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Brassica vegetables contain a multitude of bioactive compounds that prevent and suppress cancer and promote health. Evidence suggests that the gut microbiome may be essential in the production of these compounds; however, the relationship between specific microbes and the abundance of metabolites produced during cruciferous vegetable digestion are still unclear. We utilized an ex vivo human fecal incubation model with in vitro digested broccoli sprouts (Broc), Brussels sprouts (Brus), a combination of the two vegetables (Combo), or a negative control (NC) to investigate microbial metabolites of cruciferous vegetables. We conducted untargeted metabolomics on the fecal cultures by LC-MS/MS and completed 16S rRNA gene sequencing. We identified 72 microbial genera in our samples, 29 of which were significantly differentially abundant between treatment groups. A total of 4499 metabolomic features were found to be significantly different between treatment groups (q ≤ 0.05, fold change > 2). Chemical enrichment analysis revealed 45 classes of compounds to be significantly enriched by brassicas, including long-chain fatty acids, coumaric acids, and peptides. Multi-block PLS-DA and a filtering method were used to identify microbe–metabolite interactions. We identified 373 metabolites from brassica, which had strong relationships with microbes, such as members of the family Clostridiaceae and genus Intestinibacter, that may be microbially derived.

We show that skeletal muscle nitrate concentration is higher with supplementation at rest and was lower in groups with increasing exercise duration in a zebrafish model. The higher availability of nitrate at rest is associated with upregulation of key nutrient-sensing genes and greater availability of energy-producing metabolites (i.e., ATP, phosphocreatine, glycolytic intermediates). Overall, nitrate supplementation may lower oxygen cost of exercise through improved fuel availability resulting from metabolic programming of muscle prior to exercise.

Scope: Nitrate supplementation is a popular ergogenic aid that improves exercise performance by reducing oxygen consumption during exercise. We investigated the effect of nitrate exposure and exercise on metabolic pathways in zebrafish liver.Materials and methods: Fish were exposed to sodium nitrate (606.9 mg/L), or control water, for 21 days and analyzed at intervals during an exercise test. We utilized untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and measured gene expression of 24 genes central to energy metabolism and redox signaling.Results: We observed a greater abundance of metabolites involved in endogenous nitric oxide (NO) metabolism and amino acid metabolism in nitrate-treated liver at rest, compared to rested controls. In the absence of exercise, nitrate treatment upregulated expression of genes central to nutrient sensing (pgc1a), protein synthesis (mtor) and purine metabolism (pnp5a and ampd1) and downregulated expression of genes involved in mitochondrial fat oxidation (acaca and cpt2).Conclusion: Our data support a role for sub-chronic nitrate treatment in the improvement of exercise performance, in part, by improving NO bioavailability, sparing arginine, and modulating hepatic gluconeogenesis and glycolytic capacity in the liver.

Zinc (Zn) deficiency impairs bone growth. However, the precise skeletal effects of varying levels of Zn deficiency and response to subsequent Zn repletion on the growing skeleton are incompletely understood. To address this gap in knowledge, we investigated the effects of dietary Zn ((severe deficiency ( 0.5 mg Zn/kg diet) and short-term Zn repletion (30 mg/kg diet), marginal deficiency (6 mg Zn/kg diet)) on bone mass, density, and cortical and cancellous bone microarchitecture in growing male Sprague Dawley rats. Marginal Zn intake for 42 days had no effect on bone mass or cortical and cancellous bone microarchitecture. Twenty-one days of severe Zn deficiency lowered serum osteocalcin and C terminal telopeptide of type I collagen (CTX-1), decreased tibial bone mineral content and density, and lowered cross-sectional volume, cortical volume, and cortical thickness in tibial diaphysis as compared to both Zn-adequate (30 mg/kg diet) and pair-fed controls. Severe Zn deficiency similarly lowered cancellous bone volume in proximal tibial metaphysis. Zn repletion (10 days) accelerated weight gain, indicative of catch-up growth, normalized CTX-1 and osteocalcin, but did not normalize bone mass (unadjusted and adjusted for body weight) or cortical and cancellous bone microarchitecture. In summary, severe but not marginal Zn deficiency in rapidly growing rats impaired acquisition of cortical and cancellous bone, resulting in abnormalities in bone microarchitecture. Zn repletion accelerated weight gain compared to Zn-adequate controls but absence of a compensatory increase in serum osteocalcin or bone mass suggests Zn repletion may be insufficient to fully counteract the detrimental effects of prior Zn deficiency on skeletal growth.

Robust evidence shows that phytochemicals from cruciferous vegetables, like broccoli, are associated with numerous health benefits. The anti-cancer properties of these foods are attributed to bioactive isothiocyanates (ITCs) and indoles, phytochemicals generated from biological precursor compounds called glucosinolates. ITCs, and particularly sulforaphane (SFN), are of intense interest as they block the initiation, and suppress the progression of cancer, through genetic and epigenetic mechanisms. The efficacy of these compounds is well-demonstrated in cell culture and animal models, however, high levels of inter-individual variation in absorption and excretion of ITCs is a significant barrier to the use of dietary glucosinolates to prevent and treat disease. The source of inter-individual ITC variation has yet to be fully elucidated and the gut microbiome may play a key role. This review highlights evidence that the gut microbiome influences the metabolic fate and activity of ITCs. Human feeding trials have shown inter-individual variations in gut microbiome composition coincides with variations in ITC absorption and excretion, and some bacteria produce ITCs from glucosinolates. Additionally, consumption of cruciferous vegetables can alter the composition of the gut microbiome and shift the physiochemical environment of the gut lumen, influencing the production of phytochemicals. Microbiome and diet induced changes to ITC metabolism may lead to the decrease of cancer fighting phytochemicals such as SFN and increase the production of biologically inert ones like SFN-nitrile. We conclude by offering perspective on the use of novel “omics” technologies to elucidate the interplay of the gut microbiome and ITC formation.

