Effects of in vitro metabolism of a broccoli leachate, glucosinolates and S-methylcysteine sulphoxide on the human faecal microbiome.

PURPOSE: Brassica are an important food source worldwide and are characterised by the presence of compounds called glucosinolates. Studies indicate that the glucosinolate derived bioactive metabolite sulphoraphane can elicit chemoprotective benefits on human cells. Glucosinolates can be metabolised in vivo by members of the human gut microbiome, although the prevalence of this activity is unclear. Brassica and Allium plants also contain S-methylcysteine sulphoxide (SMCSO), that may provide additional health benefits but its metabolism by gut bacteria is not fully understood. METHODS: We examined the effects of a broccoli leachate (BL) on the composition and function of human faecal microbiomes of five different participants under in vitro conditions. Bacterial isolates from these communities were then tested for their ability to metabolise glucosinolates and SMCSO. RESULTS: Microbial communities cultured in vitro in BL media were observed to have enhanced growth of lactic acid bacteria, such as lactobacilli, with a corresponding increase in the levels of lactate and short-chain fatty acids. Members of Escherichia isolated from these faecal communities were found to bioconvert glucosinolates and SMCSO to their reduced analogues. CONCLUSION: This study uses a broccoli leachate to investigate the bacterial-mediated bioconversion of glucosinolates and SMCSO, which may lead to further products with additional health benefits to the host. We believe that this is the first study that shows the reduction of the dietary compound S-methylcysteine sulphoxide by bacteria isolated from human faeces.

4-Week consumption of anthocyanin-rich blood orange juice does not affect LDL-cholesterol or other biomarkers of CVD risk and glycaemia compared with standard orange juice: a randomised controlled trial.

Elevated circulating cholesterol levels are a risk factor for CVD which is also associated with sub-optimal vascular function. There is emerging evidence that anthocyanins can cause beneficial cardio-protective effects by favourably modulating lipoprotein profiles. We compared the effects of blood orange juice which is rich in anthocyanins and blonde orange juice without anthocyanins on LDL-cholesterol and other biomarkers of CVD risk, vascular function and glycaemia. In all, forty-one participants (aged 25-84 years) with a waist circumference >94 cm (men) and >80 cm (women) completed a randomised, open label, two-arm cross-over trial. For 28 d participants ingested (i) 500 ml blood orange juice providing 50 mg anthocyanins/d and (ii) 500 ml blonde orange juice without anthocyanins. There was a minimum 3-week washout period between treatments. LDL-cholesterol and other biomarkers associated with CVD risk and glycaemia were assessed at the start and end of each treatment period. No significant differences were observed in total, HDL- and LDL-cholesterol, TAG, glucose, fructosamine, nitric oxide, C-reactive protein, aortic systolic blood pressure and diastolic blood pressure or carotid-femoral and brachial-ankle pulse wave velocity after 28 d ingestion of blood orange juice compared with standard orange juice. The lack of effect on LDL-cholesterol may be due to the modest concentration of anthocyanins in the blood orange juice.

The contribution of alliaceous and cruciferous vegetables to dietary sulphur intake.

Despite its importance in many areas of human metabolism, there are no recommended daily intake guide lines for sulphur. It is generally assumed that most dietary sulphur originates from intake of methionine and cysteine. We estimated sulphur intake from food diaries, and validated the results with the use of a duplicate diet analyses. Sulphur intake estimations were highly correlated with that obtain through an elemental analysis of duplicate diets, with a mean±sd daily intakes of 956±327.9mg estimated from diet diary analyses and 935±329.9mg estimated by a duplicate diet analyses. Sulphur intake from alliaceous and cruciferous vegetables contributed up to 42% of total sulphur intake. Daily intake estimation comparisons through diet diary analyses and duplicate diet for other elements showed good agreement, except for sodium and zinc, in which analyses of 24h diet dairies overestimated intake by 35% and 52%, respectively.

Consumption of a diet rich in Brassica vegetables is associated with a reduced abundance of sulphate-reducing bacteria: A randomised crossover study.

