroxide-induced genetic damage in primary rat and human colon cells and in human colon tumour cell lines.^33,34) The theory of butyrate's efficacy in reducing cancer risks, however, has several flaws, the major one being that there is virtually no evidence available that these mechanisms also occur in vivo.³⁵⁾ Moreover, little is known on 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 syner- gistically increase the activities that butyrate has been shown to extend as an individual compound. Newer studies are, therefore addressing the possibility of these combination effects.

Some have, for instance, shown that butyrate's impact on cell proliferation, is additive with propionate (but not with acetate).

Thus it was shown that propionate also inhibits cell growth at low concentrations and mixtures of propionate and butyrate were more effective than each individual compound on its own.²⁰⁾ The combination of SCFA (as present in the complex fermentation mixture) was, however, less effective than the complete fermentation mixture itself. Therefore, probably a range of diverse other components are involved, which can inhibit growth 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 for the measured parameters. Examples for these other SCFA are valerate, hexanoate and branched short chain fatty acids (isobutyrate and isovalerate),^28,36) for which only very little is known in terms of potential biological activities. Qualities (relative molar composition) and quantities (absolute molar concentrations) of these SCFA are highly different for different fibres and thus must be considered when elucidating why different fibres have different impacts in the gut.³⁷⁾

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 phy- toprotectants, which have antioxidative potential²²⁾ and which are concentrated in the aleuron layer.³⁸⁾ These include phytic acid,^39,40) alkylresorcinols,⁴¹⁾ apigenin,²²⁾ lignans,⁴²⁾ lipids⁴³⁾ and hydroxycinnamic acids,⁴⁴⁾ in particular ferulic acids. These have been shown to have diverse biological activities such as sca- venging of radicals,⁴⁵⁾ acting anti-inflammatory,⁴⁶⁾ and anticar- cinogenic in the rat colon⁴⁷⁾ and mouse skin.⁴⁸⁾ Diferulic acids are antioxidants.⁴⁹⁾ Both ferulic acid and hydroxycinnamic acids have been shown to inhibit tumour cell growth and have antimutagenic potential.⁵⁰⁾

It is interesting to consider Soya products in this context as well. The high consumption of soy foods in Asia, e.g., has been associated with a decreased risk for the development of hormonal related tumours in breast and prostate. The related mechanisms have been attributed to the plant isoflavonoids genistein and daidzein - which are contained in Soya in high concentrations as glycosides. Upon ingestion of soy foods, the aglycons genistein and daidzein are released by the gut flora and also further metabolised. First experiments have now shown that genistein was more bioactive than its metabolites in breast and prostate cells, whereas daidzein was less effective

than one of its metabolites, namely equol (Steiner et al, submitted, Raschke et al, submitted). Since equol is exclusively a product of intestinal bacterial metabolism of dietary isoflavones and since it was more active than its parent compound in terms of estrogenic and antioxidant activity, the study of gut fermentation in relation to soy products is of particular importance. Interestingly, equol is not produced in all healthy adults in response to dietary challenge with soy or daidzein and there appears to be people who are good

“equol-producers.”, whereas others are not. On this basis, it has been proposed that the clinical effectiveness of soy protein in cardiovascular, bone and menopausal health may be a function of the ability of the individual’s gut flora to biotransform soy isoflavones to the more potent estrogenic isoflavone, equol.⁵¹⁾ Such interindividual differences may also exist for other types of dietary foods and for other potentially beneficial, anticancer gut metabolites.

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.

EXPERIMENTAL APPROACHES 1. Characterisation of the fibre sources

Samples have to be analysed for contents of fibres and of secondary plant ingredients. The method for the determination of total dietary fibre requires the enzyme digestion of protein and non-resistant starch, followed by the precipitation of soluble fibre with alcohol and weighing.⁵²⁾ The method of Van Soest et al.⁵³⁾ can be used to determine neutral-detergent fibre and acid-detergent fibre in different food supplements by using the Fibretece 1,020 System M6 (FOSS Tecator, Hillerød, Denmark). Depending on the source of the dietary fibres used, secondary plant ingredients are determined by a wide range of different methods, only few of which are exemplified in.²⁰⁾ 2. Predigestion of fibre sources from vegetables Dietary fibres, which contain ingredients that do not fully reach the colon because they may be digested and absorbed

in upper regions of the gastrointestinal tract, can be pre- digested using conditions, which simulate the upper regions of the gastro intestinal tract. This can be done according to an in vitro-batch-technique which simulates the enzymatic degra- dation of starch and proteins in the upper gastrointestinal tract (stomach and duodenum), using pepsin and α-amylase.⁵⁴⁾

3. Preparation of gut flora fermentation supernatants

Dietary fibres, which completely reach the colon in an undigested manner, can be worked up directly. 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.⁵⁵⁾ For this, fermentations are conducted in vitro under anaerobic conditions (86 % nitrogen, 10 % carbon-dioxide, 4 % hydrogen at 37^oC).

Fresh human faeces are used as a bacterial source and they are fermented with the food ingredient (to provide 20 g/L fibre).

