Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations.

Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects.

Increasing fish consumption does not affect genotoxicity markers in the colon in an intervention study.

Observational studies suggest that fish consumption is associated with a decreased colorectal cancer (CRC) risk. A possible mechanism by which fish could reduce CRC risk is by decreasing colonic genotoxicity. However, concerns have also been raised over the levels of toxic compounds found in mainly oil-rich fish, which could increase genotoxicity. Therefore, the objective was to investigate the effects of fish on genotoxicity markers in the colon in a randomized controlled parallel intervention study. For a period of 6 months, subjects were randomly allocated to receive two extra weekly portions of (i) oil-rich fish (salmon), (ii) lean fish (cod) or (iii) just dietary advice (DA). The Comet Assay was used to measure the DNA damage-inducing potential of fecal water (n = 89) and DNA damage in colonocytes (n = 70) collected pre- and post-intervention as markers of genotoxicity. Genotoxicity of fecal water was not markedly changed after fish consumption: 1.0% increase in tail intensity (TI) [95% confidence interval (CI) -5.1; 7.0] in the salmon group and 0.4% increase in TI (95% CI -5.3; 6.1) in the cod group compared with the DA group. DNA damage in colonocytes was also not significantly changed after fish consumption, in either the salmon group (-0.5% TI, 95% CI -6.9; 6.0) or cod group (-3.3% TI, 95% CI -10.8; 4.3) compared with the DA group. Measurements of genotoxicity of fecal water and DNA damage in colonocytes did not correlate (r = 0.06, n = 34). In conclusion, increasing consumption of either oil-rich or lean fish did not affect genotoxicity markers in the colon.

Effects of fatty acids on metabolism and cell growth of human colon cell lines of different transformation state.

Epidemiological studies suggest that high fish intake is associated with a decreased risk of colorectal cancer which has been linked to the high content of the n - 3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in some fish. In this study, two different cell lines are compared in relation to their response to EPA and DHA versus the plant derived PUFAs, linoleic acid (LA), gamma-linolenic acid (GLA), and alpha-linolenic acid (ALA) and to the ubiquitous arachidonic acid (ARA). The uptake of 100 microM of each fatty acid (FA) was determined using GC. The 4',6-diamidino-2-phenylindole assay for DNA quantification and the Cell-Titer-Blue assay were used to determine cell survival and metabolic activity at 2-72 h after treatment. All FAs were utilized more efficiently by the human colon adenoma cell line LT97 than by the adenocarcinoma cell line HT29. LT97 were more susceptible than HT29 cells to the growth inhibitory activities of all FAs except for DHA where both were equally sensitive. Inhibition of survival and metabolic activity by EPA and DHA increased with treatment time in both cell lines. ALA or GLA were less growth inhibitory than EPA or DHA and ARA had intermediary activity. The data show that the tested FAs are incorporated into colon cells. Furthermore, adenoma cells are more susceptible than the adenocarcinoma cells.

Influence of inorganic and organic iron compounds on parameters of cell growth and survival in human colon cells.

Increased risk for development of colon cancer is associated with red meat intake and iron toxicity is discussed for one underlying mechanism. Anyhow, for iron itself only limited evidence is found. In this study, effects of different iron compounds on proliferation of HT29 carcinoma and LT97 adenoma human colon cells were investigated. After treatment of cells with inorganic (ferrous sulfate: FeSO4 and ferric nitrilotriacetate: FeNTA) and organic (hemoglobin and hemin) iron sources (24-72 h), number of cells and metabolic activity were measured. Under normal cell culture conditions, neither iron compound elevated cell growth in either cell line with the exception of FeNTA which induced LT97 cell growth significantly. Distinct inhibition of cell proliferation was measured for organic iron. Serum-free incubation of HT29 cells revealed growth promoting properties of iron under deficiency. Even though organic iron, especially hemin, was a potent growth factor, both substances showed also dose-dependent cytotoxic effects. In conclusion, these data emphasize that not iron itself, but merely organic iron may promote carcinogenic events. Since promotion of proliferation was only detectable under deficiency, cytotoxic properties of organic iron may be of more importance in colon carcinogenesis.

