Mammalian Lignans Enterolactone Research Articles

Supplementing flaxseed meal with sucrose, flaxseed oil, or both: Effects on milk enterolactone, ruminal microbiota profile, production performance, and nutrient utilization in dairy cows

Flaxseed is the richest source of secoisolariciresinol diglucoside, which is converted by ruminal microorganisms primarily to the mammalian lignan enterolactone. Our objective was to investigate the effect of diets containing soybean meal or flaxseed meal (FM) supplemented with sucrose, flaxseed oil, or both, on milk enterolactone concentration yield, diversity, and relative abundance of ruminal bacterial taxa, ruminal fermentation profile, production performance, milk fatty acids (FA) yield, and nutrient utilization in dairy cows. Sixteen Holstein cows (8 multiparous [4 ruminally cannulated] and 8 primiparous) averaging (mean ± SD) 134 ± 54.1 DIM and 679 ± 78.9 kg of BW in the beginning of the study were assigned to treatment sequences in a replicated 4 × 4 Latin square design. Each experimental period lasted 25 d, with 18 d for diet adaptation and 7 d for data and sample collection. Diets were formulated to contain a 60:40 forage:concentrate ratio and included the following (DM basis): (1) 8% soybean meal and 23% ground corn (SBM); (2) 15% FM, 10.7% ground corn, and 5% sucrose (FLX+S); (3) 15% FM, 15.4% ground corn, and 3% flaxseed oil (FLX+O); and (4) 15% FM, 10.2% ground corn, 5% sucrose, and 3% flaxseed oil (FLX+SO). Compared with SBM, the concentration and yield of milk enterolactone increased in cows fed the FM diets but did not differ among FLX+S, FLX+O, and FLX+SO. The relative abundances of the phyla Firmicutes, Verrucomicrobiota, and Actinobacteriota, and those of the bacterial genera Lachnospiraceae NK3A20 group, Eubacterium coprostanoligenes group, Anaeromusa-Anaeroarcus, WCHB1-41, and p-251-o5 decreased, whereas Prevotella and NK4A214 group increased when comparing SBM against at least 1 diet containing FM. Furthermore, the relative abundances of Firmicutes and Actinobacteriota and those of Prevotella, Lachnospiraceae NK3A20 group, Eubacterium coprostanoligenes group, Acetitomaculum, Lachnospiraceae unclassified, NK4A214 group, and Anaeromusa-Anaeroarcus changed (increased or decreased) across the FLX+S, FLX+O, and FLX+SO diets. However, all these changes in the relative abundance of the ruminal bacterial taxa were not conclusively associated with the effect of diets on milk enterolactone. Diets did not affect ruminal pH and concentrations of NH3-N and total VFA. Dry matter intake and yields of milk, milk fat, and milk true protein all decreased in cows fed FLX+O or FLX+SO. Yields of milk total odd-chain FA, branched-chain FA, total <16C FA, and total 16C FA decreased with feeding FLX+O and FLX+SO. The apparent total-tract digestibilities of DM and OM were lowest in the FLX+S and FLX+O diets, with CP and ADF digestibilities lowest in cows receiving FLX+S or FLX+O, respectively. Urinary excretion of total N was lowest with feeding SBM. Contrarily, diets did not affect the urinary excretion of total purine derivatives. In brief, despite the effect of diets on the relative abundance of several ruminal microbiota phyla and genera, we were unable to conclusively associate these changes with increased milk enterolactone in FM-containing diets versus SBM.

Anti-osteoporosis effects of mammalian lignans and their precursors from flaxseed and safflower seed using zebrafish model.

