Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

Preclinical Demonstration of a Novel Treatment with High Efficacy and No Detectable Toxicity for Inflammatory Skin Conditions including Psoriasis.

Although the management options for psoriasis have progressed with the use of systemic agents, there are few efficacious nonsteroidal topical therapies for patients with limited or lower grade disease. The effects of allopurinol (Allo) and glutathione (GSH) were examined in two different in vitro models for psoriasis. In the first model, human immortalized keratinocytes (HaCaT) were treated with M5 cocktail (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α) in four interventional groups (control, Allo, oxypurinol (Oxy), and methotrexate (MTX)). The number of live and dead cells was determined after treatment for 48 and 72 hrs. Allo decreased cell proliferation (total cells) without increasing cell death compared to both its xanthine oxidase inhibiting metabolite Oxy and a standard agent in clinical use, MTX. In the second model, a human psoriatic skin equivalent (PSE) culture system, cells were treated with vehicle control, Allo and GSH (as monotherapies and in combination), and vitamin D (VitD) for 2 and 6 days followed by histological analysis and altered gene expression. The combined exposure to Allo and GSH was equivalent to a standard antipsoriasis agent VitD in the inhibition of both proliferative and replicative markers. Histologic examination of the tissue at 6 days of exposure to VitD resulted in loss of the integrity of the squamous/epithelial continuity whereas tissue integrity was preserved with Allo and GSH exposure. The additional exposure of GSH to Allo reversed the increased thickness of the dermis layer caused by Allo exposure alone. Taken together, this data shows that topical Allo and GSH may have a synergistic effect with low toxicity and constitute a therapeutic advantage over current nonsteroidal therapies in the treatment of inflammatory skin conditions marked by increased cell proliferation such as psoriasis.

Toxicity and chemical composition of commercial road palliatives versus oil and gas produced waters.

Across the United States, road palliatives are applied to roads for maintenance operations that improve road safety. In the winter, solid rock salts and brine solutions are used to reduce the accumulation of snow and ice, while in the summer, dust suppressants are used to minimize fugitive dust emissions. Many of these products are chloride-based salts that have been linked to freshwater salinization, toxicity to aquatic organisms, and damage to infrastructure. To minimize these impacts, organic products have been gaining attention, though their widespread adoption has been limited due to their higher cost. In some states, using produced water from conventionally drilled oil and gas wells (OGPWs) on roads is permitted as a cost-effective alternative to commercial products, despite its typically elevated concentrations of heavy metals, radioactivity, and organic micropollutants. In this study, 17 road palliatives used for winter and summer road maintenance were collected and their chemical composition and potential human toxicity were characterized. Results from this study demonstrated that liquid brine solutions had elevated levels of trace metals (Zn, Cu, Sr, Li) that could pose risks to human and environmental health. The radium activity of liquid calcium chloride products was comparable to the activity of OGPWs and could be a significant source of radium to the environment. The organic fractions of evaluated OGPWs and chloride-based products posed little risk to human health. However, organic-based dust suppressants regulated toxicity pathways related to xenobiotic metabolism, lipid metabolism, endocrine disruption, and oxidative stress, indicating their use could lead to environmental harm and health risks to operators handing these products and residents living near treated roads.

Accumulating evidence to support the safe and efficacious use of a proprietary blend of capsaicinoids in mediating risk factors for obesity.

