Study on Human Urinary Metabolic Profiles after Consumption of Kale and Daikon Radish using a High-resolution Mass Spectrometry-Based Non-targeted and Targeted Metabolomic Approach.

In the present study, urine samples were collected from healthy human volunteers to determine the metabolic fates of phenolic compounds and glucosinolates after a single meal of kale and daikon radish. The major glucosinolates and phenolic compounds in kale and daikon radish were measured. The urinary metabolome after feeding at different time periods was investigated. A targeted metabolite analysis method was developed based on the known metabolic pathways for glucosinolates and phenolic compounds. Using a targeted approach, a total of 18 metabolites were found in urine: 4 from phenolic compounds and 14 from glucosinolates. Among these metabolites, 4-methylsulfinyl-3-butenyl isothiocyanate, 4-methylsulfinyl-3-butenyl isothiocyanate-cysteine, and 4-methylsulfinyl-3-butenylglucosinolate-N-acetyl cysteine were reported for the first time in human urine. The combination of non-targeted and targeted metabolomic approaches can gain a full metabolite profile for human dietary intervention studies.

BMI Is Associated With Increased Plasma and Urine Appearance of Glucosinolate Metabolites After Consumption of Cooked Broccoli.

Introduction: Preclinical studies suggest that brassica vegetable diets decrease cancer risk, but epidemiological studies show varied effects, resulting in uncertainty about any health impact of brassicas. Factors controlling absorption of glucosinolate metabolites may relate to inconsistent results. We reported previously that subjects with BMI > 26 kg/m2 (HiBMI), given cooked broccoli plus raw daikon radish (as a source of plant myrosinase) daily for 17 days, had lower glucosinolate metabolite absorption than subjects given a single broccoli meal. This difference was not seen in subjects with BMI < 26 kg/m2 (LoBMI). Our objective in this current study was to determine whether a similar response occurred when cooked broccoli was consumed without a source of plant myrosinase. Methods: In a randomized crossover study (n = 18), subjects consumed no broccoli for 16 days or the same diet with 200 g of cooked broccoli daily for 15 days and 100 g of broccoli on day 16. On day 17, all subjects consumed 200 g of cooked broccoli. Plasma and urine were collected for 24 h and analyzed for glucosinolate metabolites by LC-MS. Results: There was no effect of diet alone or interaction of diet with BMI. However, absorption doubled in HiBMI subjects (AUC 219%, plasma mass of metabolites 202% compared to values for LoBMI subjects) and time to peak plasma metabolite values and 24-h urinary metabolites also increased, to 127 and 177% of LoBMI values, respectively. Conclusion: BMI impacts absorption and metabolism of glucosinolates from cooked broccoli, and this association must be further elucidated for more efficacious dietary recommendations. Clinical Trial Registration: This trial was registered at clinicaltrials.gov (NCT03013465).

Broccoli consumption affects the human gastrointestinal microbiota.

The human gastrointestinal microbiota is increasingly linked to health outcomes; however, our understanding of how specific foods alter the microbiota is limited. Cruciferous vegetables such as broccoli are a good source of dietary fiber and phytonutrients, including glucosinolates, which can be metabolized by gastrointestinal microbes. This study aimed to determine the impact of broccoli consumption on the gastrointestinal microbiota of healthy adults. A controlled feeding, randomized, crossover study consisting of two 18-day treatment periods separated by a 24-day washout was conducted in healthy adults (n=18). Participants were fed at weight maintenance with the intervention period diet including 200 g of cooked broccoli and 20 g of raw daikon radish per day. Fecal samples were collected at baseline and at the end of each treatment period for microbial analysis. Beta diversity analysis indicated that bacterial communities were impacted by treatment (P=.03). Broccoli consumption decreased the relative abundance of Firmicutes by 9% compared to control (P=.05), increased the relative abundance of Bacteroidetes by 10% compared to control (P=.03) and increased Bacteroides by 8% relative to control (P=.02). Furthermore, the effects were strongest among participants with body mass index <26 kg/m2, and within this group, there were associations between bacterial relative abundance and glucosinolate metabolites. Functional prediction revealed that broccoli consumption increased the pathways involved in the functions of the endocrine system (P=.05), transport and catabolism (P=.04), and energy metabolism (P=.01). These results reveal that broccoli consumption affects the composition and function of the human gastrointestinal microbiota.