Secoisolariciresinol diglucoside (SDG) and tracheloside (TCL) are the main lignan components of flaxseed cake and safflower seed cake, which are by-products of oil extraction. Both SDG and TCL are metabolized into mammalian lignan enterolactone (EL) with the involvement of intestinal bacteria. In this research, we evaluated the anti-osteoporosis effects of SDG and the in vivo metabolites EL and enterodiol (ED) prepared in our previous work, as well as the newly isolated chemical constituents from safflower seed, including TCL, the lactone ring opening product of TCL (OTCL) and two alkaloids on the alloxan-induced zebrafish model. All the compounds showed significant anti-osteoporosis effects at 80µM, with p<0.05 for EL and p<0.001 for other compounds compared with the model. SDG and TCL showed the most significant and concentration-dependent effects, with p<0.001 compared with model at 20µM. The alkaloids, N-coumaroylserotonin glucoside and N-feruloylserotonin glucoside, also showed anti-osteoporosis at 20µM with p<0.01, whereas EL, ED, and OTCL showed no significant effects. Quantitative real-time polymerase chain reaction revealed that SDG and TCL upregulated the expression of osteogenic genes Runx2, SP7, OPG, Col1a1a, Alp, ON, OPN, and OCN in alloxan-treated zebrafish. The in vivo metabolite of lignans, EL, showed significant anti-inflammatory effect (p<0.01) at 20µM, which might also help to combat osteoporosis and other complications caused by excessive immune response in the body. The results provided scientific data for using the oil extraction by-products as sources of anti-osteoporosis compounds. PRACTICAL APPLICATION: This study found that lignans in flaxseed cake and safflower seed cake exhibited anti-osteoporosis effects by upregulating the expression of osteogenic genes, making the oil extraction by-products sources of anti-osteoporosis compounds.

FLAXSEED MEAL FEEDING TO DAIRY COWS AS A STRATEGY TO IMPROVE MILK ENTEROLACTONE CONCENTRATION: A LITERATURE REVIEW

ABSTRACT: Flaxseed (Linum usitatissimum) is the richest source of the plant lignan secoisolariciresinol diglucoside (SDG). In mammals, including bovine, SDG is converted to the mammalian lignans enterolactone (EL) and enterodiol (ED) by the action of gastrointestinal microbes. There is a great deal of interest in promoting increased intakes of lignans in humans’ diet due to the potential health benefits of mammalian lignans, especially in the prevention of cardiovascular diseases, hypercholesterolaemia, breast and prostate cancers, and osteoporosis. Consumption of milk and dairy products enriched in EL could be an excellent strategy to increase the intake of lignans by humans. This literature review will focus on presenting feeding strategies capable to improve milk enterolactone concentration. Research has demonstrated the potential of flaxseed meal (FM) feeding to dairy cows as a strategy to improve milk EL concentration, therefore enhancing milk nutraceutical proprieties. A considerable number of studies have demonstrated that feeding vegetable lignans-rich sources, such as FM, to dairy cows improves EL in milk. Additionally, it has been reported that changes in the carbohydrate profile of FM-based diets fed to dairy cows can alter the output of milk EL. The application of animal nutrition as a tool to increase nutraceutical properties of milk (i.e. increased EL concentration) is a valuable strategy for promoting the association of milk with humans’ health benefits and is of great interest in contemporary society.&#x0D; Keywords: nutraceutical proprieties, bioactive compounds, lignans, dairy cows, disease risk reduction

Oral Pharmacokinetics of Enriched Secoisolariciresinol Diglucoside and Its Polymer in Rats.

Secoisolariciresinol diglucoside (SDG) is the principal lignan of flaxseed and precursor of its aglycone, secoisolariciresinol (SECO), and the mammalian lignans enterolactone (EL) and enterodiol (ED), the putative bioactive forms of oral administration of SDG. SDG is present in the seed hull as an ester-linked polymer. Although extraction and purification of SDG monomer is costly, the use of naturally occurring SDG in polymer form may offer a more economical approach for delivery of this precursor. The extent of SDG release from the polymer and subsequent bioavailability of SDG metabolites are unknown. To understand the relative bioavailability of SDG polymer, this study examined the comparative bioavailability of enriched SDG and SDG polymer in rats after a single oral SDG equivalent dose (40 mg/kg). A validated LC-MS/MS method quantified SDG and its metabolites in rat plasma following serial blood collections. SDG remained undetectable in rat plasma samples. Unconjugated SECO was detected in plasma after 0.25 h. Unconjugated ED was observed after 8 h (3.4 ± 3.3 ng/mL) and 12 h (6.2 ± 3.3 ng/mL) for enriched SDG and SDG polymer, respectively. Total (conjugated and unconjugated) ED and EL resulting from enriched SDG and SDG polymer reached similar maximal concentrations between 11 and 12 h and demonstrated similar total body exposures (AUC values). These data suggest a similar pharmacokinetic profile between the enriched and polymer form of SDG, providing support for the use of SDG polymer as a more economical precursor for SECO, ED, and EL in applications of chronic disease management.