Obesity is a significant public health concern, and finding safe and effective means for combating this condition is needed. This study investigates the safety and efficacy of supplementation of a blend of capsaicinoids on weight gain, fat mass, and blood chemistry in a high-fat diet (HFD) model of obesity in mice and on adipocyte differentiation and gene expression in 3T3-L1 preadipocytes. High-fat diet (HFD)-fed mice were treated with a proprietary capsaicinoid concentrate (Capsimax®; OmniActive Health Technologies Ltd., India) and compared to orlistat (ORL) and normal chow-fed mice (NC). Mice fed a high-fat diet showed significantly lower weight gain upon Capsimax® (CAP) administration than their HFD counterparts and similar to that observed with ORL animals. In addition, CAP decreased the high-fat diet-induced increases in adipose tissue and epididymal fat pad mass and hypertrophy after 52 days of treatment. Both the CAP and ORL groups had increased plasma concentrations of leptin. CAP extracts decreased triacylglycerol content in 3T3-L1 preadipocytes and decreased markers of adipogenesis including peroxisome proliferator-activated receptor (PPAR-É£) and fatty acid-binding protein 4 (FABP4). Expression of genes involved in lipogenesis such as stearoyl-CoA desaturase (SCD) and fatty acid synthase (FSN) was decreased by CAP in a dose-dependent manner. Thermogenic genes and markers of brown adipose tissue including uncoupling protein 1 (UCP1) and PR domain-containing 16 (Prdm16) were induced by CAP in the preadipocyte cells. These in vivo and in vitro data support that this proprietary capsaicinoid concentrate reduces weight gain and adiposity at least in part through decreasing lipogenesis and increasing thermogenesis.

Lipoxygenase catalyzed metabolites derived from docosahexaenoic acid are promising antitumor agents against breast cancer.

Docosahexaenoic acid (DHA) is known to inhibit breast cancer in the rat. Here we investigated whether DHA itself or select metabolites can account for its antitumor action. We focused on metabolites derived from the lipoxygenase (LOX) pathway since we previously showed that they were superior anti-proliferating agents compared to DHA; 4-OXO-DHA was the most potent. A lipidomics approach detected several LOX-metabolites in plasma and the mammary gland in rats fed DHA; we also identified for the first time, 4-OXO-DHA in rat plasma. In a reporter assay, 4-OXO-DHA and 4-HDHA were more effective activators of PPARÉ£ than DHA. In breast cancer cell lines, 4-OXO-DHA induced PPARÉ£ and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) but inhibited the activity of NF-κB and suppressed PI3K and mTOR signaling. Because of the structural characteristics of 4-OXO-DHA (Michael acceptor), not shared by any of the other hydroxylated-DHA, we used MS and showed that it can covalently modify the cysteine residue of NF-κB. We have also shown that the chemopreventive effect of DHA is associated with significant reduction of PGE2 levels, in both rat mammary tumors induced by MNU and non-involved mammary tissues. Collectively, our results indicate that 4-OXO-DHA is the metabolite of choice in future chemoprevention studies.

Detection and removal of biologically active organic micropollutants from hospital wastewater.

The presence of contaminants of emerging concern (CECs), such as antibiotics, antimicrobial disinfectants, nonprescription drugs, personal care products, pharmaceuticals, and steroids, in water resources can impact aquatic and human health. A large portion of the CECs entering regional wastewater treatment plants originate from hospitals. The purposes of this study were to conduct exploratory analytical work to characterize two hospital wastewaters and to evaluate treatment of CECs at hospitals before dilution with domestic wastewater. A 24-h batch reaction with biogenic manganese oxides coated onto coir fiber was used to treat the wastewaters. Organic contaminants in the wastewaters were concentrated by both liquid-liquid extraction (LLE) and solid-phase extraction (SPE). LLE extracts were analyzed by Comprehensive Two-Dimensional Gas Chromatography/Time-of-Flight Mass Spectrometry (GC × GC-TOFMS) while SPE extracts were analyzedby UltraHigh Performance Liquid Chromatography/Time-of-Flight Mass Spectrometry (UHPLC-TOFMS). Fifty-two organic micropollutants were detected (26 by GC × GC-TOFMS, 25 by UHPLC-TOFMS, 1 by both) in the wastewaters, while 29 were removed by >90% and six were degraded by <50% after treatment. Control experiments revealed that sorption to coir fiber and oxidation by manganese oxides were the primary contaminant removal mechanisms. Both the LLE and SPE extracts were used to evaluate potential human toxicity of the hospital wastewaters before and after treatment. Twenty-eight human cell-based bioreceptor assays were used to screen the wastewaters, and secondary tests were run to quantify toxicity equivalents to activated receptors. The wastewaters initially contained organic micropollutants that strongly activated the Androgen Receptor, Estrogen Receptor ß, and the Mineralocorticoid Receptor but no bioactive compounds were detected after treatment. Point-of-entry treatment of hospital wastewater should reduce bioactive compounds from entering the environment.