Does an extract of carob (Ceratonia siliqua L.) have chemopreventive potential related to oxidative stress and drug metabolism in human colon cells?

Phenolic ingredients of an aqueous carob extract are well characterized and consist of mainly gallic acid (GA). In order to assess possible chemopreventive mechanisms of carob, which can be used as a cacao substitute, effects on expression of genes related to stress response and drug metabolism were studied using human colon cell lines of different transformation state (LT97 and HT29). Stress-related genes, namely catalase (CAT) and superoxide dismutase (SOD2), were induced by carob extract and GA in LT97 adenoma, but not in HT29 carcinoma cells. Although corresponding protein products and enzyme activities were not elevated, pretreatment with carob extract and GA for 24 h reduced DNA damage in cells challenged with hydrogen peroxide (H(2)O(2)). In conclusion, carob extract and its major phenolic ingredient GA modulate gene expression and protect colon adenoma cells from genotoxic impact of H(2)O(2). Upregulation of stress-response genes could not be related to functional consequences.

Modulation of gene expression in eicosapentaenoic acid and docosahexaenoic acid treated human colon adenoma cells.

Epidemiological studies suggest that high fish intake is associated with a decreased risk of colorectal cancer which has been linked to the high content of the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) in some fish. The aim of the study was to compare the modulation of gene expression in LT97 colon adenoma cells in response to EPA and DHA treatment. Therefore, we used custom-designed cDNA arrays containing probes for 306 genes related to stress response, apoptosis and carcinogenesis and hybridised them with cDNA from LT97 cells which were treated for 10 or 24 h with 50 muM EPA or DHA. There was a marked influence of n-3 PUFA on the expression of several gene types, such as detoxification, cell cycle control, signaling pathways, apoptosis and inflammation. DHA and EPA generally modulated different sets of genes, although a few common effects were noted. In our approach, we used preneoplastic adenoma cells which are a relevant model for target cells of chemoprevention. If verified with real time PCR, these results identify genes and targets for chemoprevention of colon cancer.

Use of Comet-FISH in the study of DNA damage and repair: review.

The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest, including whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA regions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how Comet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global damage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different sensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene regions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH helps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the measured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least possible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest.

Intervention with cloudy apple juice results in altered biological activities of ileostomy samples collected from individual volunteers.

BACKGROUND: Apple juice is considered to be an important component of the healthy diet, with anticancer activities in colon cancer animal models and key ingredients have numerous chemoprotective activities in human colon cells in vitro. AIM OF THE STUDY: Since only little is known on comparable activities in the human colon in vivo, here a pilot study was performed to assess related mechanisms caused by ileostomy samples from volunteers that had consumed apple juice. METHODS: Ileostomy samples were collected after intervention (0-8 h) with cloudy apple juice (1 l). They were characterized analytically for major apple polyphenols and biologically in HT29 colon cells for their potential to cause genotoxic damage, protect from the genotoxic insult by hydrogen peroxide (H(2)O(2)) and modulate the expression of GSTT2, an enzyme related to antioxidative defence against different peroxides. RESULTS: The analytical determination of polyphenols in the ileostomy samples revealed that the majority of the compounds were recovered in the samples collected 2 h after intervention. The comparison of genotoxic effects of samples before intervention and 2 h after intervention revealed a considerable variation of genotoxic response, but there was a trend for reduced genotoxicity in three of eight persons (P) after intervention. Samples collected at 2 h protected HT29 cells from genotoxic damage by H(2)O(2) (for 4 of 8 persons), resulted in an increased GSTT2 expression (for 2 of 6 persons) and of GSTT2 promotor activity (2 of 6 persons). CONCLUSIONS: The intervention with apple juice results in bioavailable concentrations of related polyphenols in the gut lumen, which could contribute to reduced genotoxicity, enhanced antigenotoxicity and favorable modulation of GSTT2 gene expression in some individuals. The pilot study for the first time used this combination of faecal biomarkers which in larger cohorts may either reveal overall significant alterations of chemoprotection or may be used to identify individuals which could particularly benefit from a personalized nutrition.

