Isoflavonoids and lignans have different potentials to modulate oxidative genetic damage in human colon cells

Beatrice L. Pool-Zobel5, Herman Adlercreutz1, Michael Glei, Ute M. Liegibel, Julie Sittlingon2, Ian Rowland2, Kristiina Wähälä4 and Gerhard Rechkemmer3

Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University, Dornburger Straße 25, 07743 Jena, Germany,
1 Institute for Preventive Medicine, Nutrition and Cancer, Folkhälsan Research Center and Department of Clinical Chemistry, University of Helsinki, PO Box 60, Mannerheimintie 97, 00014 Helsinki, Finland,
2 Northern Ireland Centre for Diet and Health, University of Ulster, Coleraine BT521AA, UK,
3 Institute for Nutritional Physiology, Federal Research Centre for Nutrition, Haid-und-Neu-Straße 9, 76131 Karlsruhe, Germany and
4 Organic Chemistry Laboratory, Department of Chemistry, University of Helsinki, PO Box 55, 00014 Helsinki, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polyphenolic compounds, including isoflavonoids and lignans, have been suggested to be chemopreventive on account of antioxidative properties. In this context it is of importance to have knowledge of their ability to reduce oxidative stress within target cells of tumorigenesis. Therefore, we investigated isoflavonoids and lignans for modulation of oxidative genetic damage in mammalian cells. H2O2-induced damage as well as endogenous DNA strand breaks and oxidized bases were determined after 30 min incubation of human colon cells with polyphenols using various modifications of the microgel electrophoresis assay (Comet assay). Enterolactone, a mammalian metabolite of plant lignans, was additionally investigated for modulation of intracellular oxidative stress in NIH 3T3 cells using laser scanning microscopy. In vivo effects of rye crispbread (a source of lignans) were investigated in 12 human volunteers by determining genetic damage in lymphocytes and antioxidant activity in plasma (FRAP assay). Genistein induced DNA breaks in the human tumour cell line HT29 clone 19A (12.5–100 µM). The polyphenols (100 µM) did not reduce damage induced by 150 µM H2O2, indicating that they lacked antioxidative potential. At this concentration enterolactone also had no effect on intracellular oxidative stress induced by 31.25 and 125 µM H2O2. In contrast, enterolactone, dihydrogenistein and formononetin reduced endogenous oxidative DNA damage at 100 µM. Daily ingestion of nine slices (76.5 g/day) of rye crispbread per day (containing 41.8 and 33.0 µg/100 g dry weight secoisolariciresinol and matairesinol, respectively) for 2 weeks did not significantly reduce genetic damage in blood lymphocytes, nor was there a modulation of plasma antioxidant capacity. The moderate effects of high concentrations of the tested compounds on endogenous oxidative DNA damage and failure to prevent H2O2- induced damage are indicative of only marginal protective potential by antioxidant mechanisms. The genotoxic effects of genistein deserve further investigation.

Abbreviations: carbDCF, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate; CLSM, confocal laser scanning microscopy; FCS, fetal calf serum; FRAP, ferric acid reducing activity of plasma; HBSS, Hank's balanced salt solution; LSM, laser scanning microscopy; ROI, regions of interest; ROS, reactive oxygen species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the major scientific achievements in the field of biomedical research of the past years has been the recognition of molecular events involved in the process of carcinogenesis. It is expected that exogenous risk factors are responsible for the induction of these molecular events, which include mutations and alterations of proto-oncogenes, tumour suppressor genes and DNA repair genes. Different patterns of risk factors may explain different patterns of cancer incidence on a global basis (1). Tobacco smoke and diet are the most important environmental factors, each responsible for 30–35% of all human cancers (2). The major diet-related tumours are located in the gastrointestinal tract (colon, stomach and oesophagus), although tumours originating in other epithelial tissues (lung, bladder and pancreas) may also have dietary associations. Tumours found in hormone-dependent organs (breast and prostate) are associated with hormonal risk factors, but dietary influences probably also play an important role (3).