Pancreatic beta cells produce and release insulin, a hormone that regulates blood glucose levels, and their dysfunction contributes to the development of diabetes mellitus. Zinc deficiency and inorganic arsenic exposure both independently associate with the development of diabetes, although the effects of their combination on pancreatic beta cell health and function remain unknown. We hypothesized zinc deficiency increases the toxicity associated with arsenic exposure, causing an increased susceptibility to DNA damage and disruption of insulin production. Zinc deficiency decreased cell proliferation by 30 percent in pancreatic INS-1 rat insulinoma cells. Arsenic exposure (0, 50 or 500 ppb exposures) significantly decreased cell proliferation, and increased mRNA levels of genes involved in stress response (Mt1, Mt2, Hmox1) and DNA damage (p53, Ogg1). When co-exposed to both zinc deficiency and arsenic, zinc deficiency attenuated this response to arsenic, decreasing the expression of Mt1, Hmox1, and Ogg1, and significantly increasing DNA double-strand breaks 2.9-fold. Arsenic exposure decreased insulin expression, but co-exposure did not decrease insulin levels beyond the arsenic alone condition, but did result in a further 33 percent decline in cell proliferation at the 500 ppb arsenic dose, and a significant increase in beta cell apoptosis. These results suggest zinc deficiency and arsenic, both independently and in combination, adversely affect pancreatic beta cell health and both factors should be considered in the evaluation of health outcomes for susceptible populations.

Background Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. Objectives We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. Methods We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. Results Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., β-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. Conclusions Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

There continues to be interest in targeting epigenetic “readers, writers, and erasers” for the treatment of cancer and other pathologies. However, a mechanistic understanding is frequently lacking for the synergy observed when combining deacetylase and bromodomain inhibitors. Here we identify cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane. N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl “reader” proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. Treatment with the BET inhibitor JQ1 synergized with sulforaphane in colon cancer cells and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with sulforaphane+JQ1 in combination implicated a BET/BRD9 acetyl switch and a shift in the pool of acetyl “reader” proteins in favor of BRD9-regulated target genes. Significance: These results highlight the competition that exists among the “readers” of acetylated histone and nonhistone proteins and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments.

Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.

Dietary nitrate supplementation improves exercise performance by reducing the oxygen cost of exercise and enhancing skeletal muscle function. However, the mechanisms underlying these effects are not well understood. The purpose of this study was to assess changes in skeletal muscle energy metabolism associated with exercise performance in a zebrafish model. Fish were exposed to sodium nitrate (60.7 mg/L, 303.5 mg/L, 606.9 mg/L), or control water, for 21 days and analyzed at intervals (5, 10, 20, 30, 40 cm/s) during a 2-h strenuous exercise test. We measured oxygen consumption during an exercise test and assessed muscle nitrate concentrations, gene expression, and the muscle metabolome before, during, and after exercise. Nitrate exposure reduced the oxygen cost of exercise and increased muscle nitrate concentrations at rest, which were reduced with increasing exercise duration. In skeletal muscle, nitrate treatment upregulated expression of genes central to nutrient sensing (mtor), redox signaling (nrf2a), and muscle differentiation (sox6). In rested muscle, nitrate treatment increased phosphocreatine (P = 0.002), creatine (P = 0.0005), ATP (P = 0.0008), ADP (P = 0.002), and AMP (P = 0.004) compared with rested-control muscle. Following the highest swimming speed, concentration of phosphocreatine (P = 8.0 × 10(−5)), creatine (P = 6.0 × 10(−7)), ATP (P = 2.0 × 10(−6)), ADP (P = 0.0002), and AMP (P = 0.004) decreased compared with rested nitrate muscle. Our data suggest nitrate exposure in zebrafish lowers the oxygen cost of exercise by changing the metabolic programming of muscle prior to exercise and increasing availability of energy-rich metabolites required for exercise. NEW & NOTEWORTHY We show that skeletal muscle nitrate concentration is higher with supplementation at rest and was lower in groups with increasing exercise duration in a zebrafish model. The higher availability of nitrate at rest is associated with upregulation of key nutrient-sensing genes and greater availability of energy-producing metabolites (i.e., ATP, phosphocreatine, glycolytic intermediates). Overall, nitrate supplementation may lower oxygen cost of exercise through improved fuel availability resulting from metabolic programming of muscle prior to exercise.

IntroductionDietary nitrate lowers blood pressure and improves athletic performance in humans, yet data supporting observations that it may increase cerebral blood flow and improve cognitive performance are mixed. Here we tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We also explored the extent to which nitrate and nitrite treatment affected the brain metabolome in order to understand how nitrate and nitrite supplementation may affect indices of cognitive function.MethodsFish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2-4 weeks and free swim, startle response, innate predator avoidance, social cohesion, and shuttle box assays were performed.ResultsNitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. Nitrate- and nitrite-treated fish initially experienced more negative reinforcement and increased time to decision in the shuttle box assay, which is consistent with a decrease in associative learning or executive function however, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite treatment significantly increased anxiety-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted LC-MS/MS analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. An untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment including an 18-19% reduction in the neurotransmitter γ-aminobutyric acid (GABA), and 17-22% reduction in its precursor, glutamine, which may contribute to the increased anxiety-like behavior.ConclusionNitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health but was associated with mild anxiety-like behavior, changes in the brain metabolome, and caused a short-term decrease in executive function or associative learning.

Gut microbiome research increasingly utilizes zebrafish (Danio rerio) given their amenability to high-throughput experimental designs. However, the utility of zebrafish for discerning translationally relevant host-microbiome interactions is constrained by a paucity of knowledge about the biological functions that zebrafish gut microbiota can execute, how these functions associate with zebrafish physiology, and the degree of homology between the genes encoded by the zebrafish and human gut microbiomes. To address this knowledge gap, we generated a foundational catalog of zebrafish gut microbiome genomic diversity consisting of 1,569,102 non-redundant genes from twenty-nine individual fish. We identified hundreds of novel microbial genes as well as dozens of biosynthetic gene clusters of potential clinical interest. The genomic diversity of the zebrafish gut microbiome varied significantly across diets and this variance associated with altered expression of intestinal genes involved in inflammation and immune activation. Zebrafish, mouse, and human fecal microbiomes shared > 50% of their total genomic diversity and the vast majority of gene family abundance for each individual metagenome (∼99%) was accounted for by genes that comprised this shared fraction. These results indicate that the zebrafish gut houses a functionally diverse microbial community that manifests extensive homology to that of humans and mice despite substantial disparities in taxonomic composition. We anticipate that the gene catalog developed here will enable future mechanistic study of host-microbiome interactions using the zebrafish model.ImportanceZebrafish have emerged as an important model system for defining host-microbiome interactions. However, the utility of this model is blunted by limited insight into the functions that are carried by zebrafish gut microbiota, their relationship with zebrafish physiology, and their consistency with the functions carried by human gut microbiota. To address these limitations, we constructed the first genomic database of zebrafish gut microbiome diversity. We use this novel resource to demonstrate that the genomic diversity of the zebrafish gut microbiome varies with diet and this variance links with altered intestinal gene expression. We also identify substantial homology between zebrafish, human, and mouse metagenomic diversity, indicating that these microbiomes may operate similarly.