Antioxidant effectiveness of phenolic apple juice extracts and their gut fermentation products in the human colon carcinoma cell line caco-2.

Apples represent a major dietary source of antioxidative polyphenols. Their metabolic conversion by the gut microflora might generate products that protect the intestine against oxidative damage. We studied the antioxidant effectiveness of supernatants of fermented apple juice extracts (F-AEs, 6 and 24 h fermentation) and of selected phenolic degradation products, identified by HPLC-DAD-ESI-MS. Cell free antioxidant capacity of unfermented apple juice extracts (AEs) was decreased after fermentation by 30-50%. In the human colon carcinoma cell line Caco-2, F-AEs (containing <0.5% of original AE-phenolics) decreased the reactive oxygen species (ROS) level more efficiently than the F-blank (fermented without AE) but were less effective than the respective AEs. Similarly, antioxidant effectiveness of individual degradation products was lower compared to respective AE constituents. Glutathione level was slightly increased and oxidative DNA damage slightly decreased by fermented AE03, rich in quercetin glycosides. In conclusion, F-AEs/degradation products exhibit antioxidant activity in colon cells but to a lesser extent than the respective unfermented AEs/constituents.

Effects of carrot and tomato juice consumption on faecal markers relevant to colon carcinogenesis in humans.

High intakes of carotenoid-rich fruits and vegetables are associated with a reduced risk of various cancers including colon cancer. A human intervention study with carrot and tomato juice should show whether a diet rich in carotenoids, especially high in beta-carotene and lycopene, can modify luminal processes relevant to colon carcinogenesis. In a randomised cross-over trial, twenty-two healthy young men on a low-carotenoid diet consumed 330 ml tomato or carrot juice per d for 2 weeks. Intervention periods were preceded by 2-week depletion phases. At the end of each study period, faeces of twelve volunteers were collected for chemical analyses and use in cell-culture systems. Consumption of carrot juice led to a marked increase of beta-carotene and alpha-carotene in faeces and faecal water, as did lycopene after consumption of tomato juice. In the succeeding depletion phases, carotenoid contents in faeces and faecal water returned to their initial values. Faecal water showed high dose-dependent cytotoxic and anti-proliferative effects on colon adenocarcinoma cells (HT29). These effects were not markedly changed by carrot and tomato juice consumption. Neither bile acid concentrations nor activities of the bacterial enzymes beta-glucosidase and beta-glucuronidase in faecal water changed after carrot and tomato juice consumption. Faecal water pH decreased only after carrot juice consumption. SCFA were probably not responsible for this effect, as SCFA concentrations and profiles did not change significantly. In summary, in the present study, 2-week interventions with carotenoid-rich juices led only to minor changes in investigated luminal biomarkers relevant to colon carcinogenesis.

Overview of experimental data on reduction of colorectal cancer risk by inulin-type fructans.

Colorectal cancer is related to diet, lifestyle, physical inactivity, and obesity. The responsible carcinogens cause mutations or enhance cell growth. Inulin-type fructans may counteract the effects via their gut flora-mediated fermentation products in vitro and in vivo. Important products formed by fermentation of inulin-type fructans with human gut flora are short-chain fatty acids. Of these, butyrate and propionate inhibit growth of colon tumor cells and histone deacetylases. Butyrate also causes apoptosis, reduces metastasis in colon cell lines, and protects from genotoxic carcinogens by enhancing expression of enzymes involved in detoxification. Fermentation supernatants of inulin have similar growth-inhibitory effects on colon adenoma and carcinoma cells and induce histone hyperacetylation by inhibiting histone deacetylases. In animal models inulin-type fructans prevent and retard colorectal carcinogenesis. Several studies reported the reduction of chemically induced preneoplastic lesions or tumors in the colon of rodents treated with inulin-type fructans. The human intervention study (SYNCAN project) sought to provide the experimental evidence for risk reduction by inulin-type fructans in humans. One group of polypectomized people at high risk for colon cancer and another of colon cancer volunteers after curative resection were given a synbiotic preparation. There were clear functional effects of the synbiotic because numerous different cancer risk markers were favorably altered. In conclusion, there is considerable experimental evidence that inulin modulates parameters of colon cancer risks in human colon cells, in animals, and in a human intervention trial. The involved mechanisms possibly include reduction of exposure to risk factors and suppression of tumor cell survival.