Short communication: The mammalian lignan enterolactone is absorbed by newborn dairy calves fed enterolactone-enriched milk

Flaxseed is the richest source of the plant lignan secoisolariciresinol diglucoside, which is converted to the mammalian lignans enterolactone (EL) and enterodiol by the gut microbiota of ruminants and humans. Enterolactone has been associated with improved animal and human health due to its antioxidant and anticarcinogenic properties. The objective of this study was to determine the pharmacokinetics of EL in newborn dairy calves fed milk replacer or EL-enriched milk. We hypothesized that newborn Holstein calves fed EL-enriched milk would have greater area under the curve and plasma concentration of EL compared with those fed milk replacer. On d 5 of life, calves were administered 2 L of milk replacer (n = 10; low-EL treatment: 123 nmol/L of EL) or 2 L of EL-enriched milk (n = 10; high-EL treatment: 481 nmol/L of EL) during the morning feeding (0700 h). Blood samples were taken from the jugular vein before (0 h) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, and 48 h after oral administration of treatments. The area under the curve for the plasma concentration of EL was analyzed according to the trapezoidal rule between 0 and 12 h after treatment administration, and it was greater in high- (26 nmol/L × h) than low-EL calves (4.30 nmol/L × h). Similarly, the maximum concentration of EL in plasma was greater in high- (5.06 nmol/L) versus low-EL calves (1.95 nmol/L). Furthermore, the time after treatment intake to reach maximum plasma concentration of EL was faster in high- (4.31 h) compared with low-EL (4.44 h) treatment. Calves were able to absorb EL, indicating that EL-enriched milk can potentially be used as source of EL to pre-weaned ruminants.

Dietary flax meal and abomasal infusion of flax oil on microbial β-glucuronidase activity and concentration of enterolactone in ruminal fluid, plasma, urine and milk of dairy cows

This study was performed to evaluate the effects of dietary flax meal (FM) and abomasal infusion of flax oil (FO) and their interaction on activity of β-glucuronidase in ruminal fluid and feces, and concentration of the mammalian lignan enterolactone (EL) in ruminal fluid, plasma, urine, and milk. Rumen fermentation characteristics and the pH in ruminal fluid and feces were also investigated to determine any potential interference with β-glucuronidase activity and EL concentration. Eight rumen fistulated lactating Holstein cows were assigned to a double 4×4 Latin square design with a 2×2 factorial arrangement of treatments: (1) control diet with no FM (CON); (2) diet containing 124g/kg FM (FMD) in the dry matter (DM); (3) CON and 250g FO/day infused in the abomasum; and (4) FMD and 250g FO/day infused in the abomasum. Abomasal infusion of FO had no effect on β-glucuronidase activity in the gastrointestinal tract and EL concentration of ruminal fluid, plasma, urine, and milk. Dietary FM and abomasal infusion of FO had no effect on fecal pH and microbial activity of β-glucuronidase in ruminal fluid and feces. Dietary FM increased concentrations of EL in ruminal fluid, plasma, urine, and milk. Molar proportion of propionate was increased and that of isovalerate was decreased with FM supplementation. Abomasal infusion of FO had no effect on ruminal concentration of ammonia N. Dietary FM had no effect on ruminal pH. As abomasal infusion of FO had no effect on β-glucuronidase activity in the gastro-intestinal tract and EL concentration, our results suggest that polyunsaturated fatty acids such as n-3 do not interfere with the absorption of mammalian lignans. Moreover, feeding 124g/kg DM FM with a source of FA rich in n-3 bypassing the rumen increases EL concentration in physiological fluids of dairy cows, especially milk, which can be used as a strategy to optimize the concentration of mammalian lignans in milk and their potential beneficial effects on human health.