Fresh faeces from healthy human volunteers is collected as the source of gut floras. The samples are immediately weighed and combined in a large homogenizing bag. Pre-warmed potassium phosphate buffer is added (5：1 v/w) and the mixture is homogenized thoroughly in a Stomacher’400 (Seward, UK). 20 ml aliquots of the faecal homogenate are measured into 50 ml

centrifuge tubes in an anaerobic cabinet. 20 ml of each food sample are then added to the separate tubes to give a final fibre content of 10 g/L and faecal suspension of 10% as recommended by Barry et al.⁵⁶⁾ Potassium phosphate buffer is added to one tube, as a negative control and 40 ml phosphate buffer is included as a blank. Each tube is vigorously shaken to mix well. The fermentations are performed for 24 hours.

Each tube is manually mixed periodically throughout the procedure (every hour for the first eight hours and every hour for the final eight hours). Placing the suspensions on ice then stops the fermentations. Each tube is centrifuged at 6,000×g at 4^oC for 30 minutes and the respective supernatants are then aliquoted into appropriately labelled tubes and stored at -20^oC.

Samples are sterilized by filtration (pore size 0.22μm) before they are added to the cell culture medium.

4. Three stage fermentation model

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).⁵⁷⁾

5. Markers of chemoprotection

Some of the major parameters that are associated with cancer

Fig. 1. Schematic presentation of mechanisms for chemoprotective/

cancer preventive activities of plant foods (modified according to [9;80]). These types of functional can be studied in appropriate hu- man colon cells and may provide test indication.

chemoprotection in the colon are shown in Fig. 1. These para- meters are indicative of secondary cancer prevention, in that neoplastic cells are suppressed from further progression. Or they are related to primary cancer prevention, since they may prevent cancer initiation by blocking carcinogens from dama- ging the cells. Most blocking types of activities are determined using cell-free in vitro systems in the presence of the putative phytoprotectants (prevention of formation of genotoxic inter- mediates during gut fermentation, antioxidative activities using different commercial kits). Effects of the fermentation samples on mechanisms of gene expression or on activities related to suppression of tumourigenesis can be determined in human gut mucosal cells with a diverse range of different methods. The determination of the biological activities can be performed, for example, with “normal” human colon cells obtained from surgical samples,⁵⁸⁾ with human colon adenoma cells LT97,⁵⁹⁾ and with tumour cells (HT29 and/or CaCo-2).^60,61) For this, the cells are treated in vitro with fermentation supernatants and different biological activities are determined, such as growth inhibition, induction of apoptosis or expression of genes related to detoxification and toxification of carcinogens. Compounds from plant foods and their metabolites (secondary plant in- gredients, gut fermentation products,) are known to affect drug metabolism and stress response, and they are known for their growth inhibitory and apoptosis-inducing activities in tumour cells. The more detailed knowledge on how complex products formed after fermentation of plant foods by gut bacteria promises to identify plant foods with possible chemoprotective potential.

SOME KEY RESULTS 1. Inulin type fructans

Inulin is a prebiotic food ingredient, which suppresses colon tumour cell growth and cancer development in rats.¹⁹⁾ It consists of a mixture of fructans with different degrees of polymerization linked by means of β (2-1) bonds. This linkage cannot be hydrolysed by digestive enzymes in the upper intestinal tract of humans and therefore arrives unchanged in the colon lumen.⁶²⁾ Here, the carbohydrates are fermented by bifidobacterium spp. and by other lactic acid producing bacteria, thus enhancing their relative populations in the gut.⁶³⁾ Inulin type fructans, themselves are fermented to lactic acid and short chain fatty acids (SCFA) with total yields of 70∼75

mM in rats, which were fed with a long chain inulin supplemented with oligofructose (SYNERGY^Ⓡ).⁶⁴⁾ Interestingly, when incubating the same type of inulin with gut flora in vitro, SCFA-yields were comparable in both batch culture and colon vessel fermentation systems (60∼80 mM).⁶⁵⁾ Of all SCFA, butyrate has been shown to be most efficient in suppressing growth of tumour cells, but little was known concerning cellular responses to complex fermentation samples. Therefore, the fermentation samples were investigated for their effects on growth of colon tumour cells. HT29 or CaCo-2 cells were incubated with supernatants of the fermented samples (2.5∼

25% v/v, 24∼72 hours), as described above, and cellular parameters of survival, differentiation, tumour progression, and invasive growth were determined using a panel of different biological systems. The studies showed that fermentation supernatants derived from Synergy1 plus faecal slurries had higher SCFA contents, and distinct cellular functions, in comparison to the faecal and medium controls. The supernatant derived from the gut model vessel representing the distal colon, was most effective for all functional parameters, possibly on account of its higher butyrate-concentrations. The comparison of biological effects by isolated SCFA and of inulin-derived fermentation products for the first time showed that not only lactic acid bacteria and/or butyrate are mediators of the investigated biological effects but that additional other bacteria and products of the gut lumen contribute as well. In addition, this study also reported for the first time that butyrate and supernatants of inulin-derived fermentation samples inhibited metastases properties (invasive growth) of colon tumour cells, an important mechanism of tumour suppression and possibly of therapy.⁶¹⁾