Fecal water as a non-invasive biomarker in nutritional intervention: comparison of preparation methods and refinement of different endpoints.

The assessment of cellular effects by the aqueous phase of human feces (fecal water, FW) is a useful biomarker approach to study cancer risks and protective activities of food. In order to refine and develop the biomarker, different protocols of preparing FW were compared. Fecal waters were prepared by 3 methods: (A) direct centrifugation; (B) extraction of feces in PBS before centrifugation; and (C) centrifugation of lyophilized and reconstituted feces. Genotoxicity was determined in colon cells using the Comet assay. Selected samples were investigated for additional parameters related to carcinogenesis. Two of 7 FWs obtained by methods A and B were similarly genotoxic. Method B, however, yielded higher volumes of FW, allowing sterile filtration for long-term culture experiments. Four of 7 samples were non-genotoxic when prepared according to all 3 methods. FW from lyophilized feces and from fresh samples were equally genotoxic. FWs modulated cytotoxicity, paracellular permeability, and invasion, independent of their genotoxicity. All 3 methods of FW preparation can be used to assess genotoxicity. The higher volumes of FW obtained by preparation method B greatly enhance the perspectives of measuring different types of biological parameters and using these to disclose activities related to cancer development.

Blood mononucleocytes are sensitive to the DNA damaging effects of iron overload--in vitro and ex vivo results with human and rat cells.

Iron exposure enhances colorectal carcinogeneis, by producing reactive oxygen species, which damage lipids, proteins and DNA. We recently demonstrated that ferric-nitrilotriacetate (Fe-NTA) damages DNA of human colon cells in different stages of malignant transformation. Opposed to this, little is known on systemic effects of iron and it is still difficult to determine the border between essential iron supplementation and iron overload in humans. The aim of this study was to determine whether Fe-NTA causes global and specific DNA damage in peripheral leucocytes. Human leucocytes were treated in vitro with Fe-NTA for 30 min at 37 degrees C. Male Sprague Dawley rats were fed (6 weeks) with an iron-overload diet (9.9 g Fe/kg DM) and whole blood was collected. DNA damage was measured in human and rat blood cells using the alkaline version of the Comet Assay with repair specific enzymes. In human cells the distribution of TP53 in the comet images was detected using fluorescence in situ hybridization (Comet FISH) to measure DNA damage in the region of the TP53 gene. Fe-NTA (10-500 microM) was clearly genotoxic in human leucocytes in vitro, and also in leucocytes of rats fed the iron overload diet. The induced damage in human leucocytes was approximately two-fold that observed previously in human colon cells. Oxidized bases were induced by iron in rat leucocytes in vivo, while they were not induced in human leucocytes in vitro. Fe-NTA enhanced the migration of TP53 signals into the comet tail of human leucocytes, indicating a high susceptibility of this tumour-relevant gene towards DNA damage induced by iron overload. In conclusion, iron markedly induced DNA damage in human and rat leucocytes, which shows that these white blood cells are sufficiently sensitive to assess exposure to iron. The measurement of DNA damage in human leucocytes could be used as a sensitive biomarker to study iron overload in vivo in humans and thus to determine whether supplementation results in genotoxic risk.

Influence of prebiotics and antioxidants in bread on the immune system, antioxidative status and antioxidative capacity in male smokers and non-smokers.