While recent research reveals that the gut microbiome drives vertebrate health, little is known about whether the mechanisms these microbes employ to interact with physiology are consistent across host species. To help close this knowledge gap, we compared gut metagenomes across 10 vertebrate species, including biomedical animal models, to define the inter-species variation in the biochemical pathways encoded by gut microbiota. Doing so revealed gut-enriched pathways conserved across vertebrates, as well as pathways that vary concordantly with host evolutionary history. Overall, the functional capacity of the non-human gut microbiome generally reflects that of humans, though a subset of the pathways encoded by human gut microbiota are not well represented in non-human microbiomes. Collectively, these results support the use of animal models to study the mechanisms through which gut microbes impact human health, but suggest that researchers should cautiously consider which model will optimally represent a specific mechanism of interest.SignificanceEfforts to understand how the gut microbiome interacts with human physiology frequently relies on the use of animal models. However, it is generally not understood if the biochemical pathways encoded in gut microbiomes of these different animal models – which define the routes of interaction between gut microbes and their hosts – reflect those found in the human gut. To address this question, we compared gut metagenomes generated 10 different vertebrate lineages. In so doing, our study revealed that non-human gut metagenomes generally encode a set of pathways that are consistent with those found in the human gut. However, some human metagenome pathways are poorly represented in non-human guts, including pathways implicated in disease. Moreover, our analysis identified pathways that appear to be conserved across vertebrates, as well as pathways that are linked to the evolutionary history of their hosts, observations that hold potential to clarify the basis for phylosymbiosis.

OBJECTIVES: Dietary nitrate contributes to optimal cardiovascular health, exercise performance, and has been hypothesized to improve cognitive performance and affect cerebral blood flow in specific brain regions. While the mechanisms responsible are not fully understood, we tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We also explored the extent to which treatment caused changes in the brain metabolome. METHODS: Fish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2–4 weeks and free swim, startle response, innate predator avoidance, social cohesion, and shuttle box assays were performed. RESULTS: Nitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. All treatment groups habituated to a repetitive startle, but nitrate-treated fish moved 10% less distance. Data from the shuttle box learning assay is consistent with a decrease in associative learning or executive function with nitrate and nitrite treatment but, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite-treatment also significantly increased anxiety-like behavior, but did not alter epinephrine, norepinephrine or dopamine levels. Targeted LC-MS/MS analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. An untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment including an 18–19% reduction in the neurotransmitter gamma-aminobutyric acid, and 17–22% reduction in its precursor glutamine, which may contribute to the increase anxiety-like behavior in the fish. CONCLUSIONS: Nitrate and nitrite treatment did not adversely affect multiple parameters of health but was associated with mild anxiety-like behavior, changes in brain metabolome, and caused a decrease in executive function or associative learning. FUNDING SOURCES: Celia Strickland and G. Kenneth Austin III Endowment, the Oregon Agricultural Experimental Station, and National Institutes of Health.

The role of epigenetic alterations during cancer has gained increasing attention and has resulted in a paradigm shift in our understanding of mechanisms leading to cancer susceptibility. Sulforaphane (SFN) is a naturally occurring isothiocyanate derived from the precursor glucosinolate, glucoraphanin (GFN), which is found in cruciferous vegetables such as broccoli. Sulforaphane has been shown to suppress tumour growth by several mechanisms including inhibiting histone deacetylases. The objective of this study was to provide a detailed analysis of sulforaphane absorption following a single oral dose of a broccoli sprout supplement in normal dogs. A single dose of broccoli sprout supplement (with active myrosinase) was orally administered to 10 healthy adult dogs. Blood and urine samples were collected prior to dosing, and at various time points post‐dosing. Plasma total SFN metabolite levels peaked at 4 h post‐consumption and were cleared by 24 h post‐consumption. Urinary SFN metabolites peaked at 4 h post‐consumption, and remained detectable at 24 and 48 h post‐supplement consumption. A trend for decrease in histone deacetylase activity was observed at 1 h post‐consumption and a significant decrease was observed at 24 h post‐consumption. The data presented herein indicate that oral SFN is absorbed in dogs, SFN metabolites are detectable in plasma and urine post‐dosing, and SFN and its metabolites have some effect on histone deacetylase activity following a single dose.

The high prevalence of zinc deficiency is a global public health concern, and suboptimal maternal zinc consumption has been associated with adverse effects ranging from impaired glucose tolerance to low birthweights. The mechanisms that contribute to altered development and poor health in zinc deficient offspring are not completely understood. To address this gap, we utilized the Danio rerio model and investigated the impact of dietary zinc deficiency on adults and their developing progeny. Zinc deficient adult fish were significantly smaller in size, and had decreases in learning and fitness. We hypothesized that parental zinc deficiency would have an impact on their offspring’s mineral homeostasis and embryonic development. Results from mineral analysis showed that parental zinc deficiency caused their progeny to be zinc deficient. Furthermore, parental dietary zinc deficiency had adverse consequences for their offspring including a significant increase in mortality and decreased physical activity. Zinc deficient embryos had altered expression of genes that regulate metal homeostasis including several zinc transporters (ZnT8, ZnT9) and the metal-regulatory transcription factor 1 (MTF-1). Zinc deficiency was also associated with decreased expression of genes related to diabetes and pancreatic development in the embryo (Insa, Pax4, Pdx1). Decreased expression of DNA methyltransferases (Dnmt4, Dnmt6) was also found in zinc deficient offspring, which suggests that zinc deficiency in parents may cause altered epigenetic profiles for their progeny. These data should inform future studies regarding zinc deficiency and pregnancy and suggest that supplementation of zinc deficient mothers prior to pregnancy may be beneficial.

Previous studies suggest compounds such as sulforaphane (SFN) derived from cruciferous vegetables may prevent prostate cancer development and progression. This study evaluated the effect of broccoli sprout extract (BSE) supplementation on blood histone deacetylase (HDAC) activity, prostate RNA gene expression, and tissue biomarkers (histone H3 lysine 18 acetylation (H3K18ac), HDAC3, HDAC6, Ki67, and p21). A total of 98 men scheduled for prostate biopsy were allocated into either BSE (200 µmol daily) or a placebo in our double-blind, randomized controlled trial. We used nonparametric tests to evaluate the differences of blood HDAC activity and prostate tissue immunohistochemistry biomarkers between treatment groups. Further, we performed RNA-Seq analysis on the prostate biopsies and identified 40 differentially expressed genes correlated with BSE treatment, including downregulation of two genes previously implicated in prostate cancer development, AMACR and ARLNC1. Although urine and plasma SFN isothiocyanates and individual SFN metabolites were statistically higher in the treatment group, our results did not show a significant difference in HDAC activity or prostate tissue biomarkers. This study indicates BSE supplementation correlates with changes in gene expression but not with several other prostate cancer biomarkers. More research is required to fully understand the chemopreventive effects of BSE supplementation on prostate cancer.