2. Vegetables as sources of dietary fibres

In another study,²⁰⁾ we compared the efficacy of fermentation samples from different vegetables as dietary sources. We were also interested in determining the activities of butyrate, propionate and acetate compared to the complex fermentation supernatants and to mixtures of SCFA that mimicked the concentrations found in the complete samples. To investigate such interactions, a variety of dietary fibre sources was fer- mented with human faecal slurries in vitro, analysed for SCFA, and corresponding SCFA mixtures were prepared. HT29 colon tumour cells were treated for 72 h with individual SCFA or complex samples. The growth of cells, GST activity, and

chemoresistance towards 4-hydroxynonenal were determined.

Fermentation products inhibited cell growth more than the corresponding SCFA mixtures, and the SCFA mixtures were more active than butyrate, probably due to phytoprotectants and to propionate, respectively, which also inhibited cell growth. Only butyrate induced GSTs, whereas chemoresistance was caused by selected SCFA mixtures, but not by all cor- responding fermentation samples. Altogether, the results of these studies allowed us to conclude that supernatant fractions of fermentation samples contain additional compounds that: (1) enhance the anti-proliferative properties of butyrate (propio- nate, phytochemical fraction); (2) do not seem to enhance butyrate’s capacity to induce GSTs; (3) prevent chemoresistance in tumour cells. Thus, fermented dietary fibre sources are more potent inhibitors of tumour cell growth than butyrate alone, and also contain ingredients, which counteract the undesired positive selection pressures that higher concentrations of but- yrate induced in tumour cells.

3. Arabinoxylans from wheat

The final example is a new study, in which we have now been able to demonstrate that wheat (triticum aestivum) bran arabinoxylans and their gut flora-mediated fermentation pro- ducts protect human colon cells from genotoxic activities of 4-hydroxynonenal and hydrogen peroxide.⁶⁶⁾ These cellular parameters associated with chemoprevention were studied in human colon HT29 cells treated with water extractable (WeAx) and alkali extractable arabinoxylans (AeAx), isolated from wheat bran.⁶⁷⁾ The samples were also fermented with gut flora in vitro, which resulted in a 3-fold increase of SCFA. Both SCFA and the fermentation samples were used to treat the cells as well. Cell growth, cytotoxicity, antigenotoxic activities ag- ainst hydrogen peroxide (H2O2) and 4-hydroxy-nonenal (HNE), and activity of glutathione S-transferases (GST) and other parameters were determined. Non-fermented WeAx decreased H2O2-induced DNA damage by 64%, thus demonstrating chemoprotective properties by this non-fermented wheat bran fibre. The fermentation of WeAx and AeAx resulted in 3 fold increases of SCFA, but all FS (including the control without arabinoxylans) inhibited growth of the HT29 cells, reduced the genotoxicity of HNE, and enhanced activity of GSTs (FS WeAx 2-fold, FS AeAx 1.7-fold, control FS 1.4 fold) which detoxify HNE. Thus, increases in SCFA were not reflected by enhanced functional effects. The conclusion is that fermentation

mixtures contained modulatory compounds, such as bile acids that arise from the faeces and that might add on to the effectiveness of butyrate. Altogether, the results demonstrated for the first time that wheat bran extracts and metabolites have chemoprotective properties in human colon cells, and that several types of different mechanisms may be involved.

4. Comparison of different fermentation samples

Table 1 summarises selected results of different studies that we have performed using (1) inulin (mixture of long chain inulin and oligofructose, Synergy^Ⓡ), (2) inulin (Synergy^Ⓡ) together with probiotics, (3) long chain inulin and wheat flour (4) water extractable (WeAx) and alkali extractable (AeAx) arabinoxylans isolated from wheat bran. Fermentation of all of these substrates with human gut flora led to a significant increase of the SCFA content in comparison to the respective faeces controls; for instance, fermentation of 20 g inulin-type fructans (Studies 1, 2 and 3) resulted in total SCFA amounts of approximately 90∼120 mM. The increase was between 2.5 to 7 folds the concentrations found in the faeces controls (fermentation suspension without added dietary fibre). All four investigated inulin-type samples (±probiotics) were of similar efficacy for inhibiting growth of human colon cells with EC50

values of 7.1% to 10% fermentation sample in the cell culture medium. Compared to this the fermentation sample of whole wheat (study 3) resulted in the highest yield of total SCFA (178.4 mM) and was most effective in inhibiting growth of tumour cells (EC50=2.8%).