Individual dietary factors which may contribute to enhancing risks for various cancers are heterogeneous. In general, diets high in total fat or in saturated/animal fat are considered causative for tissues such as breast, prostate, colon and pancreas (4). In contrast, it is well recognized that vegetables and fruits are protective for many tissues (5,6; reviewed in refs 7,8). The dietary ingredients that are implicated as being the actual chemopreventive factors are phytoprotectants (micronutrients and secondary plant ingredients) and products formed during the fermentation of non-digestible components in the gut. According to Wattenberg (9), the major mechanisms are preventing carcinogens from exerting their mutagenic effects (blocking agent activity) or preventing the initiated cell from developing further into a transformed or cancerous cell (suppressing activity).

The aim of the work described here was to determine blocking agent activity of several polyphenols, namely lignans, isoflavonoids and a few of their mammalian metabolites. These compounds have received considerable attention as potential cancer-preventing agents via their anti-oestrogenic activity (10,11). However, there are also indications that several representatives of these groups may act via antioxidant mechanisms, especially in cells not expressing oestrogen receptors (12,13).

Therefore, we have studied their effects on DNA damage (which precedes mutations and may be reduced by blocking agents) and oxidative DNA damage (which may be an indicator of oxidative stress prevailing within cells) in human colon cells, which are a major target for diet-related cancer. The compounds were investigated for their potential to protect against H2O2-induced endogenous damage using the single cell microgel electrophoresis (Comet assay) (14).

Additional studies were performed in order to increase our knowledge of basic mechanisms. One mechanism for the observed reduction in oxidative genetic damage could be a reduced level of oxidative stress within cells. In order to test this hypothesis, we have used a technique to detect reactive oxygen species (ROS) within intact cells using confocal laser scanning microscopy (CLSM) (15) with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carbDCF) as marker (16). The mouse embryo fibroblasts (NIH 3T3 cells) used in these experiments have recently been demonstrated to be a sensitive tool for the detection of oxidative stress induced by H2O2 (31) and can also be employed to study possible chemopreventive mechanisms.

Finally, to assess the relevance of in vitro results for humans, a small dietary study was performed to determine the impact of ingested polyphenols on DNA damage in humans. The study was performed with rye crispbread, since it is expected to contain relatively high amounts of lignans (17). Peripheral lymphocytes were analysed for genetic damage, as described previously (14), and plasma antioxidant capacity was determined with an assay to detect ferric acid reducing activity of plasma (FRAP assay) (18).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Metabolites of plant-derived lignans (enterodiol and enterolactone) as well as isoflavonoids and their metabolites (genistein, biochanin A, formononetin, daidzein, O-desmethylangolensin, equol, dihydrodaidzein and dihydrogenistein) were used. Enterolactone, enterodiol, genistein, biochanin A, formononetin, daidzein, O-desmethylangolensin and equol were synthesized as described previously (1922). Daidzein and genistein were reduced to dihydrodaidzein (82% yield) and dihydrogenistein (60% yield) with 10% Pd/C and ammonium formate in methanol (23,24). The glycoside of genistein (genistin) was obtained from Carl Roth GmbH (Karlsruhe, Germany). All these chemicals were 99% pure and dissolved in DMSO (added at a maximum of 0.1%) for cell incubation experiments. N-methyl-N-nitrosoguanidine and hydrogen peroxide (H2O2) were from Fluka Chemie AG (Deisenhofen, Germany) and Merck KGaA (Darmstadt, Germany), respectively. They were added (at 10 µl/ml) to the cell culture medium in NaCl (0.85%).

Primary colon cells
The human colon mucosal cells were obtained from healthy sigmoid colon tissue as described elsewhere (25,26). The study was approved by the Ethikkommission der Landesärztekammer Baden-Württemberg. Donors gave their informed consent and all procedures complied with ethical standards. Briefly, six biopsies from one donor were pooled, minced and suspended in a digestion mixture containing 6 mg proteinase K and 3 mg collagenase in 3 ml Hank's balanced salt solution (HBSS). The suspension was incubated for 30 min at 37°C and then supplemented with fresh HBSS to yield a volume of 15 ml. The suspension was centrifuged for 8 min at 139 g. The pellet was resuspended in fresh HBSS for further processing (cytotoxicity and genotoxicity).