Short communication: Concentrations of the mammalian lignan enterolactone in preovulatory follicles and the correlation with intrafollicular estradiol in dairy cows fed extruded flaxseed

Flaxseed is a rich source of lignans that can be metabolized to the mammalian lignan enterolactone (EL), which may elicit weak estrogenic or antiestrogenic effects. The objectives of this study were to examine the effects of feeding an extruded flaxseed supplement to dairy cows on concentrations of EL in plasma and preovulatory follicles and the association with intrafollicular estradiol (E2). Twenty-four multiparous 256-d-pregnant Israeli Holstein cows were fed either a standard diet both pre- and postpartum (control; n=12) or provided with an extruded flaxseed supplement (n=12), at 7.9 and 9.2% of dry matter, pre- and postpartum, respectively. Follicular fluid (FF) aspirations were conducted at 84±16 d in lactation as follows: 7 to 8 d following behavioral estrus, cows were injected with prostaglandin F2α and 48h later follicles >7mm were aspirated. Follicles were regarded as preovulatory when the E2-to-progesterone ratio was >1. Plasma EL concentrations were not different between treatment groups; however, concentrations of EL in FF of preovulatory follicles were 1.7 times higher in extruded flaxseed-supplemented cows than in control. Across-treatment analysis revealed a positive correlation between concentrations of EL in plasma and in FF. In addition, intrafollicular EL concentrations were positively correlated with E2 concentrations (r=0.50), and with the intrafollicular E2-to-progesterone ratio. In conclusion, supplementing dairy cows with extruded flaxseed increased EL concentrations in preovulatory follicles. Intrafollicular EL was correlated with E2 concentrations; therefore, the possible effects of EL from flaxseed on follicular steroidogenesis should be considered.

Abstract 5542: Flaxseed and its lignan component protect from asbestos-induced inflammation in mice

Abstract Introduction/Background: Malignant mesothelioma (MM) is a highly lethal form of thoracic cancer with high mortality and poor treatment options. Development of MM has been linked directly to exposure to asbestos fibers, but with a long latency period. Recent studies have indicated that the pathogenesis of asbestos-induced cancers is due, in part, to chronic inflammation and oxidative tissue damage caused by persistent asbestos fibers. Whole grain flaxseed (FS) has known antioxidant, anti-inflammatory and cancer chemopreventive properties. Rationale: As a prelude to future chemoprevention studies, we tested the ability of oral FS and its lignan component, FLC which is enriched in the lignan secoisolariciresinol diglucoside (SDG), to prevent acute asbestos-induced inflammation and inflammatory cytokine release in MM-prone Nf2+/mut;Cdkn2a+/mut mice. Methods: Mice were given a single ip bolus of 400 mM of crocidolite asbestos. They were then placed on 10% FS or 10% FLC supplemented diets 1 day prior (-1) to or after (+1) asbestos instillation. All mice were evaluated 3 days after injection of asbestos for abdominal inflammation and proinflammatory cytokine release. The Nf2+/mut;Cdkn2a+/mut model was selected as it develops an accelerated form of MM when exposed to asbestos. Results: Using liquid chromatography and tandem mass spectrometry (LC/MS/MS), we showed that systemic levels (plasma) of flaxseed lignan metabolites (such as the mammalian lignans Enterolactone (EL) and Enterodiol (ED)) were comparable to those in other mouse models where FS was shown to be an effective chemopreventive agent. The numbers of macrophages (MF) and neutrophils (PMN) in peritoneal lavage fluid indicated that both FS and FLC blunted acute abdominal inflammation induced by asbestos. In addition, the levels of pro-inflammatory cytokines TNF alpha and IL-1 beta in lavage fluid were also decreased by the dietary agents. Conclusions: Our findings suggest that the known chemopreventive properties of FS and its lignan component appear to reduce short-term asbestos-induced inflammation and may thus prove to be a promising dietary agent in the chemoprevention of MM. Funding: This study was supported in part by Penn's SRP Center Grant (P42 ES027320), “Asbestos fate, exposure, remediation and adverse health effects” from the National Institute of Environmental Health (NIEHS) and 1R03CA180548-02 (MCS). Citation Format: Steven M. Albelda, Ralph A. Pietrofesa, Craig W. Menges, Joseph R. Testa, Melpo Christofidou-Solomidou. Flaxseed and its lignan component protect from asbestos-induced inflammation in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5542. doi:10.1158/1538-7445.AM2015-5542

Phytoestrogens and their metabolites in bulk-tank milk: effects of farm management and season.