The arabinoxylans from wheat (Study 4), in contrast, did not reduce cell growth more than the faeces control, despite the increase of SCFA, which was ≥3 fold. These samples were available for analysis of bile acids, where we found relatively high concentrations in the faeces control (22μM total bile acids), but low concentrations in the samples with AeAx and WeAx (4.8 and 3.7μM, respectively). Since bile acids are known to cause apoptosis and by this mechanisms reduce cell number, they may have been responsible to the low EC50

valued of the faeces controls, whereas the SCFA (mainly but- yrate and propionate) were probably responsible for the reduc- tion of cell survival in the WeAX and AeAx fermentation supernatants.⁶⁸⁾ Future studies with fermentation samples will include the determinations of bile acids on a regular basis, as their levels may be important for better understanding mech- anisms of growth control by these complex samples.

The relative proportions of the individual SCFA in the inulin-derived samples was not as different as has been shown previously for wheat or for resistant starch.^69,70) For instance, the fermentation of inulin-type fructans did not result in an elevated butyrate concentration in comparison to the faeces control, whereas the fermentation of whole wheat (study 3) did, thus confirming the previous reports.⁷⁰⁾ The higher production of butyrate is interesting in the context of health benefits and anticancer activities, since butyrate has numerous biological activities related to chemoprevention, as described above. Also, there has been one report showing a significantly higher ratio of acetate to total SCFA and lower ratio of butyrate to total SCFA in polyp-colon cancer subjects compared to normal subjects.⁷¹⁾ However, on account of the numerous health benefits also observed for inulin-type of fructans, it seems sufficient if there is a significant increase of total SCFA (including butyrate and propionate) and not necessarily only of

the proportion of butyrate to mediate some of the beneficial effects, like inhibition of tumour cell growth.

Fermentation samples of inulin-type fructans and of whole wheat did not modulate the activity of glutathione S-trans- ferases in HT29 colon tumour cells, compared to the faeces controls. The obtained values on GST-activity, which amounted to 49, 47.1, 50.7 and 49.3 mmoles/min per 10⁶ cells, were not significantly higher than the values of the faeces controls, which amounted to 41.6, and 65.1 mmoles/min per 10⁶ cells.

Opposed to this, fermentation of dietary fibres from wheat (Study 4) had a significantly higher content of SCFA and caused a marked increase of GST activity from 44 (faeces control) to 57 and 71 mmoles/min per 10⁶ cells for AeAx and WeAx, respectively. This is equivalent to an increase of 1.3 and 1.6, which were induction levels previously also observed for butyrate, tested on its own.⁷²⁾ On the basis of these findings, that cover only a limited number of different sample types, Table 1. Fermentation of dietary fibre with human gut flora leads to formation of short-chain fatty acids (SCFA)

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SCFA EC50 (%) GST

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Specific Absolute (mmol) Relative (%)

Study Sample conditions Acetateꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ treat- per 10Propionate Butyrate Total change tate onate rateFold Ace- Propi- Buty- 72 h (mmol/minment cells)⁶

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1 Medium 10% v/v 0 0 0 0.00 - 13.5 31.9±9.4

Faeces HT29 25.9 4.8 4.7 35.5 - 73 14 13 11.3 41.6±4.6

Synergy^Ⓡ 24 h* 68.3 14.3 10.9 93.5 3 73 18 12 7.1 49.0± 7.6

2 Glucose 10% v/v 68.3±8.4 7.6±3.4 4.4±3.1 80.3 2.3 85 10 5 12.9 46.2±3.6 Synergy^Ⓡa HT29 70.0±4.8 8.3±4.2 9.1±6.0 87.4 2.5 80 10 10 10.2 47.1±5.6 Synergy^Ⓡb 24 h* 70.1±9.0 18.8±7.2 11.5±4.9 100.4 2.8 69 19 11 7.1 50.7±3.0

3^† Faeces 10% v/v 10.2 3.9 3.5 17.60 - 58 22 20 53.6 65.1±28.3

Wheat HT29 117.5 11.2 49.7 178.40 10.1 66 6 28 2.8 44.1±4.9

Inulin 48 h* 87.2 13.6 21.7 122.50 7.0 71 11 18 7.5 49.3±4.7

4 Faeces 10% v/v 14.6±1.8 5.6±0.9 3.8±0.5 24 - 61 23 16 11.1 44.3±2.9

AeAx HT29 54.0± 1.7 20.9± 3.0 10.7± 0.9 85.6 3.6 63 24 13 10.6 57.3± 3.0 WeAx 24 h* 48.4± 5.4 14.7± 1.9 11.7± 1.9 74.8 3.1 65 20 16 9 70.7± 7.2 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

*time between seeding of cells and treatment, ^†determination was performed only once, therefore SEM values are not available.