Replication Study: Systematic identification of genomic markers of drug sensitivity in cancer cells.

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Vanden Heuvel et al., 2016), that described how we intended to replicate selected experiments from the paper 'Systematic identification of genomic markers of drug sensitivity in cancer cells' (Garnett et al., 2012). Here we report the results. We found Ewing's sarcoma cell lines, overall, were more sensitive to the PARP inhibitor olaparib than osteosarcoma cell lines; however, while the effect was in the same direction as the original study (Figure 4C; Garnett et al., 2012), it was not statistically significant. Further, mouse mesenchymal cells transformed with either the EWS-FLI1 or FUS-CHOP rearrangement displayed similar sensitivities to olaparib, whereas the Ewing's sarcoma cell line SK-N-MC had increased olaparib sensitivity. In the original study, mouse mesenchymal cells transformed with the EWS-FLI1 rearrangement and SK-N-MC cells were found to have similar sensitivities to olaparib, whereas mesenchymal cells transformed with the FUS-CHOP rearrangement displayed a reduced sensitivity to olaparib (Figure 4E; Garnett et al., 2012). We also studied another Ewing's sarcoma cell line, A673: A673 cells depleted of EWS-FLI1 or a negative control both displayed similar sensitivities to olaparib, whereas the original study reported a decreased sensitivity to olaparib when EWS-FLI1 was depleted (Figure 4F; Garnett et al., 2012). Differences between the original study and this replication attempt, such as the use of different sarcoma cell lines and level of knockdown efficiency, are factors that might have influenced the outcomes. Finally, where possible, we report meta-analyses for each result.

Effects of cranberry extracts on gene expression in THP-1 cells.

Cranberry contains high levels of nutrients and bioactive molecules that have health-promoting properties. The purpose of the present studies was to determine if cranberry extracts (CEs) contain phytochemicals that exert anti-inflammatory effects. The human monocytic cell line THP-1 was treated with two CEs (CE and 90MX) and subsequently challenged with Lipopolysaccharides (LPS). Tumor necrosis factor α (TNF α) expression was decreased in the CE-treated cells, indicative of an anti-inflammatory effect. Gene expression microarrays identified several immune-related genes that were responsive to CEs including interferon-induced protein with tetratricopeptide repeats 1 and 3 (IFIT 1 and 3), macrophage scavenger receptor 1 (MSR1) and colony-stimulating factor 2 (CSF2). In addition, in the CE-treated cells, metallothionein 1F and other metal-responsive genes were induced. Taken together, this data indicates that CEs contain bioactive components that have anti-inflammatory effects and may protect cells from oxidative damage.

Biliverdin Reductase A Attenuates Hepatic Steatosis by Inhibition of Glycogen Synthase Kinase (GSK) 3ß Phosphorylation of Serine 73 of Peroxisome Proliferator-activated Receptor (PPAR) α.