OBJECTIVES: Dietary nitrate supplementation shows protective effects against cardio-metabolic disease, decreases pulmonary oxygen uptake, and improves exercise performance in animal models and humans. However, the biological effect of nitrate on energy metabolism in the liver is not well understood. The objective of this study was to elucidate changes in liver metabolism associated with nitrate treatment and exercise. METHODS: Fish were exposed to sodium nitrate (606.9 mg/L), or control water, for 21 days and analyzed at intervals during a strenuous exercise test. We utilized untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine the effect of nitrate treatment and exercise on the liver metabolome. We measured gene expression of 31 genes linked to energy metabolism and redox signaling. RESULTS: In the absence of exercise, nitrate treatment upregulated expression of genes central to nutrient sensing (pgc1a and sirt3), protein synthesis (mtor) and purine metabolism (pnp5a and ampd1) and downregulated expression of genes involved in mitochondrial fat oxidation (acaca, cpt2 and hadh). Upregulation of these genes was associated with an increased abundance of metabolites involved in endogenous nitric oxide metabolism, dopamine biosynthesis, branched chain amino acid metabolism, and lipid metabolism in nitrate-treated livers at rest, compared to rested controls. As expected, the availability of these metabolites was diminished in nitrate-treated livers relative to rested controls. We found no significant change in gene of metabolites directly linked to glycolysis. CONCLUSIONS: The main novel finding of this study was that sub-chronic nitrate treatment altered dopamine biosynthesis, protein synthesis and lipid metabolism in zebrafish liver without exercise. This is significant because dietary nitrate is emerging as an interesting therapeutic modality for metabolic syndrome and non-alcoholic fatty liver disease by preventing lipid accumulation in the liver. FUNDING SOURCES: Celia Strickland and G. Kenneth Austin III Endowment and National Institutes of Health.

OBJECTIVES: Dietary nitrate (NO(3)(−)) supplementation improves exercise performance by reducing the oxygen cost of exercise and enhancing skeletal muscle function. However, the mechanisms underlying the beneficial effects on exercise performance are not well understood and may be supported by changes in metabolism within the skeletal muscle. The purpose of this study was to elucidate nitrate-induced changes in skeletal muscle energy metabolism associated with improvements in exercise performance that may reflect enhanced metabolic flexibility. METHODS: Fish were exposed to sodium nitrate (60.7 mg/L, 303.5 mg/L, and 606.9 mg/L), or control water, for 21 days and analyzed at intervals during a strenuous exercise test. Nitrate storage in muscle was measured using chemiluminescence. We utilized nuclear magnetic resonance spectroscopy (NMR), liquid-chromatography tandem mass spectrometry (LC-MS/MS) untargeted metabolomics and real-time quantitative polymerase chain reaction (RT-qPCR) to determine changes in muscle metabolism with nitrate and exercise. RESULTS: Nitrate treatment significantly increased muscle nitrate concentrations, while muscle nitrate levels declined with increasing exercise duration, and nitrate treatment was associated with a decrease in the oxygen cost of exercise. In skeletal muscle, nitrate treatment upregulated expression of genes central to nutrient sensing (mtor), glucose (hk2) and lipid metabolism (acaca), redox signaling (nrf2a) and muscle differentiation (sox6). Nitrate treatment caused rested skeletal muscle to have significantly increased metabolites directly linked to energy production (phosphocreatine (PCr), creatine (Cr), adenosine nucleosides, purines, glycolytic, fatty acid and tricarboxylic acid cycle (TCA) intermediates) and a concomitant decrease in these metabolites after exercise, compared to rested-control fish. CONCLUSIONS: Our data suggest that nitrate exposure may improve exercise performance by changing the metabolic programming of muscle prior to exercise, thus increasing the availability of energy producing metabolites required for exercise such as ATP and phosphocreatine. FUNDING SOURCES: Celia Strickland and G. Kenneth Austin III Endowment and National Institutes of Health.

OBJECTIVES: Nitrate, found abundantly in green leafy vegetables, may improve exercise performance by increasing availability and utilization of metabolic fuels that require less oxygen for energy production. However, it is not known if the performance effect occurs at the peak exercise intensity. We hypothesize that supplemental nitrate treatment will promote the metabolism of specific fuels (carbohydrates versus fatty acids) during exercise that require less oxygen to produce ATP. Metabolic analysis will quantify if a net change in these fuels are linked to an improvement in exercise performance with nitrate treatment during submaximal exercise conditions. METHODS: Adult zebrafish were exposed to sodium nitrate (606.9 mg NaNO(3)/L water) or control water for 21 days (n = 54). Fish were sampled at three conditions during a graded exercise test: 1) rested, 2) peak speed, and 3) post-exercise. Whole fish tissue was homogenized and analyzed using high-pressure liquid chromatography Triple Q-ToF mass spectrometry based untargeted metabolomics. RESULTS: Metabolomics analysis resulted in the detection of 12,135 and 10,604 features in positive and negative ion mode respectively. Preliminary results show succinate levels significantly increased in nitrate-treated rested fish as compared to control rested fish. Likewise, a significant increase in methylmalonate, which serves as a vital intermediate in the catabolism of lipids and protein, was detected in nitrate-treated fish at rest relative to rested controls. Nitrate treatment both at rest and at peak exercise intensity, significantly increased the abundance of various acyl carnitines relative to control fish at the same exercise intensity, and these metabolites function to transfer long-chain fatty acids to mitochondria for β-oxidation, relative to control fish at the same exercise intensity. Work is ongoing to further identify metabolites that significantly changed with nitrate treatment at various exercise intensities. CONCLUSIONS: Our data are consistent with the hypothesis that nitrate treatment may alter lipid and carbohydrate metabolism of zebrafish. FUNDING SOURCES: Celia Strickland and G. Kenneth Austin III Endowment, the Oregon Agricultural Experimental Station, and National Institutes of Health.