The table shows absolute (mmol) and relative (%) concentrations of the three major SCFA produced after fermenting different sources of dietary fibre. The data on cell growth inhibitory concentrations (EC50) of the fermentation supernatants and their influence on glutathione S- transferase activity (mmol/min per 10⁶ cells) are derived from studies in preparation (Studies 1 and 4) or as previously reported (study 2⁶¹⁾; study 3²⁰⁾). Shown are individual values or the means±SEM from 3 independently reproduced experiments. Bold letters indicate significant findings in comparison to the corresponding faeces controls (studies 1, 3 & 4). The data of study 2 was compared to the faeces control of study 1. In study 2, Synergy^Ⓡ was investigated as prebiotic preparations together with probiotics, namely together with ^aLactobacillus rhamnosus with ^bBifidibacteria lactis.

which are related, we may still conclude that there are quite different patterns of biological activities exerted by the different dietary fibre sources. Whereas inulin-type fructans±probiotics and whole wheat result in an increase of SCFA and inhibit cell growth, the fermentation of isolated wheat arabinoxylans also leads to SCFA production without inhibiting tumour cell growth, but at the investigated concentrations modulates GST- activity. The decreased growth of tumour cells is a straight- forward mechanism of chemoprevention, but an enhanced GST activity in tumour cells needs to be considered as a double edged sword.⁷³⁾ GSTs are involved in phase II metabolism, possibly facilitating deactivation of reactive carcinogen inter- mediates.⁷⁴⁾ This property would be considered of advantage for the tissue if it occurs for primary cells. In tumour cells, it may cause an undesired growth advantage that needs further elucidation. This is being addressed in further studies.⁷³⁾

SUMMARY AND CONCLUSIONS

Research is self evidently needed to enhance understanding on the efficacy of plant foods as contributors to colonic health, and on this basis to develop functional foods with added health benefits. The most straightforward approach is to investigate these plant ingredients and foods using experimental in vitro and in vivo approaches. The type of in vitro methods presented here offer the possibility of screening biological activities of gut flora-mediated fermentation products from dietary fibres derived from diverse food sources. The most effective measured biological parameters (SCFA formation, tumour cell growth inhibition, antigenotoxicity, modulation of GST activities) can then be used as “functional” biomarkers ( by measuring effects in colonic mucosa and faecal water activities) in subjects participating in intervention trials with the particular food.^75,76) The final goals could then be to provide seniors with these pre-selected functional foods and to assess how intervention may improve their gut flora and minimise discomforts arising from bowel problems.⁷⁷⁾ Clinical applications could be seen in providing the foods to patients with inflammatory bowel disease, as a way to reduce the symptoms.⁷⁸⁾ Another hope is that intervention with pre-selected foods that have a particular high functionality will reduce recurrence of disease in poly- pectomised patients or in patients with suspected CRC.⁷⁹⁾ Finally, as long terms measure of public health the functional foods could be included in a healthy diet to more strongly

contribute to the reduction of colorectal cancer risks. The types of methods described in this review will be of value for the continuous identification and development of such functional foods.

ACKNOWLEDGEMENT

Authors’ research on dietary fibres and fermentation products is supported by EU proposal (SEAFOOD plus, FQS-506359; SYNCAN, QLRT-1999-00346), by a National Network of Molecular Nutrition Research (BMBF Nr. FKZ:

01EA0103), by the German Research Foundation, Deutsche Forschungsgemeinschaft (DFG PO 284/8-1), by the Fors- chungskreis der Ernahrungsindustrie e.V., Bonn), and the AiF and the Ministry of Economics and Labour. Project No.:

AiF-FV 13065 BG.

REFERENCES

1) World Cancer Research Fund and American Institute for Cancer Research. Food, nutrition and the prevention of cancer: a global perspective. American institute for cancer research, Washington DC, 1997.

2) Potter JD. Colorectal cancer: molecules and populations. J Natl Cancer Inst 91, 916-932, 1999.

3) Heavey PM, McKenna D, Rowland IR. Colorectal cancer and the relationship between genes and the environment. Nutr Cancer 48, 124-141, 2004.

4) Benno Y, Mitsuoka T. Effect of diet and aging on human fecal microflora. Bifidobacteria Microflora 10, 89-96, 1991.

5) Kleesen B, Sykura B, Zunft HJ, Blaut M. Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. Am J Clin Nutr 65, 1397- 1402, 1997.

6) Fodde R, Smits R, Clevers H. APC signal transduction and genetic instability in colorectal cancer. Nat Rev Cancer 1, 55- 67, 2001.

7) Ferguson LR. Natural and man-made mutagens and carcino- gens in the human diet. Mutat Res 443, 1-10, 1999.

8) Rieger MA, Parlesak A, Pool-Zobel BL, Rechkemmer G, Bode C. Diets high in fat and meat, but low in fiber increase the genotoxic potential of fecal water. Carcinogenesis 20, 2317- 2326, 1999.

9) Johnson IT, Williamson G, Musk SRR. Anticarcinogenic fac- tors in plant foods: a new class of nutrients? Nutrition Research Reviews 7, 175-204, 1994.

10) Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. J Nutr 125, 1401-1412, 1995.

11) Macfarlane GT, Cummings JH. Probiotics and prebiotics: can regulating the activities of intestinal bacteria benefit health?

BMJ 318, 999-1003, 1999.

12) Renner HW, Munzner R. The possible role of probiotics as dietary antimutagen. Mutat Res 262, 239-245, 1991.

13) Wollowski I, Ji S, Bakalinsky AT, Neudecker C, Pool-Zobel BL. Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats.

J Nutr 129, 77-82, 1999.