Interest in functional foods is increasing. The aim of the present study was to investigate breads supplemented with functional components. One was bread supplemented with inulin, linseed and soya fibre (prebiotic bread). The other was a prebiotic antioxidant bread (pre-aox-bread), which additionally contained green tea powder, herbs and tomato paste. The effects of these two breads on immunological and antioxidative parameters were compared with control bread (placebo). Twenty smokers and eighteen non-smokers were enrolled in the randomised parallel study, which consisted of a control period and an intervention period, each lasting for 5 weeks. Daily intake of bread and nutrients did not differ between the intervention and the control period. Most of the twenty-three investigated immunological parameters measured in peripheral blood were unaffected. However, the percentage of CD19 increased after intervention with prebiotic bread, whereas intercellular adhesion molecule-1 (ICAM-1) and CD3+NK+ (P < 0.05) decreased in both intervention arms. The ferric reducing ability of plasma (FRAP) was increased after consumption of the pre-aox-bread for non-smokers (1256 v. 1147 micromol/l; P = 0.019) and remained unchanged for smokers consuming the pre-aox-bread. All analysed carotenoids (P

Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes.

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system.

Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 microM) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 microM), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.

Mixtures of SCFA, composed according to physiologically available concentrations in the gut lumen, modulate histone acetylation in human HT29 colon cancer cells.

Intake of fibre has beneficial properties on gut health. Butyrate, a product of bacterial gut fermentation, is thought to contribute to positive effects by retarding growth and enhancing apoptosis of tumour cells. One mechanism is seen in its capacity to modulate histone acetylation and thereby transcriptional activity of genes. Next to butyrate, propionate and acetate are also major products of gut fermentation and together they may exert different potencies of cellular effects than butyrate alone. Since virtually nothing is known on combination effects by SCFA mixtures, here we had the aim to assess how physiological relevant concentrations and mixtures of SCFA modulate histone acetylation in human colon cells. HT29 colon cancer cells were incubated with mixtures of butyrate, acetate and propionate and with the individual compounds as controls. Histone acetylation was determined with acid-urea gel electrophoresis and immunoblotting. Acetylated histones slowly increased over 24 h and persisted up to 72 h in butyrate-treated HT29 cells. Butyrate (5-40 mM) and propionate (20-40 mM) enhanced histone acetylation significantly after 24 h incubation, whereas acetate (2.5-80 mM) was ineffective. Mixtures of these SCFA also modulated histone acetylation, mainly due to additive effects of butyrate and propionate, but not due to acetate. In conclusion, physiological concentrations of propionate together with butyrate could have more profound biological activities than generally assumed. Together, these SCFA could possibly mediate important processes related to an altered transcriptional gene activation and thus contribute to biological effects possibly related to cancer progression or prevention.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro.

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.

Reduced isoflavone metabolites formed by the human gut microflora suppress growth but do not affect DNA integrity of human prostate cancer cells.

Dietary isoflavones, such as genistein and daidzein, are metabolised by the human gut microflora. Case-control studies have disclosed a link between the formation of the daidzein metabolite equol and prostate cancer risk. We evaluated the effects of genistein, daidzein and five metabolites on two prostate cancer cell lines by determining DNA integrity and cell growth. LNCaP cells contain the T877A androgen receptor mutation whereas Los Angeles prostate cancer (LAPC)-4 cells express the wild-type receptor, both of which may affect responses to isoflavones. DNA integrity was determined using the comet assay. Cell growth was assessed by staining DNA with 4',6'-diamidino-2-pheylindole hydrochloride. Endogenous steroid hormones, but not isoflavones, induced DNA strand breaks. Dihydrotestosterone stimulated the growth of both cell lines. 17beta-Oestradiol increased the growth of LNCaP but not LAPC-4 cells, pointing to an involvement of the T877A androgen receptor. Isoflavones did not stimulate growth in either prostate cancer cell line. However, the growth of LNCaP and LAPC-4 cells was suppressed by genistein (inhibitory concentration 50 % (IC50) 39.7 mumol/l, 37.2 mumol/l) and by equol (IC50 53.8 mumol/l, 35.1 mumol/l). O-desmethylangolensin inhibited the growth of LAPC-4 cells (IC50 45.2 mumol/l), but not of LNCaP cells. In conclusion, isoflavones do not damage DNA or promote growth of androgen-dependent prostate cancer cells. Several isoflavones, including the reduced daidzein metabolites equol and O-desmethylangolensin, suppress cancer cell growth. Taken together, these data suggest a contribution of gut-formed isoflavone metabolites to the beneficial effects of dietary isoflavones on prostate cancer risk.