Human tumour cell line HT29 clone 19A
The human colon cell line HT29 was established by J.Fogh (Memorial Sloan Kettering Cancer Centre, New York, NY) in 1964 (27). Its subclone 19A was terminally differentiated with 5 mM sodium butyrate and was a kind gift of C.L.Laboisse (28). Cells were grown in DMEM (Gibco BRL, Eggenstein, Germany) supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin at 37°C in a humidified incubator (5% CO2, 95% air). The culture was trypsinized with 1.5 ml trypsin/versene (1:10 v/v) for up to 10 min and subcultured at a dilution of 1:8 with supplemented DMEM. The medium was changed on days 2 and 5.

NIH 3T3 cells
The mouse embryo fibroblast cell line was obtained from the tissue culture collection at the German Cancer Research Centre (Heidelberg, Germany). NIH 3T3 cells were cultured in RPMI 1640 including 10% (v/v) FCS, 1% (v/v) penicillin/streptomycin and 2 mM glutamine. Dilution after trypsinization was 1:10. The medium was changed every 2–3 days.

Detection of DNA damage in primary colon mucous cells (Comet assay)
Each test consisted of two pairs of tubes (each with 0.5–2x106 cells/ml). One pair contained medium + 10 µl/ml NaCl (0.85%) and the other medium + phenolic phytoprotectant (100 µM). The tubes were placed in a shaking water bath (37°C) and incubated for 15 min (29). Determination of cell number and viability was performed with the trypan blue exclusion test. Subsequently, the suspension was mixed with agarose and distributed onto microscope slides. The slides were treated with NaCl (0.85%) or with 150 µM H2O2 for 5 min, placed in lysis solution and further processed in the microgel electrophoresis assay. Alternatively, for determination of endogenous damage they were only treated with NaCl (0.85%) on the slides for 5 min and, after lysis, the DNA was digested with endonuclease III, to detect levels of oxidized pyrimidine bases, or with formamidopyrimidine glycosylase, which recognizes altered purines, for 45 and 30 min, respectively. Subsequently, microgel electrophoresis was carried out as usual. Staining with ethidium bromide and microscopical analysis revealed images with damaged DNA (`comets'). The comets are a consequence of DNA migration within the electrical field (30). The proportion and extent of DNA migration were determined to evaluate the assay. Fifty DNA spots were evaluated per slide. Tail intensity (the intensity of fluorescence in the comet tail) is the evaluation criterion presented in the graphs.

Cultivation of NIH 3T3 cells on coverslips
For the CLSM experiments, NIH 3T3 cells were seeded in 5 ml of medium in culture dishes (55mm diameter) containing a sterile coverslip (33mm diameter) and cultured for 3–5 days until they had reached the optimal cell density (80% confluence). The medium was changed 24 h before the experi- ment and enterolactone was added to give a final concentration of 100 µM.

Treatment with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carbDCF) for CLSM
CarbDCF (Molecular Probes Europe BV, Leiden, The Netherlands) is a non-fluorescent, oxidant-sensitive dye. Upon oxidation, carbDCF becomes fluorescent. It was stored as a 5 mM stock solution in DMSO at -20°C. A 10 µM working solution, supplemented with 5 mg BSA/ml, was prepared in HBSS (including Ca2+ and Mg2+) freshly before use. To load the cells with carbDCF, the coverslips were fixed in special perfusion chambers which were fitted to the microscope table. The cells were incubated with the working solution (1.5 ml) for 15 min at 30°C. The carbDCF was then replaced by fresh HBSS (including Ca2+ and Mg2+), the chamber was fixed on the microscope table and perfusion with 37°C HBSS (1.5 ml/min, 37°C) using a peristaltic pump (Abimed Gilson, Villiers-le-Bel, France) was started.