14) Bingham SA, Riboli E. Diet and cancer - the European pro- spective investigation into cancer and nutrition. Nat Rev Cancer 4, 206-215, 2004.

15) Bingham SA, Norat T, Moskal A, Ferrari P, Slimani N, Clavel-Chapelon F, Kesse E, Nieters A, Boeing H, Tjonneland A, Overvad K, Martinez C, Dorronsoro M, Gonzalez CA, Ardanaz E, Navarro C, Quiros JR, Key TJ, Day NE, Trichopoulou A, Naska A, Krogh V, Tumino R, Palli D, Panico S, Vineis P, Bueno-de-Mesquita HB, Ocke MC, Peeters PHM, Berglund G, Hallmans G, Lund E, Skeie G, Kaaks R, Riboli E. Is the association with fiber from foods in colorectal cancer confounded by folate intake? Cancer Epidemiol Biomarkers Prev 14, 1552-1556, 2005.

16) Hung HC, Joshipura KJ, Jiang R, Hu FB, Hunter D, Smith- Warner SA, Colditz GA, Rosner B, Spiegelman D, Willett WC. Fruit and vegetable intake and risk of major chronic disease. JNCI Cancer Spectrum 96, 1577-1584, 2004.

17) Schatzkin A, Kipnis V. Could exposure assessment problems give us wrong answers to nutrition and cancer questions?

JNCI Cancer Spectrum 96, 1564-1565, 2004.

18) Ferguson LR, Harris PJ. The dietary fibre debate: more food for thought - commentary. The Lancet, 361, 1487-1488, 2003.

19) Pool-Zobel BL. Inulin-type fructans and reduction in colon cancer risk: Review of experimental and human data. Br J Nutr 93, S73-S90, 2005.

20) Beyer-Sehlmeyer G, Glei M, Hartmann F, Hughes R, Persin C, Bohm V, Rowland IR, Schubert R, Jahreis G, Pool-Zobel BL. Butyrate is only one of several growth inhibitors produced during gut flora-mediated fermentation of dietary fibre sources. Br J Nutr 90, 1057-1070, 2003.

21) Guarner F. Inulin and oligofructiose: impact on intestinal disearses and disorders. Br J Nutr 93, S61-S65, 2005.

22) Ferguson LR, Harris PJ. Protection against cancer by wheat bran: role of dietary fibre and phytochemicals. Eur J Cancer Prev 8, 17-25, 1999.

23) Harris PJ, Ferguson LR. Dietary fibre: its composition and role in protection against colorectal cancer. Mutat Res 290, 97-110, 1993.

24) Hill MJ. Introduction: dietary fibre, butyrate and colorectal cancer. Eur J Cancer Prev 4, 341-343, 1995.

25) Baghurst PA, Baghurst KI, Record SJ. Dietary Fibre, non- starch polysaccharides and resistant starch-A review. Sup- plement to Food Australia 48, S3-S35, 1996.

26) Ryden P, Robertson JA. The consequences of fruit and ve-

getable fibre fermentation on their binding capacity for MeIQx and the effects of soluble fibre sources on the binding affinity of wheat bran preparations. Carcinogenesis 16, 1711- 1716, 1995.

27) Kestell P, Zhao L, Zhu S, Harris PJ, Ferguson LR. Studies on the mechanism of cancer protection by wheat bran: effects on the absorption, metabolism and excretion of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Car- cinogenesis 20, 2253-2260, 1999.

28) Cummings J. Short-chain fatty acids in the human colon.

GUT 22, 763-779, 1981.

29) Silvester KR, Bingham SA, Pollock JRA, Cummings J, O’Neill I. Effect of meat and resistant starch on fecal excretion of apparent N-nitroso compounds and ammonia from the human large bowel. Nutr Cancer 29, 13-23, 1998.

30) Kruh J. Effects of sodium butyrate, a new pharmacological agent on cells in culture. Mol Cell Biochem 42, 65-82, 1982.

31) Gamet L, Daviaud D, Denis-Pouxviel C, Remesy C, Murat JC. Effects of short-chain fatty acids on growth and differ- entiation f the human colon cancer cell line HT29. Int J Cancer 52, 286-289, 1992.

32) Hague A, Paraskeva C. The short-chain fatty acid butyrate induces apoptosis in colorectal tumour cell lines. Eur J Cancer Prev 4, 359-364, 1995.

33) Abrahamse SL, Pool-Zobel BL, Rechkemmer G. Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration in isolated rat colon cells. Carcinogenesis 20, 629-634, 1999.

34) Rosignoli P, Fabiani R, De Bartolomeo A, Spinozzi F, Agea E, Pelli MA, Morozzi G. Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells. Carcinogenesis 22, 1675- 1680, 2001.

35) Lupton JR. Butyrate and colonic cytokinetics: differences between in vitro and in vivo studies. Eur J Cancer Prev 4, 373-378, 1995.

36) Cummings JH, Pomare EW, Branch WJ, Naylor CPE, Macfarlane GT. Short chain fatty acids in human large intes- tine, portal, hepatic and venous blood. GUT 28, 1221-1227, 1987.