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3ß (GSK3ß) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3ß phosphorylates serine 73 (Ser(P)73) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3ß activity and Ser(P)73 of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKß-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

Registered report: Systematic identification of genomic markers of drug sensitivity in cancer cells.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about the reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Systematic identification of genomic markers of drug sensitivity in cancer cells" by Garnett and colleagues, published in Nature in 2012 (Garnett et al., 2012). The experiments to be replicated are those reported in Figures 4C, 4E, 4F, and Supplemental Figures 16 and 20. Garnett and colleagues performed a high throughput screen assessing the effect of 130 drugs on 639 cancer-derived cell lines in order to identify novel interactions for possible therapeutic approaches. They then tested this approach by exploring in more detail a novel interaction they identified in which Ewing's sarcoma cell lines showed an increased sensitivity to PARP inhibitors (Figure 4C). Mesenchymal progenitor cells (MPCs) transformed with the signature EWS-FLI1 translocation, the hallmark of Ewing's sarcoma family tumors, exhibited increased sensitivity to the PARP inhibitor olaparib as compared to MPCs transformed with a different translocation (Figure 4E). Knockdown mediated by siRNA of EWS-FLI1 abrogated this sensitivity to olaparib (Figure 4F). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

Therapeutic potential of GW501516 and the role of Peroxisome proliferator-activated receptor ß/δ and B-cell lymphoma 6 in inflammatory signaling in human pancreatic cancer cells.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a member of the nuclear receptor superfamily and a ligand-activated transcription factor that is involved in the regulation of the inflammatory response via activation of anti-inflammatory target genes and ligand-induced disassociation with the transcriptional repressor B-cell lymphoma 6 (BCL6). Chronic pancreatitis is considered to be a significant etiological factor for pancreatic cancer development, and a better understanding of the underlying mechanisms of the transition between inflammation and carcinogenesis would help further elucidate chemopreventative options. The aim of this study was to determine the role of PPARß/δ and BCL6 in human pancreatic cancer of ductal origin, as well as the therapeutic potential of PPARß/δ agonist, GW501516. Over-expression of PPARß/δ inhibited basal and TNFα-induced Nfkb luciferase activity. GW501516-activated PPARß/δ suppressed TNFα-induced Nfkb reporter activity. RNAi knockdown of Pparb attenuated the GW501516 effect on Nfkb luciferase, while knockdown of Bcl6 enhanced TNFα-induced Nfkb activity. PPARß/δ activation induced expression of several anti-inflammatory genes in a dose-dependent manner, and GW501516 inhibited Mcp1 promoter-driven luciferase in a BCL6-dependent manner. Several pro-inflammatory genes were suppressed in a BCL6-dependent manner. Conditioned media from GW501516-treated pancreatic cancer cells suppressed pro-inflammatory expression in THP-1 macrophages as well as reduced invasiveness across a basement membrane. These results demonstrate that PPARß/δ and BCL6 regulate anti-inflammatory signaling in human pancreatic cancer cells by inhibiting NFκB and pro-inflammatory gene expression, and via induction of anti-inflammatory target genes. Activation of PPARß/δ may be a useful target in pancreatic cancer therapeutics.

Differential nucleobase protection against 5-fluorouracil toxicity for squamous and columnar cells: implication for tissue function and oncogenesis.

PURPOSE: The goal of these studies was to test if local excess of a normal nucleobase substrate prevents the toxicity of protracted 5FU exposure used in human cancer treatment. METHODS: Messenger RNA expression studies were performed of 5FU activating enzymes in human colon cancer cells lines (CaCo-2, HT-29), primary human gingival cells (HEGP), and normal esophageal and gastric clinical tissue samples. Excess nucleobase was then used in vitro to protect cells from 5FU toxicity. RESULTS: Pyrimidine salvage pathways predominate in squamous cells of the gingiva (HEGP) and esophageal tissue. Excess salvage nucleobase uracil but not adenine prevented 5FU toxicity in HEGP cells. Pyrimidine de novo synthesis predominates in columnar Caco-2, HT-29 and gastric tissue. Excess nucleobase adenine but not uracil prevented 5FU toxicity to Caco-2 and HT-29 cells. CONCLUSION: The directed application of the normal nucleobase uracil to the squamous cells of the oral mucosa and palms and soles together with the delivery of the normal nucleobase adenine to the columnar cells of the GI tract may enable the safe delivery of higher 5FU dose intensity. These results also suggest a feature of tissue function where squamous cells grow largely by recycling overlying tissue cell components. Columnar cells use absorbed surface nutrients for de novo growth. A disruption of this tissue function can result in growth derived from an underlying nutrient source. That change would also cause the loss of the region of cell turnover at the tissue surface. Subsequent cell proliferation with limiting nutrient availability could promote oncogenesis in such initiated tissue.