Laser scanning microscopy (LSM)
As described previously (31), the detection system was a Zeiss Confocal Laser Scanning Microscope (LSM 410 invert; Carl Zeiss, Jena, Germany). The Ar 488 nm and HeNe 543 nm laser lines were used for fluorescence detection and transmission microscopy, respectively. For the Ar 488 nm laser an attenuation of 1:1000 was used. Fluorescence and transmitted light were detected after passage through a 515 nm long-pass emission filter. The magnification of the objective was 40x with a numerical aperture of 1.2. Measurements of fluorescence intensity were examined using the non-confocal mode. All experiments were performed under red safety light. Six regions of interest (ROI) in the cell layer were defined and one was chosen to define the background. The chambers were perfused for 300 s with HBSS at 37°C, then switched to H2O2 (in HBSS) for another 600 s. The controls received HBSS for the whole 900 s. The intensity of the fluorescent dye in all defined ROIs was monitored over time. Transmission and fluorescence images of the cells were stored before and after the H2O2 treatment (time points 0 and 900 s). For a better comparison of the diverse experiments, we subtracted the background values from the measured fluorescence values (grey levels) in the ROIs and calculated the time course for each cell (relative fluorescent intensity), time point 0 s being 100%. Each experiment (data point) was performed at least three times (on independent occasions).

Human feeding study
Twelve volunteers participated in a randomized crossover study with a fibre-free diet with consumption of nine slices (76.5 g/day) of rye crispbread per day (containing 41.8 and 33.0 µg/100 g dry weight of secoisolariciresinol and matairesinol, respectively) for 2 weeks. The protocol for the experiment was approved by the Medical committee of the University of Ulster and the subjects gave their informed consent prior to their participation in the study. Blood was collected weekly. Lymphocytes were isolated and subjected to analysis of cell viability, level of strand breaks and levels of oxidized DNA bases (14). Blood samples (10 ml) were centrifuged at 400 g for 15 min within 4 h of collection. The plasma was removed and stored on ice until analysed for antioxidant activity within 1 h of centrifugation. Antioxidant activity was determined using an automated FRAP assay on Cobas Bio (F. Hoffman La Roche Ltd, Switzerland) using the method described by Benzie and Strain (18). Aliquots were stored deep frozen for determination of plasma levels of lignans. These were measured by isotope dilution gas chromatography–mass spectrometry in the selected ion monitoring mode using synthesized deuterated internal standards (32).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA damage in the Comet assay
Hydrogen peroxide (150 µM) induced DNA damage in human colon cells (per cent fluorescence in the comet tail was 39 ± 11, mean ± SD of eight independent experiments, versus 2 ± 1 in the NaCl controls, n = 17). Additional incubation of the cells with 100 µM phytoprotectant, however, did not significantly modulate the degree of induced DNA damage (n = 2–3, results not shown). In contrast, incubation of colon tumour cells with individual phenolic compounds alone (100 µM) did show some effects (Figure 1Go). Genistein in particular was a potent inducer of DNA damage, whereas low levels of damage were induced by genistin, its glycosylated derivative and O-desmethylangolensin, a metabolite of daidzein. The biological significance of these minor effects are debatable since in a subsequent experiment genistin did not induce DNA damage at concentrations up to 150 µM (Figure 2Go). The effects of genistein were dose related and maximal DNA damage was induced at 100 µM (Figure 2Go). The effect of genistein was also studied in primary human colon cells, because these are expected to have a different sensitivity than HT29 colon tumour cells. Lower concentrations were investigated in these delicate primary cells to exclude interference by cytotoxicity. Figure 3Go shows that concentrations of 12.5–50 µM did not induce strand breaks in the cells of three donors. This negative effect was confirmed in a further experiment with cells from two other donors, using 12.5 µM genistin (the concentration which induced a non-significant increase in strand breaks in the previous experiment; Figure 3Go). Figure 3Go also shows the results of a microgel electrophoresis assay performed with the repair-specific enzyme endonuclease III, which reveals oxidized pyrimidine bases. It is apparent from Figure 3Go that genistein was ineffective in primary cells. The ability of the various isoflavonoids and lignans to reduce oxidized pyrimidine and purine bases was studied in HT29 cells (Figure 4Go). Although genistin and genistein had no effect, many of the polyphenols reduced the levels of oxidized pyrimidine bases and enterolactone, dihydrogenistin and formononetin reduced oxidative damage to all bases. Lower concentrations of enterolactone were additionally investigated, in both HT29 clone 19A cells and in primary human colon cells, to assess the relevance of this type of protective activity. Although statistically significant results were not achieved due to the low sample sizes (n = 3), there was a trend in both cell types that lower concentrations indeed modulate steady-state levels of oxidized DNA pyrimidine bases. In HT29 clone 19A cells the net reductions (per cent fluorescence in tail) in the treatment groups were 0 ± 4, 6.8 ± 10 and 19 ± 14 for 25, 50 and 100 µM (means ± SEM, n = 3). For primary colon cells, reductions of 3 ± 6, 5 ± 8 and 13 ± 7 were achieved at 50, 75 and 100 µM.