Phytoestrogens have structures similar to endogenous steroids and may induce or inhibit the response of hormone receptors. The objectives of the present study were to compare the effects of long-term vs. short-term grassland management in organic and conventional dairy production systems, compare organic and conventional production systems and assess seasonal variation on phytoestrogen concentrations in bulk-tank milk. The concentrations of phytoestrogens were analyzed in bulk-tank milk sampled three times in two subsequent years from 28 dairy farms: Fourteen organic (ORG) dairy farms with either short-term or long-term grassland management were paired with 14 conventional (CON) farms with respect to grassland management. Grassland management varied in terms of time since establishment. Short-term grassland management (SG) was defined as establishment or reseeding every fourth year or more often, and long-term grassland management (LG) was defined as less frequent establishment or reseeding. The proportion of red clover (Trifolium pretense L.) in the herbage was positively correlated with milk concentrations of the mammalian isoflavone equol. Therefore, organically produced bulk-tank milk contained more equol than conventionally produced milk, and milk from ORG-SG farms had more equol than milk from ORG-LG farms. Milk produced during the indoor-feeding periods had more equol than milk produced during the outdoor feeding period, because pastures contained less red clover than fields intended for silage production. Organically produced milk had also higher concentrations of the mammalian lignan enterolactone, but in contrast to equol, concentrations increased in the outdoor-feeding periods compared to the indoor-feeding periods. There were no indications of fertility problems on ORG-SG farms who had the highest red clover proportions in the herbage. This study shows that production system, grassland management, and season affect milk concentrations of phytoestrogens. However, compared to soy products, milk concentrations of phytoestrogens are low and future studies are required to investigate if the intake of phytoestrogens from dairy products has physiological effects in humans.

Role of ABCG2 in Transport of the Mammalian Lignan Enterolactone and its Secretion into Milk in Abcg2 Knockout Mice

Lignans are phytoestrogens that are metabolized by the gut microbiota to enterodiol and enterolactone, the main biologically active enterolignans. Substantial interindividual variation in plasma concentration and urinary excretion of enterolignans has been reported, this being determined, at least in part, by the intake of lignan precursors, the gut microbiota, and the host's phase 2 conjugating enzyme activity. However, the role of ATP-binding cassette (ABC) transporters in the transport and disposition of enterolactone has not been reported so far. Active transport assays using parental and Madin-Darby canine kidney epithelial cells transduced with murine and human ABCG2 showed a significant increase in apically directed translocation of enterolactone in transduced cells, which was confirmed by using the selective ABCG2 inhibitor Ko143. In addition, enterolactone also inhibited transport of the antineoplastic agent mitoxantrone as a model substrate, with inhibition percentages of almost 40% at 200 μM for human ABCG2. Furthermore, the endogenous levels in plasma and milk of enterolactone in wild-type and Abcg2((-/-)) knockout female mice were analyzed. The milk/plasma ratio decreased significantly in the Abcg2((-/-)) phenotype, as compared with the wild-type mouse group (0.4 ± 0.1 as against 6.4 ± 2.6). This paper is the first to report that enterolactone is a transported substrate and therefore most probably a competitive inhibitor of ABCG2, which suggests it has a role in the interindividual variations in the disposition of enterolactone and its secretion into milk. The inhibitory activity identified provides a solid basis for further investigation in possible food-drug interactions.