Effects of whole and refined grains in a weight-loss diet on markers of metabolic syndrome in individuals with increased waist circumference: a randomized controlled-feeding trial.

BACKGROUND: Higher whole-grain (WG) intake is associated with a lower prevalence of metabolic syndrome (MetS); however, there is inconsistent clinical evidence with regard to the benefit of WGs compared with refined grains (RGs) on MetS. OBJECTIVE: We hypothesized that consuming WGs in the place of RGs would improve MetS criteria in individuals with or at risk of MetS. DESIGN: A randomized, controlled, open-label parallel study was conducted in 50 overweight and obese individuals with increased waist circumference and one or more other MetS criteria. Participants consumed a controlled weight-loss diet containing either WG or RG (control) products for 12 wk. Body composition, MetS criteria and related markers, and plasma alkylresorcinols (compliance marker of WG intake) were measured at baseline and at 6 and 12 wk. A subgroup (n = 28) underwent magnetic resonance imaging to quantify subcutaneous and visceral adipose tissue (AT). RESULTS: Baseline variables were not significantly different between groups; however, the RG group tended to have higher triglycerides and lower high-density lipoprotein (HDL) cholesterol (P = 0.06). Alkylresorcinols increased with consumption of the WG diet and did not change with consumption of the RG diet (time × treatment, P < 0.0001), which showed dietary compliance. There were no differences in anthropometric changes between groups; however, weight, body mass index, and percentage of body AT decreased at both 6 and 12 wk (P < 0.05), and reductions in percentage of abdominal AT occurred by 6 wk and did not change between 6 and 12 wk (P = 0.09). Both glucose (P = 0.02) and HDL cholesterol (P = 0.04) were lower with the consumption of the WG compared with the RG diet. However, when noncompliant individuals (n = 3) were removed, the glucose effect was stronger (P = 0.01) and the HDL-cholesterol effect was no longer significant (P = 0.14). CONCLUSIONS: Replacing RGs with WGs within a weight-loss diet does not beneficially affect abdominal AT loss and has modest effects on markers of MetS. WGs appear to be effective at normalizing blood glucose concentrations, especially in those individuals with prediabetes.

Evidence for triclosan-induced activation of human and rodent xenobiotic nuclear receptors.

The bacteriostat triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether) (TCS) decreases rat serum thyroxine via putative nuclear receptor (NR) interaction(s) and subsequent transcriptional up-regulation of hepatic catabolism and clearance. However, due to the evolutionary divergence of the constitutive androstane and pregnane-X receptors (CAR, PXR), TCS-mediated downstream effects may be species-dependent. To test the hypothesis that TCS activates xenobiotic NRs across species, cell-based NR reporter assays were employed to assess potential activation of rat, mouse, and human PXR, and rat, mouse, and three splice variants of human CAR. TCS activated hPXR, acted as an inverse agonist of hCAR1, and as a weak agonist of hCAR3. TCS failed to activate rPXR in full-length receptor reporter assays, and instead acted as a modest inverse agonist of rCAR. Consistent with the rat data, TCS also failed to activate mPXR and was a modest inverse agonist of mCAR. These data suggest that TCS may interact with multiple NRs, including hPXR, hCAR1, hCAR3, and rCAR in order to potentially affect hepatic catabolism. Overall these data support the conclusion that TCS may interact with NRs to regulate hepatic catabolism and downstream thyroid hormone homeostasis in both rat and human models, though perhaps by divergent mechanisms.

Role of Peroxisome Proliferator-Activated Receptor ß/δ and B-Cell Lymphoma-6 in Regulation of Genes Involved in Metastasis and Migration in Pancreatic Cancer Cells.