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Fig. 1. DNA strand breaks induced by 100 µM polyphenolic compounds in HT29 clone 19A human colon tumour cells (**P < 0.01; *P < 0.05; significant in two tailed paired t-test, n = 7–8).

 


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Fig. 2. Concentration effect of genistein- and genistin-induced strand breaking activities in HT29 clone 19A human colon tumour cells (means and extremes, n = 2–3 slides/experiment).

 


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Fig. 3. Effect of genistein on endogenous damage (strand breaks and oxidized pyrimidine bases) detected after in vitro incubation of primary human colon cells from human biopsy samples (n = 3).

 


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Fig. 4. Net reduction (control minus treatment) in oxidized DNA bases in HT29 clone 19A human colon tumour cells: (A) oxidized pyrimidine bases; (B) oxidized purine bases (means ± SEM, n = 6, *P < 0.05, means are significantly different from 0; control values for oxidized pyrimidine and purine bases were 43 ± 8 and 45 ± 5% fluorescence in tail, respectively, n = 6).

 
Oxidative stress determined by LSM
CarbDCF effectively decomposes to yield a fluorescent intermediate upon oxidative stress in NIH 3T3 cells. These mouse embryo fibroblasts react only weakly towards endogenous stress occurring within 900 s of incubation but H2O2 effectively induced oxidative stress beginning at 7.8 up to 125 µM (15). As can be seen in Figure 5Go, H2O2 effectively induced oxidative stress at 31.25 and 125 µM, where a relative intensity of greater than 700 was achieved. Treatment with 100 µM enterolactone 24 h prior to the CLSM experiment, however, did not decrease the activity of H2O2 in this system (Figure 5Go).



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Figure 5. Microscopical determination of oxidative stress in mouse embryo fibroblasts (NIH 3T3) by carbDCF oxidation. Cells were incubated with 100 µM enterolactone in culture medium for 24 h, controls with 1 µl DMSO/ml culture medium. Subsequently, they were perfused with HBSS (0–300 s) and then with 31.25 or 125 µM H2O2 (300–900 s) (means ± SEM, n = 3–4 independent experiments, *P < 0.05, two-tailed unpaired t-test; t = 900 s, solvent control versus 24 h enterolactone).

 
Human feeding study
Since rye bread contains lignans, its consumption could lead to reduced genotoxic damage in somatic cells. We have investigated this possibility in 12 human volunteers. Lymphocytes were isolated and genetic damage was determined after a fibre-free diet with consumption of nine slices of rye crispbread per day. A similar study with male volunteers consuming carrot juice had previously shown a reduction in both lymphocyte DNA strand breaks and oxidized DNA bases (14). In our study, however, we observed no such effects. The switch from a rye diet to a fibre-free diet did not increase cytotoxicity or genetic damage. Also, there was no reduction in these parameters when the rye bread intervention period followed the fibre-free diet. Table IGo shows a summary of the results obtained for all subjects immediately following a fibre-free diet or a diet supplemented with rye crispbread. Although there was a trend showing that less oxidative stress seems to be present after the fibre-free diet and also a higher antioxidative capacity was achieved, significant results were not obtained. The analysis of enterolactone in the plasma of these subjects revealed only a small, non-significant increase after rye bread consumption (Table IGo), although there was full compliance by the subjects of this trial. The original enterolactone levels were very low and it is possible that 2 weeks consumption of rye bread did not yet cause full adaptation of the intestinal microflora to rye consumption. We recently observed that subjects with very low plasma enterolactone values need a long time to adapt to a high fibre intake before increased enterolactone production by the microflora can be observed (unpublished results). There were insignificant differences in lymphocyte viability, DNA strand breaks and oxidized bases between the two dietary protocols (Table IGo). Antioxidative capacity in the plasma also was not significantly influenced by rye bread consumption.