37) Ferguson LR, Chavan RR, Harris PJ. Changing concepts of dietary fiber: Implications for carcinogenesis. Nutr Cancer 39, 155-169, 2001.

38) Buri RC, von Reding W, Gavin MH. Description and charac- terisation of wheat aleuron. Cereal Foods World 49, 275-281, 2004.

39) Shamsuddin AM, Ullah A. Inositol hexaphosphate inhibits large intestinal cancer in F344 rats 5 months afer induction by azoxymethane. Carcinogenesis 10, 625-626, 1989.

40) Shamsuddin AM. Anti-cancer function of phytic acid. Int J Food Sci Technol 37, 769-782, 2002.

41) Ross AB, Kamal-Eldin A, Aman P. Dietary alkylresorcinols:

Absorption, bioactivities, and possible use as biomarkers of

whole-grain wheat- and rye-rich foods. Nutr Rev 62, 81-95, 2004.

42) Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 3, 364-373, 2002.

43) Reddy BS, Hirose Y, Cohen LA, Simi B, Cooma I, Rao CV.

preventive potential of wheat bran fractions against experi- mental colon carcinogenesis: implications for human colon cancer prevention. Cancer Res 60, 4792-4797, 2000.

44) Yang CS, Landau JM, Huang MT, Newmark HL. Inhibition of carcinogenesis by dietary polyphenolic compounds. Ann Rev Nutr 21, 381-406, 2001.

45) Ogiwara T, Satoh K, Kadoma Y, Murakami Y, Unten S, Atsumi T, Sakagami H, Fujisawa S. Radical scavenging activity and cytotoxicity of ferulic acid. Anticancer Res 22, 2711-2717, 2002.

46) Ozaki Y. Antiinflammatory effect of tetramethylpyrazine and ferulic acid. Chem Pharm Bull (Tokyo) 40, 954-956, 1992.

47) Hirose M, Takahashi S, Ogawa K, Futakuchi M, Shirai T.

Phenolics: Blocking agents for heterocyclic amine-induced carcinogenesis. Food Chem Toxicol 37, 985-992, 1999.

48) Huang MT, Smart RC, Wong CQ, Conney AH. Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetra- decanoylphorbol-13-acetate. Cancer Res 48, 5941-5946, 1988.

49) GarciaConesa MT, Plumb GW, Kroon PA, Wallace G, Williamson G. Antioxidant properties of ferulic acid dimers.

Redox Report 3, 239-244, 1997.

50) Ferguson LR, Lim IF, Pearson AE, Ralph J, Harris PJ.

Bacterial antimutagenesis by hydroxycinnamic acids from plant cell walls. Mutat Res 542, 49-58, 2003.

51) Setchell KDR, Brown NM, Lydeking-Olsen E. The clinical importance of the metabolite equol--a clue to the effectiveness of soy and its isoflavones. J Nutr 132, 3577-3584, 2002.

52) AOAC International. Total dietary fiber, l, official methods of analysis. Gaithersburg, MD. 1992.

53) Van Soest PJ, Robinson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 74, 3583-3589, 1991.

54) Aura AM, Harkonen H, Fabritius M, Poutanen K. Develop- ment of an in vitro enzymic digestion method for removal of starch and protein and assessment of its performance using rye and wheat breads. J Cereal Sci 29, 139-152, 1999.

55) Wang X, Gibson GR. Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. J Appl Bacteriol 75, 373-380, 1993.

56) Barry JL, Hoebler C, Macfarlane GT, Macfarlane S, Mathers J, Reed KA, Mortensen PB, Nordgard I, Rowland I, Rumney CJ. Estimation of the fermentability of dietary fibre in vitro:

a European interlaboratory study. Briti 74, 303-322, 2002.

57) Macfarlane GT, Macfarlane S, Gibson GR. Validation of a three-stage compound continuous culture system for inves- tigating the effect of retention time on the ecology and metabolism of bacteria in the human colon. Microb Ecol 35,

180-187, 1998.

58) Schaferhenrich A, Sendt W, Scheele J, Kuechler A, Liehr T, Claussen U, Rapp A, Greulich KO, Pool-Zobel BL. Endo- genously formed cancer risk factors induce damage-of p53 in human colon cells obtained from surgical samples. Food Chem Toxicol 41, 655-664, 2003.

59) Richter M, Jurek D, Wrba F, Kaserer K, Wurzer G, Karner- Hanusch J, Marian B. Cells obtained from colorectal micro- adenamas mirror early premalignant growth patterns in vitro.

Eur J Cancer 38, 1937-1945, 2002.

60) Fogh J, Trempe X. Human tumor cells in vitro. In: ed, by Fogh J. Human tumor cells in vitro. Plenum Press, New York, pp 115-159.

61) Klinder A, Gietl E, Hughes R, Jonkers N, Karlsson P, McGlyn H, Pistoli S, Tuohy KM, Rafter J, Rowland IR, Van Loo J, Pool-Zobel BL. Gut fermentation products of inulin- derived prebiotics inhibit markers of tumour progression in human colon tumour cells. Int J Cancer Prev 1, 19-32, 2004.