PPARß/δ is a ligand-activated transcription factor that regulates various cellular functions via induction of target genes directly or in concert with its associated transcriptional repressor, BCL-6. Matrix remodeling proteinases are frequently over-expressed in pancreatic cancer and are involved with metastasis. The present study tested the hypothesis that PPARß/δ is expressed in human pancreatic cancer cells and that its activation could regulate MMP-9, decreasing cancer cells ability to transverse the basement membrane. In human pancreatic cancer tissue there was significantly higher expression of MMP-9 and PPARß/δ, and lower levels of BCL-6 mRNA. PPARß/δ activation reduced the TNF α -induced expression of various genes implicated in metastasis and reduced the invasion through a basement membrane in cell culture models. Through the use of short hairpin RNA inhibitors of PPARß/δ, BCL-6, and MMP-9, it was evident that PPARß/δ was responsible for the ligand-dependent effects whereas BCL-6 dissociation upon GW501516 treatment was ultimately responsible for decreasing MMP-9 expression and hence invasion activity. These results suggest that PPARß/δ plays a role in regulating pancreatic cancer cell invasion through regulation of genes via ligand-dependent release of BCL-6 and that activation of the receptor may provide an alternative therapeutic method for controlling migration and metastasis.

Marine- and plant-derived ω-3 fatty acids differentially regulate prostate cancer cell proliferation.

Fish oil contains the marine ω-3 polyunsaturated fatty acids (ω-3 PUFAs) docosahexaenoic (DHA) and eicosapentaenoic acid (EPA). The consumption of diets rich in these fatty acids is associated with a decreased incidence of prostate cancer. However, there is limited knowledge regarding the non-marine ω-3 PUFA α-linolenic acid (ALA). To study which ω-3 PUFAs are more effective in prostate cancer prevention, and whether the mechanisms of action are conserved between them, we investigated the effect of DHA, EPA and ALA on the human prostate cancer cell lines PC-3 and LNCaP. Different trends of inhibition of PC-3 cell proliferation were observed for the three ω-3 PUFA, with DHA having the most pronounced effects on cell proliferation, while ALA had the minimum effects of the three ω-3 PUFAs. All the ω-3 PUFAs decreased fatty acid synthase (FASN) mRNA. Concerning genes involved in inflammation, cell cycle and apoptosis, DHA regulated the most genes in all categories, followed by EPA and then ALA. In addition, DHA and EPA increased the gene expression of the pro-apoptotic protein activating transcription factor 3 mRNA. Moreover, these two fatty acids significantly induced apoptosis. In conclusion, while some mechanisms of cancer cell inhibition are conserved among ω-3 PUFA, the extent, magnitude, and duration of transcriptional changes vary for each individual fatty acid.

Mechanistic examination of walnuts in prevention of breast cancer.

Walnuts contain bioactive molecules that may contribute to their beneficial effects, including alpha-linolenic acid (ALA) and phytosterols. In these studies, extracts of walnut, purified compounds, or postprandial serum were examined for effects on breast cancer cell proliferation and gene expression. Extracts derived from walnut oil decreased proliferation of MCF-7 cells, as did ALA and ß-sitosterol. The gene expression response of ALA in the mouse breast cancer cell line TM2H indicates this molecule has multiple cellular targets with peroxisome proliferator-activated receptor (PPAR) target genes, liver X receptor (LXR), and farnesoid X receptor (FXR) target genes being affected. In transactivation assays, walnut oil extracts increased activity of FXR to a greater extent than the other tested nuclear receptors. When examined separately, walnut components ALA and ß-sitosterol were the most efficacious activators of FXR. When serum from individuals fed walnut components were applied to MCF-7 cells, there was a correlation between body mass index and breast cancer cell proliferation in vitro. Taken together, these data support an effect of walnut and its bioactive constituents on mammary epithelial cells and that multiple molecular targets may be involved.