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Table I. Effect of a diet enriched with rye crispbread on functional parameters in the blood of female volunteers (means ± SD, n = 11–12)
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a number of model systems, isoflavonoids and lignans exert antioxidant effects. For example, soy isoflavonoids are effective in vitro in inhibiting oxidation of ß-carotene linoleate, with the aglycones being more effective than the corresponding glycosides and genistein more effective than daidzein and glycitein (13). Genistein at 5–50µM was also effective in inhibiting tumour promoter-induced H2O2 formation in vitro and in vivo (33) and UV light- and Fenton reaction-induced oxidative DNA damage (34). In serum, 1µM genistein and daidzein and 0.1 µM equol and O-demethylangolensin were efficient inhibitors of in vitro lipoprotein oxidation (35). Also, in an assay aimed at determining antioxidant activity in the aqueous phase in vitro, genistein was more effective than daidzein > genistin > biochanin = daidzin > formononetin ~ ononin, the latter of which was not an antioxidant (12). In a variety of ex vivo/extracellular assays for antioxidative capacity, Mitchell et al. confirmed the activity of the isoflavonoids, although overall the latter appeared less effective than flavonoids and the synthetic antioxidant trolox. In spite of these relatively low levels of antioxidant properties, a 4 week soy supplementation in healthy volunteers resulted in a decrease in oxidative DNA damage detected with the Comet assay (36).

In contrast, our studies have shown that neither genistein nor daidzein reduced the endogenous levels of oxidized DNA bases in human colon cells and did not provide protection against H2O2-induced genotoxicity. In fact, genistein in the concentration range 12.5–100 µM was itself an inducer of DNA strand breaks. This is well above physiological concentrations, which have been determined to be ~1 µM in plasma following soy consumption (11,36). However, our genotoxic effects were obtained after only 15 min incubation of the cells with the compounds. Since toxicity is a function of time and concentration, it will be important to assess the genotoxicity of this compound at physiological concentrations after longer incubation times. In any case, studies of the genotoxicity of mammalian lignans and isoflavonoids using mutational assays are of importance for assessing risk–benefit associations. Thus, previous studies by Kulling et al. have shown no indication of genotoxicity by lignan compounds, whereas other phytoestrogens, especially genistein but not daidzein, did induce genetic damage in vitro (37,38). The genotoxic potency of genistein is of importance to the reported findings of Rao et al. (39). Genistein, fed at a dietary level of 250 p.p.m. to rats, enhanced azoxymethane-induced non-invasive and total adenocarcinoma multiplicity in the colon, without affecting adenocarcinoma incidence or multiplicity of invasive adenocarcinoma. The authors concluded that this effect may be related to the inhibition of prostaglandin-inactivating enzymes, which were also significantly affected by genistin in the study. In this context of genotoxic potential, it should be borne in mind that the ingestion of phytoestrogens with the diet leads to high endogenous concentrations, which exceed the plasma levels of physiological hormones 10- to 1000-fold. Furthermore, as a result of the finding of protective effects of plant ingredients, food-related products or supplements containing phytoestrogens may become available, which will allow a much higher intake of these bioactive compounds than is possible with normal dietary components.