OBJECTIVES: Whereas phytochemicals derived from cruciferous vegetables have demonstrated health benefits, the data linking intake of these vegetables to health outcomes are inconsistent. These inconsistencies may stem from methodological limitations in accurately assessing cruciferous vegetable exposure. Goals of this study were to explore the use of endogenously deuterium-labeled collard greens combined with untargeted metabolomics to identify unique plant-derived and host-derived metabolites following vegetable consumption as potential biomarkers of cruciferous vegetable intake in humans. METHODS: 26 participants (16 women, 10 men) were fed a breakfast including 100 g of collard greens grown with or without deuterium-labeled water. Plasma was sampled prior to ingestion and 4 h post-ingestion; 24 h urine samples were also collected. High-pressure liquid chromatography Triple Q-ToF mass spectrometry-based untargeted metabolomics was performed. RESULTS: Analysis of collard greens confirmed deuterium-labeled compounds, including glucosinolates (glucobrassicin, sinigrin), and flavonols (quercetin). The maximum abundance of stable isotopologues was (2)H(4) (M(4)) for glucobrassicin and (2)H(12) (M(12)) for 1,2,2'-trisinapoylgentiobiose. Consumption of collard greens was associated with a significant increase in 199 compounds in plasma, including a 5.26-fold increase in the antioxidant hydroferulic acid (q

OBJECTIVES: Treatment with nitrate, but not nitrite, improves exercise performance but the mechanisms responsible are not fully understood. Thus, we tested the hypothesis that nitrate and nitrite treatment alter exercise performance through regulation of genes related to glucose and lipid metabolism in skeletal muscle and liver. Furthermore, we tested the hypothesis that nitrate treatment caused increased abundance and utilization of metabolic fuels in muscle that require less oxygen for energy production. METHODS: Adult zebrafish fish were exposed to sodium nitrate (606.9 mg NaNO(3)/L water), sodium nitrite (19.5 mg NaNO(2)/L of water), or control water for 21 days (n = 9–12/treatment). Liver and muscle gene expression were analyzed by quantitative real-time PCR and liver and muscle metabolomes were assessed by (1)H-NMR untargeted metabolomics. RESULTS: Nitrite treatment significantly increased carnitine palmitoyl transferase 1b (cpt1b) expression in the liver and significantly decreased acetyl-CoA carboxylase (acaca) expression in skeletal muscle. Nitrate treatment significantly increased expression of peroxisome proliferator activated receptor-γ (pparg) muscle while acaca significantly decreased in skeletal muscle. Nitrate treatment also induced significant increases in metabolic fuels, such as ATP and creatine phosphate, and fuel sources including β-hydroxybutyrate and glycolytic intermediates in rested skeletal muscle. After a graded exercise test, these metabolites decreased in skeletal muscle of nitrate-treated fish while they increased with exercise in the skeletal muscle of control-treated zebrafish. CONCLUSIONS: Our data are consistent with the hypothesis that nitrate treatment altered lipid and carbohydrate metabolism of zebrafish, in part, through a pparg mediated mechanism in the liver, and may improve exercise performance through utilization of fuel sources that require less oxygen during exercise. In contrast, our data indicate that nitrite may attenuate exercise performance, in part, by promoting dependence on fatty acid oxidation in the liver of zebrafish. These mechanisms may mediate improved exercise tolerance in populations with cardiovascular disease. FUNDING SOURCES: Celia Strickland and G. Kenneth Austin III Endowment and National Institutes of Health.

Sulforaphane (SFN) is a phytochemical derived from cruciferous vegetables that has multiple molecular targets and anti-cancer properties. Researchers have demonstrated several chemopreventive benefits of SFN consumption, such as reductions in tumor growth, increases in cancer cell apoptosis, and disruption of signaling within tumor microenvironments both in vitro and in vivo. Emerging evidence indicates that SFN exerts several of its chemopreventive effects by altering epigenetic mechanisms. This review summarizes evidence of the impact of SFN on epigenetic events and how they relate to the chemopreventive effects of SFN observed in preclinical and clinical studies of breast and prostate cancers. Specific areas of focus include the role of SFN in the regulation of cell cycle, apoptosis, inflammation, antioxidant defense, and cancer cell signaling and their relationships to epigenetic mechanisms. Finally, remaining challenges and research needs for translating mechanistic work with SFN into human studies and clinical intervention trials are discussed.

Scope Several lines of evidence suggest that the consumption of cruciferous vegetables is beneficial to human health. Yet, underlying mechanisms and key molecular targets that are involved with achieving these benefits in humans are still not fully understood. To accelerate this research, we conduct a human study to identify potential molecular targets of crucifers for further study. This study aims to characterize plasma metabolite profiles in humans before and after consuming fresh broccoli sprouts (a rich dietary source of bioactive sulforaphane). Methods and results Ten healthy adults consume fresh broccoli sprouts (containing 200 μmol sulforaphane equivalents) at time 0 and provide blood samples at 0, 3, 6, 12, 24, and 48 h. An untargeted metabolomics screen reveals that levels of several plasma metabolites are significantly different before and after sprout intake, including fatty acids (14:0, 14:1, 16:0, 16:1, 18:0, and 18:1), glutathione, glutamine, cysteine, dehydroepiandrosterone, and deoxyuridine monophosphate. Evaluation of all time points is conducted using paired t-test (R software) and repeated measures analysis of variance for a within-subject design (Progenesis QI). Conclusion This investigation identifies several potential molecular targets of crucifers that may aid in studying established and emerging health benefits of consuming cruciferous vegetables and related bioactive compounds.

Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions can co-exist in the human population but little is known about their interaction, and in particular, whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish) model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed defined zinc deficient and zinc adequate diets and were spawned resulting in zinc adequate and zinc deficient embryos. The embryos were treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa) were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall, the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes.

Epidemiological studies provide evidence that consumption of cruciferous vegetables, like broccoli, can reduce the risk of cancer development. Sulforaphane (SFN) is a phytochemical derived from cruciferous vegetables that induces anti-proliferative and pro-apoptotic responses in prostate cancer cells, but not in normal prostate cells. The mechanisms responsible for this cancer-specific cytotoxicity remain unclear. To examine this issue we utilized RNA sequencing and determined the transcriptomes of normal prostate epithelial cells, androgen-dependent prostate cancer cells, and androgen-independent prostate cancer cells treated with SFN. SFN treatment dynamically altered gene expression and resulted in distinct transcriptome profiles depending on prostate cell line. SFN also down-regulated the expression of genes that were up-regulated in prostate cancer cells. Network analysis of genes altered by SFN treatment revealed that the transcription factor Specificity protein 1 (Sp1) was present in an average of 90.5% of networks. Sp1 protein was significantly decreased by SFN treatment in prostate cancer cells and Sp1may be an important mediator of SFN-induced changes in expression. Overall, the data show that SFN alters gene expression differentially in normal and cancer cells with key targets in chemopreventive processes, making it a promising dietary anti-cancer agent.