SCOPE: We examined whether a Brassica-rich diet was associated with an increase in the relative abundance of intestinal lactobacilli and sulphate-reducing bacteria (SRB), or alteration to the composition of the gut microbiota, in healthy adults. METHODS AND RESULTS: A randomised crossover study was performed with ten healthy adults who were fed a high- and a low-Brassica diet for 2-wk periods, with a 2-wk washout phase separating the diets. The high-Brassica diet consisted of six 84 g portions of broccoli, six 84 g portions of cauliflower and six 300 g portions of a broccoli and sweet potato soup. The low-Brassica diet consisted of one 84 g portion of broccoli and one 84 g portion of cauliflower. Faecal microbiota composition was measured in samples collected following 2-wk Brassica-free periods (consumption of all Brassica prohibited), and after each diet, whereby the only Brassica consumed was that supplied by the study team. No significant changes to the relative abundance of lactobacilli were observed (p = 0.8019). The increased consumption of Brassica was associated with a reduction in the relative abundance of SRB (p = 0.0215), and members of the Rikenellaceae, Ruminococcaceae, Mogibacteriaceae, Clostridium and unclassified Clostridiales (p < 0.01). CONCLUSION: The increased consumption of Brassica vegetables was linked to a reduced relative abundance of SRB, and therefore may be potentially beneficial to gastrointestinal health.

Diet rich in high glucoraphanin broccoli reduces plasma LDL cholesterol: Evidence from randomised controlled trials.

SCOPE: Cruciferous-rich diets have been associated with reduction in plasma LDL-cholesterol (LDL-C), which may be due to the action of isothiocyanates derived from glucosinolates that accumulate in these vegetables. This study tests the hypothesis that a diet rich in high glucoraphanin (HG) broccoli will reduce plasma LDL-C. METHODS AND RESULTS: One hundred and thirty volunteers were recruited to two independent double-blind, randomly allocated parallel dietary intervention studies, and were assigned to consume either 400 g standard broccoli or 400 g HG broccoli per week for 12 weeks. Plasma lipids were quantified before and after the intervention. In study 1 (37 volunteers), the HG broccoli diet reduced plasma LDL-C by 7.1% (95% CI: -1.8%, -12.3%, p = 0.011), whereas standard broccoli reduced LDL-C by 1.8% (95% CI +3.9%, -7.5%, ns). In study 2 (93 volunteers), the HG broccoli diet resulted in a reduction of 5.1% (95% CI: -2.1%, -8.1%, p = 0.001), whereas standard broccoli reduced LDL-C by 2.5% (95% CI: +0.8%, -5.7%, ns). When data from the two studies were combined the reduction in LDL-C by the HG broccoli was significantly greater than standard broccoli (p = 0.031). CONCLUSION: Evidence from two independent human studies indicates that consumption of high glucoraphanin broccoli significantly reduces plasma LDL-C.

A diet rich in high-glucoraphanin broccoli interacts with genotype to reduce discordance in plasma metabolite profiles by modulating mitochondrial function.

BACKGROUND: Observational and experimental studies suggest that diets rich in cruciferous vegetables and glucosinolates may reduce the risk of cancer and cardiovascular disease (CVD). OBJECTIVE: We tested the hypothesis that a 12-wk dietary intervention with high-glucoraphanin (HG) broccoli would modify biomarkers of CVD risk and plasma metabolite profiles to a greater extent than interventions with standard broccoli or peas. DESIGN: Subjects were randomly assigned to consume 400 g standard broccoli, 400 g HG broccoli, or 400 g peas each week for 12 wk, with no other dietary restrictions. Biomarkers of CVD risk and 347 plasma metabolites were quantified before and after the intervention. RESULTS: No significant differences in the effects of the diets on biomarkers of CVD risk were found. Multivariate analyses of plasma metabolites identified 2 discrete phenotypic responses to diet in individuals within the HG broccoli arm, differentiated by single nucleotide polymorphisms associated with the PAPOLG gene. Univariate analysis showed effects of sex (P < 0.001), PAPOLG genotype (P < 0.001), and PAPOLG genotype × diet (P < 0.001) on the plasma metabolic profile. In the HG broccoli arm, the consequence of the intervention was to reduce variation in lipid and amino acid metabolites, tricarboxylic acid (TCA) cycle intermediates, and acylcarnitines between the 2 PAPOLG genotypes. CONCLUSIONS: The metabolic changes observed with the HG broccoli diet are consistent with a rebalancing of anaplerotic and cataplerotic reactions and enhanced integration of fatty acid ß-oxidation with TCA cycle activity. These modifications may contribute to the reduction in cancer risk associated with diets that are rich in cruciferous vegetables. This trial was registered at clinicaltrials.gov as NCT01114399.

The dietary isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN null preclinical murine model of prostate cancer.