Absorption and metabolism of isothiocyanates formed from broccoli glucosinolates: effects of BMI and daily consumption in a randomised clinical trial.

Sulphoraphane originates from glucoraphanin in broccoli and is associated with anti-cancer effects. A preclinical study suggested that daily consumption of broccoli may increase the production of sulphoraphane and sulphoraphane metabolites available for absorption. The objective of this study was to determine whether daily broccoli consumption alters the absorption and metabolism of isothiocyanates derived from broccoli glucosinolates. We conducted a randomised cross-over human study (n 18) balanced for BMI and glutathione S-transferase µ 1 (GSTM1) genotype in which subjects consumed a control diet with no broccoli (NB) for 16 d or the same diet with 200 g of cooked broccoli and 20 g of raw daikon radish daily for 15 d (daily broccoli, DB) and 100 g of broccoli and 10 g of daikon radish on day 16. On day 17, all subjects consumed a meal of 200 g of broccoli and 20 g of daikon radish. Plasma and urine were collected for 24 h and analysed for sulphoraphane and metabolites of sulphoraphane and erucin by triple quadrupole tandem MS. For subjects with BMI >26 kg/m2 (median), plasma AUC and urinary excretion rates of total metabolites were higher on the NB diet than on the DB diet, whereas for subjects with BMI <26 kg/m2, plasma AUC and urinary excretion rates were higher on the DB diet than on the NB diet. Daily consumption of broccoli interacted with BMI but not GSTM1 genotype to affect plasma concentrations and urinary excretion of glucosinolate-derived compounds believed to confer protection against cancer. This trial was registered as NCT02346812.

Comparison of Effects of Diet on Mammary Cancer: Efficacy of Various Preventive Agents and Metabolomic Changes of Different Diets and Agents.

To determine the effects of diet, rats were placed on a standard diet (4% fat) or on a modified Western (high-fat diet, HFD) diet (21% fat) at 43 days of age (DOA) and administered methylnitrosourea (MNU) at 50 DOA. Rats were administered effective (tamoxifen, vorozole, and Targretin) or ineffective (metformin and Lipitor) chemopreventive agents either by daily gavage or in the diet beginning at 57 DOA and continuing until sacrifice (190 DOA). Latency period of the tumors was determined by palpation, and multiplicity and cancer weights per rat were determined at final sacrifice. Rats on the HFD versus standard diet had: (i) a 6% increase in final body weights; (ii) significant decreases in tumor latency; and (iii) significant increases in final tumor multiplicity and average tumor weight. Tamoxifen, vorozole, and Targretin were highly effective preventive agents, whereas Lipitor and metformin were ineffective in rats on either diet. Serum was collected at 78 DOA and at sacrifice (190 DOA), and metabolomics were determined to identify the metabolite changes due to diets and effective agents. Rats given the HFD had increased levels of saturated free fatty acids (including myristate) and decreased levels of 2-aminooctanoate. Furthermore, rats on the HFD diet had increased levels of 2-aminobutyrate and decreases in glycine markers previously identified as indicators of prediabetes. Targretin increased long-chain glycophospholipids (e.g., oleyl-linoleoyl-glycerophosphocholine) and decreased primary bile acids (e.g., taurocholate). Tamoxifen increased palmitoyl-linoleoyl-glycophosphocholine and decreased stearoyl-arachidonyl glycophosphocholine. Finally, increased levels of methylated nucleotides (5-methylcytidine) and decreased levels of urea cycle metabolites (N-acetylcitrulline) were associated with the presence of mammary cancers.