The mammalian lignans enterolactone and enterodiol were also not effective in preventing H2O2-induced DNA damage in HT 29 cells and enterolactone did not reduce H2O2-induced intracellular oxidative stress determined by CLSM. However, we have found that enterolactone, enterodiol and formononetin reduced endogenous generation of oxidized DNA bases, indicating that these compounds do have some protective potential in colon cells. In the human study, ingestion of rye bread did not lead to a reduced level of oxidized bases in peripheral lymphocytes nor to antioxidative activity of the plasma. These systemic effects are not surprising, especially when regarding the concentrations needed to exert effects in vitro. We have shown that 50–100 µM (15 min–24 h exposure) enterolactone can modulate steady-state levels of oxidative DNA damage. Plasma levels of only 10 nM (maximum) enterolactone were detected after ingestion of rye bread, meaning that the effective and physiological concentrations are 10 000-fold different. The findings in peripheral blood lymphocytes, however, do not exclude the possibility that in the colon tissue itself, antigenotoxicity (due to the fibre content of the bread) or a reduction in oxidized bases (as seen for enterolactone in vitro) could result. In fact, recent animal experiments have shown that rye bran supplementation decreases the frequency of colon tumours (40). This effect is not due to a decrease in early preneoplastic lesions but more to prevention of the progression of larger multicrypt neoplastic alterations. The findings thus point to a higher potential of rye to act in colon cells as suppressing agents rather than as blocking agents and are also supportive of our findings. The low levels of enterolactone found both before and after rye bread consumption indicate that the subjects investigated had a microflora producing only little enterolactone.

The major support for the assumption that phytoestrogens contribute to the cancer preventive potential of plant foods comes from epidemiological studies. Asian populations that have a high intake of soy products have a lower risk for breast, uterine, prostate and colon cancer. They also have strikingly higher exposure to isoflavonoids. Their urinary excretion of genistein and daidzein is, for example, 10- to 100-fold higher than that of American or Finnish people (41). Also, plasma levels are higher and Japanese people reach levels of up to 2µM genistein. This is, however, still far from the concentrations found to be antiproliferative in cell culture systems. In fact, proliferative activity has been observed at concentrations near the plasma level ranges (42). Also, in spite of the circumstantial evidence, actual epidemiological associations do not prove that phytoestrogen-containing foods lead to a risk reduction. In a review by Messina et al. (43), the risk associations of soy products were examined by evaluating 21 case–control studies and 26 cancer sites. In 15 studies no statistically significant difference in risk was found for consumption of soy products and tumour incidence at nine cancer sites (including breast, prostate and colon). Ten reported a decreased risk for stomach and rectum while in one study (consumption of fried beans) an increased risk was observed for oesophageal cancer. The 25 studies with fermented soy products revealed no significant difference in 18 studies, a decreased risk in three and an increased risk in four. Additional studies by Yuan et al. found no relationship between breast cancer incidence and soy protein intake. Tofu was associated with a decreased risk for stomach cancer (reviewed in ref. 8).

In conclusion, genistein induced damage in human colon cells. Concentration–effect curves showed genistein to also be genotoxic at lower concentrations, suggesting that this compound should be further analysed for its potential as a risk factor. Enterolactone, in contrast, reduced oxidized bases at high, non-physiological concentrations, but had no effects on oxidative stress. Ingestion of rye bread had no systemic effects, neither in human lymphocytes nor in plasma, but further analysis will be needed to assess the protective effects of these foods in humans in the target tissue, colon. Overall, these observations lend support to the hypotheses that the failure to find unequivocal protection by isoflavonoid-containing foods in epidemiological studies may have its roots in the low power of such associations or in the fact that isoflavonoids, available as they are from the food, are not available in sufficient quantity to exert the effects they have shown in some in vitro and chronic animal cancer studies. In contrast, lignans have some protective activities in colon cells. The meaning of these findings for the overall colon cancer preventive potential of the foods in which these compounds are contained (linseed and rye, respectively) needs to be elucidated in the future by studying the colon as the target in human dietary intervention studies.


    Notes
 
5 To whom correspondence should be addressed Email: b8pobe{at}uni-jena.de Back


    Acknowledgments
 
The competent technical assistance of Ms Renate Lambertz, Mr Martin Knoll and Ms Eva Möller is gratefully acknowledged. We are thankful to Dr Wolfgang Kark and Prof. Roland Gugler (Städtisches Klinikum, Karlsruhe) and to Prof. Eberhard Günther Siegel (St Vinzentius Krankenhaus, Karlsruhe) for the human biopsy samples. We are grateful for financial support by the European Community project Phenolic Phytoprotectants—Role in Preventing Initiation, Promotion and Progression of Cancer (FAIR-CT-95-0894) and to WASA (Novartis Nutrition GmbH, Celle, Germany).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 14, 1999; revised February 18, 2000; accepted February 21, 2000.