62) Schneemann BO. Fiber, inulin and oligofructose: similarities and differences. Br J Nutr 129, 1424S-1427S, 1999.

63) Ellgard L, Andersson H, Bosaeus I, Bosaeus I. Inulin and oligofructose do not influence the absorption of cholestrol, or the excretion of cholesterol, Ca, Mg, Zn, Fe, or bile acids but increase energy excretion in ileostomy subjects. Eur J Clin Nutr 45, 451-457, 1997.

64) Femia AP, Luceri C, Dolara P, Giannini A, Biggeri A, Sal- vadori M, Clune Y, Collins KJ, Paglierani M, Caderni G.

Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in rats. Carcinogenesis 23, 1953- 1960, 2002.

65) Klinder A, Forster A, Caderni G, Femia AP, Pool-Zobel BL.

Fecal water genotoxicity is predictive of tumor preventive activities by inulin-like oligofructoses, probiotics (Lactobacillus rhamnosus and Bifidobacterium lactis) and their synbiotic com- bination. Nutr Cancer 49, 144-155, 2004.

66) Glei M, Hofmann T, Kuster K, Hollmann J, Lindhauer MG, Pool-Zobel BL. Wheat (triticum aestivum) bran arabinoxylans and their gut flora-mediated fermentation products protect human colon cells from genotoxic activites by 4-hydroxy- nonenal and hydrogen peroxide. Submitted. 2005.

67) Hollmann J, Lindhauer MG. Isolierung und Struktur von Ballaststoffen auf Arabinoxylanbasis aus der Weizenkleie.

Getreidetechnologie (Cereal Technology) 58, 343-349, 2004.

68) Glei M, Hofmann T, Kuster K, Hollmann J, Lindhauer MG, Pool-Zobel BL. Wheat (triticum aestivum) bran arabinoxylans and their gut flora-mediated fermentation products protect human colon cells from genotoxic activites by 4-hydroxy- nonenal and hydrogen peroxide. J Agr Food Chem, accepted for publication 2006.

69) Topping DL, Clifton PM. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch

polysaccharides. Physiol Rev 81, 1031-1064, 2001.

70) Muir JG, Yeow EG, Keogh J, Pizzey C, Bird AR, Sharpe K, O’Dea K, Macrae FA. Combining wheat bran with resistant starch has more beneficial effects on fecal indexes than does wheat bran alone. Am J Clin Nutr 79, 1020-1028, 2004.

71) Weaver GA, Krause JA, Miller TL, Wolin MJ. Short chain fatty acid distributions of enema samples from a sigmoid- oscopy population: an association of high acetate and low butyrate ratios with adenomatous polyps and colon cancer.

GUT 29, 1539-1543, 1988.

72) Ebert MN, Klinder A, Schaferhenrich A, Peters WHM, Sendt W, Scheele J, Pool-Zobel BL. Expression of glutathione S- transferases (GST) in human colon cells and inducibility of GSTM2 by butyrate. Carcinogenesis 24, 1637-1644, 2003.

73) Pool-Zobel BL, Veeriah S, Kautenburger T, Kiefer J, Richter KK, Sauer J, Soom M, Wolfl S. Butyrate may enhance toxicological defence in primary, adenoma and tumour human colon cells by favourably modulating expression of glutathione S-transferases genes. Carcinogenesis 26, 1064-1076, 2005.

74) Pool-Zobel BL, Veeriah S, Bohmer FD. Modulation of xeno- biotic metabolising enzymes by anticarcinogens - focus on glutathione S-transferases and their role as targets of dietary chemoprevention in colorectal carcinogenesis. Mutat Res 591,

74-92, 2005.

75) Glei M, Habermann N, Osswald K, Seidel C, Bohm V, Persin C, Jahreis G, Pool-Zobel BL. Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the comet assay: a biomarker model. Biomarkers 10, 203-217, 2005.

76) Oberreuther-Moschner D, Jahreis G, Rechkemmer G, Pool- Zobel BL. Dietary intervention with the probiotics Lactobacillus acidophilus 145 and Bifidobacterium longum 913 modulates DNA-damage inducing potential of human faecal water in HT29clone19A cells. Br J Nutr 91, 1-9, 2004.

77) Saunier K, Dore J. Gastrointestinal tract and the elderly:

Functional foods, gut microflora and healthy ageing. Digest Liver Dis 34, S19-S24, 2002.

78) Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening- Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, Lochs H. Mucosal flora in inflammatory bowel disease. Gastroenterology 122, 44-54, 2002.

79) Van Loo J, Clune Y, Bennett M, Collins KJ. The SYNCAN project: goals, set-up, first results and settings of the human intervention study. Br J Nutr 93, S91-S98, 2005.

80) Wattenberg LW. Inhibition of carcinogenesis by minor dietary constituents. Cancer Res 52(Suppl), 2085s-2091s, 1992.