Dietary Selenium Levels Affect Selenoprotein Expression and Support the Interferon-γ and IL-6 Immune Response Pathways in Mice.

Selenium is an essential element that is required to support a number of cellular functions and biochemical pathways. The objective of this study was to examine the effects of reduced dietary selenium levels on gene expression to assess changes in expression of non-selenoprotein genes that may contribute to the physiological consequences of selenium deficiency. Mice were fed diets that were either deficient in selenium or supplemented with selenium in the form of sodium selenite for six weeks. Differences in liver mRNA expression and translation were measured using a combination of ribosome profiling, RNA-Seq, microarrays, and qPCR. Expression levels and translation of mRNAs encoding stress-related selenoproteins were shown to be up-regulated by increased selenium status, as were genes involved in inflammation and response to interferon-γ. Changes in serum cytokine levels were measured which confirmed that interferon-γ, as well as IL-6, were increased in selenium adequate mice. Finally, microarray and qPCR analysis of lung tissue demonstrated that the selenium effects on immune function are not limited to liver. These data are consistent with previous reports indicating that adequate selenium levels can support beneficial immune responses, and further identify the IL-6 and interferon-γ pathways as being responsive to dietary selenium intake.

The 15kDa selenoprotein and thioredoxin reductase 1 promote colon cancer by different pathways.

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/ß-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

Opportunities for small nutrition-related cancer research grants (R03) from the National Cancer Institute.

Small research grants, or R03 grants, provide limited, short-term support for individual research projects. R03s may be an excellent means of support for projects by nutrition scientists at all stages in their careers. The National Cancer Institute (NCI) has awarded roughly one-half of all nutrition-related NIH R03 grants in the period from 2005 to 2010. A detailed review of the recent NCI grant portfolio identified potential strategies for successful applications. Projects that addressed important nutrition and cancer issues had feasible and appropriate specific aims, were innovative, and were based on sound concepts were most positively viewed by reviewers. Furthermore, applicants with suitable expertise, training, mentorship, and records of accomplishment who, when appropriate, collaborated with investigators with complementary knowledge and skills were more likely to receive higher priority scores.

Evaluation of commercial kava extracts and kavalactone standards for mutagenicity and toxicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells.

Kava (Piper methysticum) is a member of the pepper family and has been cultivated by South Pacific islanders for centuries and used as a social and ceremonial drink. Traditionally, kava extracts are prepared by grinding or chewing the rhizome and mixing with water and coconut milk. The active constituents of kava are a group of approximately 18 compounds collectively referred to as kavalactones or kava pyrones. Kawain, dihydrokawain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin are the six major kavalactones. Kava beverages and other preparations are known to be anxiolytic and are used for anxiety disorders. Dietary supplements containing the root of the kava shrub have been implicated in several cases of liver toxicity in humans, including several who required liver transplants after using kava supplements. In order to study the toxicity and mutagenicity, two commercial samples of kava, Kaviar and KavaPure, and the six pure kavalactones including both D-kawain and DL-kawain, were evaluated in L5178Y mouse lymphoma cells. Neither the kava samples nor the kavalactones induced a mutagenic response in the L5178Y mouse lymphoma mutation assay with the addition of human liver S9 activation.

A review of the interaction among dietary antioxidants and reactive oxygen species.