BACKGROUND: Dietary or therapeutic interventions to counteract the loss of PTEN expression could contribute to the prevention of prostate carcinogenesis or reduce the rate of cancer progression. In this study, we investigate the interaction between sulforaphane, a dietary isothiocyanate derived from broccoli, PTEN expression and gene expression in pre malignant prostate tissue. RESULTS: We initially describe heterogeneity in expression of PTEN in non-malignant prostate tissue of men deemed to be at risk of prostate cancer. We subsequently use the mouse prostate-specific PTEN deletion model, to show that sulforaphane suppresses transcriptional changes induced by PTEN deletion and induces additional changes in gene expression associated with cell cycle arrest and apoptosis in PTEN null tissue, but has no effect on transcription in wild type tissue. Comparative analyses of changes in gene expression in mouse and human prostate tissue indicate that similar changes can be induced in humans with a broccoli-rich diet. Global analyses of exon expression demonstrated that sulforaphane interacts with PTEN deletion to modulate alternative gene splicing, illustrated through a more detailed analysis of DMBT1 splicing. CONCLUSION: To our knowledge, this is the first report of how diet may perturb changes in transcription induced by PTEN deletion, and the effects of diet on global patterns of alternative gene splicing. The study exemplifies the complex interaction between diet, genotype and gene expression, and the multiple modes of action of small bioactive dietary components.

Identification of the Eph receptor pathway as a novel target for eicosapentaenoic acid (EPA) modification of gene expression in human colon adenocarcinoma cells (HT-29).

BACKGROUND: The health benefits of polyunsaturated fatty acids (PUFAs), particularly those of the n-3 series are well documented. The mechanisms by which these effects are mediated are not fully clarified. METHODS: We used microarrays to assess the effects on gene expression in HT29 colon adenocarcinoma cells of exposure to the n-3 fatty acid eicosapentaenoic acid (EPA). HT29 cells were cultured with EPA (150 muM) for up to 24 hr prior to harvesting and isolation of RNA. Microarray results were analyzed within the statistical package 'R', and GeneGo MetaCore was used to identify key pathways of altered gene expression. RESULTS: EphB4, Vav2 and EphA1 gene expression were identified as significantly altered by EPA treatment. Statistically significant changes in gene expression after HT29 exposure to EPA were confirmed in a second experiment by real-time RT-PCR (TaqMan), This experiment also compared the effects of exposure to EPA to arachadonic acid (AA, n-6). Corresponding changes in protein expression were also assessed by Western blotting. CONCLUSIONS: Eph receptor mediated signaling is an entirely novel signaling pathway through which EPA may promote a wide range of health benefits, in particular in relation to reduction of colorectal cancer progression.

A comparison of the effects of soya isoflavonoids and fish oil on cell proliferation, apoptosis and the expression of oestrogen receptors alpha and beta in the mammary gland and colon of the rat.

Isoflavonoids and fish oil may be protective against colorectal cancer, but the evidence in relation to breast cancer risk is ambiguous. In the present study, we have investigated the impact of soya-derived isoflavonoids and n-3 fatty acids from fish oil, both individually and in combination, on apoptosis, cell proliferation and oestrogen receptor (ER) expression in the colon and mammary gland of the rat. Female rats were fed diets high in n-3 fatty acids (80 g/kg diet) or soya protein (765 mg/kg diet isoflavones) for 2 weeks, and then killed before the removal of the colon and mammary glands. Cell proliferation and apoptosis were quantified morphologically in whole crypts and terminal end buds. The expressions of ERalpha and ERbeta were measured in colon tissue scrapes and the mammary gland. Fish oil significantly increased apoptosis and decreased mitosis in both tissues, an effect associated with a decrease in the expressions of ERalpha and ERbeta. Soya had no effect on apoptosis in either tissue, but reduced mitosis in the colon (P < 0.001) while increasing it in the mammary gland (P = 0.001). The changes in proliferation were associated with contrasting changes in the ER expression such that fish oil significantly decreased both ERbeta and ERalpha, while soya increased ERalpha and decreased ERbeta. The results may provide a novel mechanism by which n-3 fatty acids could reduce cancer risk, but the interpretation of the results in relation to soya consumption and breast cancer risk requires further investigation.

Variation in gene expression profiles of peripheral blood mononuclear cells from healthy volunteers.

The normal degree of intra- and interindividual variation in gene transcription profiles of healthy human tissues has not been extensively investigated. In the study described here, microarrays were employed to analyze gene transcription in peripheral blood mononuclear cells prepared from serial blood samples that had been obtained, at weekly intervals, from apparently healthy human volunteers. Transcript levels for the majority of genes examined were found to be remarkably consistent within samples from a single donor. Conversely, marked differences were observed in samples obtained from different donors. Genes that exhibited differential expression dependent on sex, age, body mass index, and the presence of varying proportions of different leukocyte subsets were identified. These results emphasize the important contributions of genetic and environmental factors, as well as varying representation of different cell types, in determining the overall gene transcriptional profiles of human tissues. However, the study also provides evidence that, within an individual, the gene transcription profiles of sampled tissues can be comparatively stable over time.