There continues to be interest in targeting epigenetic ‘readers, writers and erasers’ for the treatment of cancer and other pathologies. A mechanistic understanding is frequently lacking, however, for the synergy observed when combining deacetylase and bromodomain inhibitors. We identified cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane (SFN). N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 (HDAC3) and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl ‘reader’ proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. The BET inhibitor JQ1 synergized with SFN in colon cancer cells, and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with SFN+JQ1 in combination implicated a BET/BRD9 acetyl switch, and a shift in the pool of acetyl ‘reader’ proteins in favor of BRD9 regulated target genes. These results highlight the competition that exists among the ‘readers’ of acetylated histone and non-histone proteins, and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments. Citation Format: Gavin S. Johnson, Praveen Rajendran, Li Li, Ying-Shiuan Chen, W. Mohaiza Dashwood, Nhung Nguyen, Ahmet M. Ulusan, Furkan Ertem, Mutian Zhang, Deqiang Sun, Yun Huang, Shan Wang, Hon-Chiu E. Leung, David Lieberman, Laura M. Beaver, Emily Ho, Mark T. Bedford, Kyle Chang, Eduardo Vilar, Roderick H. Dashwood. CCAR2 acetylation establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2614.

Epidemiological evidence has demonstrated a reduced risk of prostate cancer associated with cruciferous vegetable intake. Follow-up studies have attributed this protective activity to the metabolic products of glucosinolates, a class of secondary metabolites produced by crucifers. The metabolic products of glucoraphanin and glucobrassicin, sulforaphane, and indole-3-carbinol respectively, have been the subject of intense investigation by cancer researchers. Sulforaphane and indole-3-carbinol inhibit prostate cancer by both blocking initiation and suppressing prostate cancer progression in vitro and in vivo. Research has largely focused on the anti-initiation and cytoprotective effects of sulforaphane and indole-3-carbinol through induction of phases I and II detoxification pathways. With regards to suppressive activity, research has focused on the ability of sulforaphane and indole-3-carbinol to antagonize cell signaling pathways known to be dysregulated in prostate cancer. Recent investigations have characterized the ability of sulforaphane and indole-3-carbinol derivatives to modulate the activity of enzymes controlling the epigenetic status of prostate cancer cells. In this review, we will summarize the well-established, “classic” non-epigenetic targets of sulforaphane and indole-3-carbinol, and highlight more recent evidence supporting these phytochemicals as epigenetic modulators for prostate cancer chemoprevention.

Summary Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.

Long noncoding RNAs (lncRNAs) have emerged as important in cancer development and progression. The impact of diet on lncRNA expression is largely unknown. Sulforaphane (SFN), obtained from vegetables like broccoli, can prevent and suppress cancer formation. Here we tested the hypothesis that SFN attenuates the expression of cancer-associated lncRNAs. We analyzed whole-genome RNA-sequencing data of normal human prostate epithelial cells and prostate cancer cells treated with 15 μM SFN or dimethylsulfoxide. SFN significantly altered expression of ~100 lncRNAs in each cell type and normalized the expression of some lncRNAs that were differentially expressed in cancer cells. SFN-mediated alterations in lncRNA expression correlated with genes that regulate cell cycle, signal transduction and metabolism. LINC01116 was functionally investigated because it was overexpressed in several cancers, and was transcriptionally repressed after SFN treatment. Knockdown of LINC01116 with siRNA decreased proliferation of prostate cancer cells and significantly up-regulated several genes including GAPDH (regulates glycolysis), MAP1LC3B2 (autophagy) and H2AFY (chromatin structure). A four-fold decrease in the ability of the cancer cells to form colonies was found when the LINC01116 gene was disrupted through a CRISPR/CAS9 method, further supporting an oncogenic function for LINC01116 in PC-3 cells. We identified a novel isoform of LINC01116 and bioinformatically investigated the possibility that LINC01116 could interact with target genes via ssRNA:dsDNA triplexes. Our data reveal that chemicals from the diet can influence the expression of functionally important lncRNAs, and suggest a novel mechanism by which SFN may prevent and suppress prostate cancer.

cope The anticancer agent sulforaphane (SFN) acts via multiple mechanisms to modulate gene expression, including the induction of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent signaling and the inhibition of histone deacetylase activity. Transcriptomics studies were performed in SFN-treated human colon cancer cells and in non-transformed colonic epithelial cells in order to pursue new mechanistic leads. Methods and results RNA-sequencing corroborated the expected changes in cancer-related pathways after SFN treatment. In addition to NAD(P)H quinone dehydrogenase 1 (NQO1) and other well-known Nrf2-dependent targets, SFN strongly induced the expression of Loc344887. This non-coding RNA was confirmed as a novel functional pseudogene for NmrA-like redox sensor 1 (NMRAL1), and was given the name NmrA-like redox sensor 2 pseudogene (NMRAL2P). Chromatin immunoprecipitation experiments corroborated the presence of Nrf2 interactions on the NMRAL2P genomic region, and interestingly, NMRAL2P also served as a co-regulator of NQO1 in human colon cancer cells. Silencing of NMRAL2P via CRISPR/Cas9 genome-editing protected against SFN-mediated inhibition of cancer cell growth, colony formation, and migration. Conclusion NMRAL2P is the first functional pseudogene to be identified both as a direct transcriptional target of Nrf2, and as a downstream regulator of Nrf2-dependent NQO1 induction. Further studies are warranted on NMRAL2P-Nrf2 crosstalk and the associated mechanisms of gene regulation. This article is protected by copyright. All rights reserved

Androgen receptor (AR) is a nuclear receptor transcription factor that plays a central role in normal prostate physiology as well as in prostate cancer biology (PCa). Phytochemicals like 3,3′-Diindolylmethane (DIM) have emerged as promising therapeutic agents against PCa. DIM has been shown to influence both AR activity and other epigenetic regulators in PCa cells. However, it is not known if DIM contributes to PCa suppression via epigenetic regulation of AR target genes. Here we assessed epigenetic regulation of important AR-target genes in LNCaP PCa cells treated with DIM. DIM led to epigenetic suppression of AR-target genes involved in DNA repair (PARP1, MRE11 and DNA-PK) that coincided with an increase in DNA damage. Decreased AR-target genes expression was accompanied by an increase in repressive chromatin marks and loss of AR occupancy and recruitment of EZH2 to their regulatory regions. In addition, decreased expression of DNA repair genes was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Together our results suggest that the dietary phytochemical DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells.