Nutrigenomics and nutrigenetics of ω3 polyunsaturated fatty acids.

Diets rich in ω3 polyunsaturated fatty acids (ω3-PUFAs) such as alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid are associated with decreased incidence and severity of several chronic diseases including cardiovascular disease (CVD) and cancer. At least some of the beneficial effects of these dietary fatty acids are via metabolites such as prostaglandins, leukotrienes, thromboxanes, and resolvins. The effects of ω3-PUFAs are in contrast to those of fatty acids with virtually identical structures, such as the ω6-PUFAs linoleic acid and arachidonic acid, and their corresponding metabolites. The purpose of this chapter is to discuss both the nutrigenomics (nutrient-gene interactions) and nutrigenetics (genetic variation in nutrition) of dietary fatty acids with a focus on the ω3-PUFAs (Gebauer et al., 2007(1)). Important in the biological response for these fatty acids or their metabolites are cognate receptors that are able to regulate gene expression and coordinately affect metabolic or signaling pathways associated with CVD and cancer. Four nuclear receptor (NR) subfamilies will be emphasized as receptors that respond to dietary and endogenous ligands: (1) peroxisome proliferator-activated receptors, (2) retinoid X receptors, (3) liver X receptors, and (4) farnesoid X receptor. In addition to the different responses elicited by varying structures of fatty acids, responses may vary because of genetic variation in enzymes that metabolize ω3- and ω6 fatty acids or that respond to them. In particular, polymorphisms in the fatty acid desaturases and the aforementioned NRs contribute to the complexity of nutritional effects seen with ω3-PUFAs. Following a brief introduction to the health benefits of ω3-PUFAs, the regulation of gene expression by these dietary fatty acids via NRs will be characterized. Subsequently, the effects of single-nucleotide polymorphisms (SNPs) in key enzymes involved in the metabolism and response to ω3-PUFAs will be described. An outline of the events to be explored is shown in Fig. 1. Understanding the nutrigenomics and nutrigenetics of dietary fatty acids is key to understanding the etiology, as well as prevention, of critically important human diseases including CVD and cancer.

The dietary fatty acid 10E12Z-CLA induces epiregulin expression through COX-2 dependent PGF(2α) synthesis in adipocytes.

Conjugated linoleic acids (CLAs) are a group of dietary fatty acids that are widely marketed as weight loss supplements. The isomer responsible for this effect is the trans-10, cis-12 CLA (10E12Z-CLA) isomer. 10E12Z-CLA treatment during differentiation of 3T3-L1 adipocytes induces expression of prostaglandin-endoperoxide synthase-2 (Cyclooxygenase-2; COX-2). This work demonstrates that COX-2 is also induced in fully differentiated 3T3-L1 adipocytes after a single treatment of 10E12Z-CLA at both the mRNA (20-40 fold) and protein level (7 fold). Furthermore, prostaglandin (PG)F(2α), but not PGE(2), is significantly increased 10 fold. In female BALB/c mice fed 0.5% 10E12Z-CLA for 10 days, COX-2 was induced in uterine adipose (2 fold). In vitro, pharmacological COX-2 inhibition did not block the effect of 10E12Z-CLA on adipocyte-specific gene expression although PGF(2α) was dose-dependently decreased. These studies demonstrate that PGF(2α) was not by itself responsible for the reduction in adipocyte character due to 10E12Z-CLA treatment. However, PGF(2α), either exogenously or endogenously in response to 10E12Z-CLA, increased the expression of the potent mitogen and epidermal growth factor (EGF) receptor (EGFR) ligand epiregulin in 3T3-L1 adipocytes. Blocking PGF(2α) signaling with the PGF(2α) receptor (FP) antagonist AL-8810 returned epiregulin mRNA levels back to baseline. Although this pathway is not directly responsible for adipocyte dependent gene expression, these results suggest that this signaling pathway may still have broad effect on the adipocyte and surrounding cells.