During normal cellular activities, various processes inside of cells produce reactive oxygen species (ROS). Some of the most common ROS are hydrogen peroxide (H(2)O(2)), superoxide ion (O(2)(-)), and hydroxide radical (OH(-)). These compounds, when present in a high enough concentration, can damage cellular proteins and lipids or form DNA adducts that may promote carcinogenic activity. The purpose of antioxidants in a physiological setting is to prevent ROS concentrations from reaching a high-enough level within a cell that damage may occur. Cellular antioxidants may be enzymatic (catalase, glutathione peroxidase, superoxide dismutase) or nonenzymatic (glutathione, thiols, some vitamins and metals, or phytochemicals such as isoflavones, polyphenols, and flavanoids). Reactive oxygen species are a potential double-edged sword in disease prevention and promotion. Whereas generation of ROS once was viewed as detrimental to the overall health of the organism, advances in research have shown that ROS play crucial roles in normal physiological processes including response to growth factors, the immune response, and apoptotic elimination of damaged cells. Notwithstanding these beneficial functions, aberrant production or regulation of ROS activity has been demonstrated to contribute to the development of some prevalent diseases and conditions, including cancer and cardiovascular disease (CVD). The topic of antioxidant usage and ROS is currently receiving much attention because of studies linking the use of some antioxidants with increased mortality in primarily higher-risk populations and the lack of strong efficacy data for protection against cancer and heart disease, at least in populations with adequate baseline dietary consumption. In normal physiological processes, antioxidants effect signal transduction and regulation of proliferation and the immune response. Reactive oxygen species have been linked to cancer and CVD, and antioxidants have been considered promising therapy for prevention and treatment of these diseases, especially given the tantalizing links observed between diets high in fruits and vegetables (and presumably antioxidants) and decreased risks for cancer.

Oxidative stress and antioxidants: a link to disease and prevention?

The thrust of this presentation takes a more programmatic approach and gives an overview of the programs at the NIH and the NCI that have a broad nutritional and basic science undercurrent and outline. Also discussed briefly are some areas of general concern that are under investigation in the nutrition group and are included in the group's outreach efforts among professional and academic organizations. The overarching focus of these efforts is to stress the importance of nutrition as a potential modulator of health/disease risks associated with genetic predisposition and environmentally induced disease from diet, lifestyle and exposure to pollutants.

Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells.

Chromium picolinate is one of the most commonly used chromium dietary supplements available in the United States, and it has been marketed to consumers for use in weight loss, increasing muscle mass, and lowering serum cholesterol. Chromium picolinate is a synthetic compound that provides a bioavailable form of Cr(III) that is absorbed better than dietary chromium. However, there are several reports that it can have adverse effects. In order to study the mechanism of observed cellular toxicity and mutagenicity, chromium picolinate and its component compounds, chromium (III) chloride and picolinic acid, were evaluated in Salmonella typhimurium and L5178Y mouse lymphoma cells. Neither chromium picolinate nor chromium chloride induced a mutagenic response in S. typhimurium. However, in the L5178Y mouse lymphoma mutation assay, chromium picolinate induced mutagenic responses without and with the addition of S9.

The antioxidant conundrum in cancer.

The health-related effects of interactions between reactive oxygen species (ROS) and dietary antioxidants and the consequences of dietary antioxidant supplementation on human health are by no means clear. Although ROS, normal byproducts of aerobic metabolism, are essential for various defense mechanisms in most cells, they can also cause oxidative damage to DNA, proteins, and lipids, resulting in enhanced disease risk. Dietary antioxidants (e.g., vitamin E, vitamin C, beta-carotene, and selenium), as well as endogenous antioxidant mechanisms, can help maintain an appropriate balance between the desirable and undesirable cellular effects of ROS. However, any health-related effects of interactions between dietary antioxidants and ROS likely depend on the health status of an individual and may also be influenced by genetic susceptibilities. Clinical studies of antioxidant supplementation and changes in either oxidative status, disease risk, or disease outcome have been carried out in healthy individuals, populations at risk for certain diseases, and patients undergoing disease therapy. The use of antioxidants during cancer therapy is currently a topic of heated debate because of an overall lack of clear research findings. Some data suggest antioxidants can ameliorate toxic side effects of therapy without affecting treatment efficacy, whereas other data suggest antioxidants interfere with radiotherapy or chemotherapy. Overall, examination of the evidence related to potential interactions between ROS and dietary antioxidants and effects on human health indicates that consuming dietary antioxidant supplements has pros and cons for any population and raises numerous questions, issues, and challenges that make this topic a fertile field for future research. Overall, current knowledge makes it premature to generalize and make specific recommendations about antioxidant usage for those at high risk for cancer or undergoing treatment.