The role of epigenetic alterations in various human chronic diseases has gained increasing attention and has resulted in a paradigm shift in our understanding of disease susceptibility. In the field of cancer research, e.g., genetic abnormalities/mutations historically were viewed as primary underlying causes; however, epigenetic mechanisms that alter gene expression without affecting DNA sequence are now recognized as being of equal or greater importance for oncogenesis. Methylation of DNA, modification of histones, and interfering microRNA (miRNA) collectively represent a cadre of epigenetic elements dysregulated in cancer. Targeting the epigenome with compounds that modulate DNA methylation, histone marks, and miRNA profiles represents an evolving strategy for cancer chemoprevention, and these approaches are starting to show promise in human clinical trials. Essential micronutrients such as folate, vitamin B-12, selenium, and zinc as well as the dietary phytochemicals sulforaphane, tea polyphenols, curcumin, and allyl sulfur compounds are among a growing list of agents that affect epigenetic events as novel mechanisms of chemoprevention. To illustrate these concepts, the current review highlights the interactions among nutrients, epigenetics, and prostate cancer susceptibility. In particular, we focus on epigenetic dysregulation and the impact of specific nutrients and food components on DNA methylation and histone modifications that can alter gene expression and influence prostate cancer progression.

Background Cruciferous vegetables have been associated with the chemoprevention of cancer. Epigenetic regulators have been identified as important targets for prostate cancer chemoprevention. Treatment of human prostate cancer cells with sulforaphane (SFN), a chemical from broccoli and broccoli sprouts, inhibits epigenetic regulators such as histone deacetylase (HDAC) enzymes, but it is not known whether consumption of a diet high in broccoli sprouts impacts epigenetic mechanisms in an in vivo model of prostate cancer. Objective In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, we tested the hypothesis that a broccoli sprout diet suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes the expression of genes regulated by HDACs. Methods TRAMP mice were fed a 15% broccoli sprout or control AIN93G diet; tissue samples were collected at 12 and 28 wk of age. Results Mice fed broccoli sprouts had detectable amounts of SFN metabolites in liver, kidney, colon, and prostate tissues. Broccoli sprouts reduced prostate cancer incidence and progression to invasive cancer by 11- and 2.4-fold at 12 and 28 wk of age, respectively. There was a significant decline in HDAC3 protein expression in the epithelial cells of prostate ventral and anterior lobes at age 12 wk. Broccoli sprout consumption also decreased histone H3 lysine 9 trimethylation in the ventral lobe (age 12 wk), and decreased histone H3 lysine 18 acetylation in all prostate lobes (age 28 wk). A decline in p16 mRNA levels, a gene regulated by HDAC3, was associated with broccoli sprout consumption, but no significant changes were noted at the protein level. Conclusions Broccoli sprout intake was associated with a decline in prostate cancer occurrence and HDAC3 protein expression in the prostate, extending prior work that implicated loss of HDAC3/ corepressor interactions as a key preventive mechanism by SFN in vivo.

Daily rhythms generated by the circadian clock regulate many life functions, including responses to xenobiotic compounds. In Drosophila melanogaster, the circadian clock consists of positive elements encoded by cycle (cyc) and Clock (Clk) and negative elements encoded by period (per) and timeless (tim) genes. The epsilon-isoform of the PAR-domain protein 1 (Pdp1epsilon) transcription factor is controlled by positive clock elements and regulates daily locomotor activity rhythms. Pdp1 target genes have not been identified, and its involvement in other clock output pathways is not known. Mammalian orthologs of Pdp1 have been implicated in the regulation of xenobiotic metabolism; therefore, we asked whether Pdp1 has a similar role in the fly. Using pesticides as model toxicants, we determined that disruption of Pdp1epsilon increased pesticide-induced mortality in flies. Flies deficient for cyc also showed increased mortality, while disruption of per and tim had no effect. Day/night and Pdp1-dependent differences in the expression of xenobiotic-metabolizing enzymes Cyp6a2, Cyp6g1, and alpha-Esterase-7 were observed and likely contribute to impaired detoxification. DHR96, a homolog of constitutive androstane receptor and pregnane X receptor, is involved in pesticide response, and DHR96 expression decreased when Pdp1 was suppressed. Taken together, our data uncover a pathway from the positive arm of the circadian clock through Pdp1 to detoxification effector genes, demonstrating a conserved role of the circadian system in modulating xenobiotic toxicity.

Certain particulate hexavalent chromium [Cr(VI)] compounds are human respiratory carcinogens that release genotoxic soluble chromate, and are associated with fibrosis, fibrosarcomas, adenocarcinomas and squamous cell carcinomas of the lung. We postulate that inflammatory processes and mediators may contribute to the etiology of Cr(VI) carcinogenesis, however the immediate (0-24 h) pathologic injury and immune responses after exposure to particulate chromates have not been adequately investigated. Our aim was to determine the nature of the lung injury, inflammatory response, and survival signaling responses following intranasal exposure of BALB/c mice to particulate basic zinc chromate. Factors associated with lung injury, inflammation and survival signaling were measured in airway lavage fluid and in lung tissue. A single chromate exposure induced an acute immune response in the lung, characterized by a rapid and significant increase in IL-6 and GRO-alpha levels, an influx of neutrophils, and a decline in macrophages in lung airways. Histological examination of lung tissue in animals challenged with a single chromate exposure revealed an increase in bronchiolar cell apoptosis and mucosal injury. Furthermore, chromate exposure induced injury and inflammation that progressed to alveolar and interstitial pneumonitis. Finally, a single Cr(VI) challenge resulted in a rapid and persistent increase in the number of airways immunoreactive for phosphorylation of the survival signaling protein Akt, on serine 473. These data illustrate that chromate induces both survival signaling and an inflammatory response in the lung, which we postulate may contribute to early oncogenesis.

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period( per) and timeless( tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock( Clk) and cycle( cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.

Background: Cruciferous vegetables have been associated with the chemoprevention of cancer. Epigenetic regulators have been identified as important targets for prostate cancer chemoprevention. Treatment of human prostate cancer cells with sulforaphane (SFN), a chemical from broccoli and broccoli sprouts, inhibits epigenetic regulators such as histone deacetylase (HDAC) enzymes, but it is not known whether consumption of a diet high in broccoli sprouts impacts epigenetic mechanisms in an in vivo model of prostate cancer. Objective: In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, we tested the hypothesis that a broccoli sprout diet suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes the expression of genes regulated by HDACs. Methods: TRAMP mice were fed a 15% broccoli sprout, or control AIN93G diet; tissue samples were collected at 12 and 28 weeks of age. Results: Mice fed broccoli sprouts had detectable levels of SFN metabolites in liver, kidney, colon, and prostate tissues. Broccoli sprouts reduced prostate cancer incidence, and progression to invasive cancer by 11- and 2.4-fold at 12 and 28 weeks of age, respectively. There was a significant decline in HDAC3 protein expression in the epithelial cells of prostate ventral and anterior lobes at 12 weeks. Broccoli sprout consumption also decreased histone H3 lysine 9 tri-methylation in the ventral lobe (12 week), and decreased histone H3 lysine 18 acetylation in all prostate lobes (28 weeks). A decline in p16 mRNA levels, a gene regulated by HDAC3, was associated with broccoli sprouts consumption, but no significant changes were noted at the protein level. Conclusions: Broccoli sprout intake caused a decline in prostate cancer occurrence and HDAC3 protein expression in the prostate, extending prior work that implicated loss of HDAC3/corepressor interactions as a key preventive mechanism by SFN in vivo.

The dysregulation of long intergenic non-coding RNA’s (lincRNAs) has become an emerging factor in cancer development. Dietary chemopreventive compounds, such as sulforaphane (SFN) from cruciferous vegetables, can alter epigenetic targets for cancer prevention but the impact of diet on lincRNA expression is unknown. Normal prostate epithelial cells and prostate cancer cells were treated with 15 μM SFN to test the hypothesis that SFN alters the expression of lincRNAs. RNA-sequencing revealed that hundreds of lincRNAs were differentially expressed in prostate cancer cells relative to normal prostate epithelial cells. SFN treatment significantly altered the expression of ~70 lincRNAs in each cell type. The SFN-induced response differed depending on cell line and was dynamic over time. In prostate cancer cells, SFN treatment reversed the aberrant expression of some lincRNAs. Preliminary guilt by association work showed that lincRNA's altered by SFN treatment correlate with genes that regulate angiogenesis, cel...

Epigenetic changes, including aberrant DNA methylation, result in altered gene expression and play an important role in carcinogenesis. Phytochemicals such as sulforaphane (SFN) and 3,3'-diindolylmethane (DIM) are promising chemopreventive agents for the treatment of prostate cancer. Both have been shown to induce re-expression of genes, including tumor suppressor genes silenced in cancer cells, via modulation of epigenetic marks including DNA methylation. However, it remained unclear the effects SFN and DIM on DNA methylation at a genomic scale. The goal of this study was to determine the genome-wide effects of SFN and DIM on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Both SFN and DIM treatment decreased DNA methyltransferase expression in normal prostate epithelial cells (PrEC), and androgen-dependent (LnCAP) and androgen-independent (PC3) prostate cancer cells. The effects of SFN and DIM on promoter methylation profiles in normal PrEC, LnCAP and PC3 prostate cancer cells were determined using methyl-DNA immunoprecipitation followed by genome-wide DNA methylation array. We showed widespread changes in promoter methylation patterns, including both increased and decreased methylation, in all three prostate cell lines in response to SFN or DIM treatments. In particular, SFN and DIM altered promoter methylation in distinct sets of genes in PrEC, LnCAP, and PC3 cells, but shared similar gene targets within a single cell line. We further showed that SFN and DIM reversed many of the cancer-associated methylation alterations, including aberrantly methylated genes that are dysregulated or are highly involved in cancer progression. Overall, our data suggested that both SFN and DIM are epigenetic modulators that have broad and complex effects on DNA methylation profiles in both normal and cancerous prostate epithelial cells. Results from our study may provide new insights into the epigenetic mechanisms by which SFN and DIM exert their cancer chemopreventive effects.

Increased consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer. Indole-3-carbinol (I3C) and 3, 3′-diindolylmethane (DIM), phytochemicals derived from crucifero...

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

Globally, prostate cancer is the second most frequently diagnosed cancer in men although the incidence of cancer varies greatly throughout the world. Nutrition and diet are important modifiable risk factors for prostate cancer development. Epidemiological studies have shown an inverse association between cruciferous vegetable intake and the risk of developing prostate cancer. Here we focus specifically on the molecular mechanisms by which phytochemicals in cruciferous vegetables, sulforaphane (SFN), indole-3-carbinol (I3C) and its derivative 3,3′-diindolylmethane (DIM), may prevent the initiation of prostate cancer and slow tumorigenesis. We have particularly emphasized a possible role for epigenetics in this process as many dietary factors can modulate epigenetic alterations and alter susceptibility to disease. We have identified known and possible epigenetic mechanisms by which these phytochemicals can alter detoxification pathways, sex hormone signaling, and genes that regulate cell cycle, apoptosis, inflammation, angiogenesis and metastasis. The ability of SFN, I3C or DIM to target aberrant epigenetic patterns, in addition to their effects on detoxification/carcinogen metabolism, may make them effective chemoprevention agents at multiple stages of the prostate carcinogenesis pathway. The identification of dietary epigenetic modulators and their use either alone or in combination, may increase efficacy of anti-cancer therapies and prevention strategies, without serious side effects.

Background Chronic inflammation is implicated in the development of several human cancers, including lung cancer. Certain particulate hexavalent chromium [Cr(VI)] compounds are well-documented human respiratory carcinogens that release genotoxic soluble chromate and are associated with fibrosis, fibrosarcomas, adenocarcinomas, and squamous cell carcinomas of the lung. Despite this, little is known about the pathologic injury and immune responses after repetitive exposure to particulate chromates. Objectives In this study we investigated the lung injury, inflammation, proliferation, and survival signaling responses after repetitive exposure to particulate chromate. Methods BALB/c mice were repetitively treated with particulate basic zinc chromate or saline using an intranasal exposure regimen. We assessed lungs for Cr(VI)-induced changes by bronchoalveolar lavage, histologic examination, and immunohistochemistry. Results Single exposure to Cr(VI) resulted in inflammation of lung tissue that persists for up to 21 days. Repetitive Cr(VI) exposure induced a neutrophilic inflammatory airway response 24 hr after each treatment. Neutrophils were subsequently replaced by increasing numbers of macrophages by 5 days after treatment. Repetitive Cr(VI) exposure induced chronic peribronchial inflammation with alveolar and interstitial pneumonitis dominated by lymphocytes and macrophages. Moreover, chronic toxic mucosal injury was observed and accompanied by increased airway pro-matrix metalloprotease-9. Injury and inflammation correlated with airways becoming immunoreactive for phosphorylation of the survival signaling protein Akt and the proliferation marker Ki-67. We observed a reactive proliferative response in epithelial cells lining airways of chromate-exposed animals. Conclusions These data illustrate that repetitive exposure to particulate chromate induces chronic injury and an inflammatory microenvironment that may promote Cr(VI) carcinogenesis.