Effects of abomasal infusion of flaxseed (Linum usitatissimum) oil on microbial β-glucuronidase activity and concentration of the mammalian lignan enterolactone in ruminal fluid, plasma, urine and milk of dairy cows

Ruminal microbiota plays an important role in the conversion of plant lignans into mammalian lignans. The main mammalian lignan present in the milk of dairy cows fed flax products is enterolactone (EL). The objectives of the present study were to investigate the effects of abomasal infusion of flax oil on the metabolism of flax lignans and concentrations of EL in biological fluids of dairy cows. A total of six rumen-cannulated dairy cows were assigned within a 2 × 3 factorial arrangement of six treatments utilising flax hulls (0 and 15·9 % of DM) and abomasal infusion of flax oil (0, 250 and 500 g/d). There were six periods of 21 d each. Samples were collected during the last 7 d of each period and subjected to chemical analysis. Flax hull supplementation increased concentrations of EL in ruminal fluid, plasma, urine and milk, while flax oil infusion had no effect. Post-feeding, β-glucuronidase activity in the ruminal fluid of cows infused with 250 g flax oil was significantly lower for cows fed hulls than for those fed the control diet. The present study demonstrated that the presence of a rich source of n-3 fatty acids such as flax oil in the small intestine does not interfere with the absorption of the mammalian lignan EL and that lower ruminal β-glucuronidase activity had no effect on the conversion of flax lignans into EL in the rumen of dairy cows.

Mammary gene expression and activity of antioxidant enzymes and concentration of the mammalian lignan enterolactone in milk and plasma of dairy cows fed flax lignans and infused with flax oil in the abomasum

The objectives of the study were to investigate the effects of dietary supplementation of flax hulls and/or flax oil on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)) in plasma and the mammary gland and the relative mRNA abundance of antioxidant genes in the mammary gland of dairy cows. A total of eight dairy cows were used in a replicated 4 × 4 Latin square design. There were four treatments: control with no flax hulls (CONT), 9·88% flax hulls in the DM (HULL), control with 500 g flax oil/d infused in the abomasum (COFO), 9·88% flax hulls in the DM and 500 g flax oil/d infused in the abomasum (HUFO). Plasma GPX activity tended to decrease with flax oil supplementation. Cows fed HULL had higher levels of CAT, GPX1 and SOD1 mRNA in the mammary gland and lower mRNA abundance of GPX3, SOD2 and SOD3 compared with those fed CONT. Abundance of CAT, GPX1, GPX3, SOD2 and SOD3 mRNA was down-regulated in the mammary gland of cows fed HUFO compared to those fed CONT. The mRNA abundance of CAT, GPX1, GPX3 and SOD3 was lower in the mammary gland of cows fed COFO than in the mammary gland of cows fed CONT. The present study demonstrates that flax hulls contribute to increasing the abundance of some antioxidant genes, which can contribute to protecting against oxidative stress damage occurring in the mammary gland and other tissues of dairy cows.

Production performance and milk composition of dairy cows fed different concentrations of flax hulls

A total of 45 lactating Holstein cows averaging 617 kg of body weight (SE = 20.6) were allotted at week 20 of lactation to five groups of nine cows blocked for similar days in milk to determine the effects of feeding different concentrations of flax hulls on dry matter (DM) intake, milk production, milk composition, digestion, and milk concentration of the mammalian lignan enterolactone. Cows within each block were assigned to one of the five isoenergetic total mixed diets containing either 0, 50, 100, 150, or 200 g/kg DM flax hulls (FHs). The experiment was carried out from wk 20 to 24 of lactation and diets were fed for ad libitum intake. Concentration of FH in the diet had no effect on milk yield, proportions of protein and fat and yields of protein, fat and lactose. There was a linear increase in proportion of lactose in milk and a quadratic effect of feeding level of flax hulls on somatic cell count. Total tract apparent digestibility of DM, acid detergent fiber (ADF), and neutral detergent fiber (aNDF) was similar among diets. Total tract apparent digestibility of ether extract increased with higher proportions of flax hulls in the diet and the increase was more important from 0 to 50 g/kg DM flax hulls. Moreover, milk proportions of total trans, monounsaturated and polyunsaturated fatty acids and concentration of the mammalian lignan enterolactone increased with greater concentration of flax hulls in the diet while the inverse was observed for proportions of saturated fatty acids. Flax hull supplementation may then contribute to the modification of milk composition for better human health with no detrimental effect on productivity of dairy cows.

Tamoxifen, Flaxseed, and the Lignan Enterolactone Increase Stroma- and Cancer Cell–Derived IL-1Ra and Decrease Tumor Angiogenesis in Estrogen-Dependent Breast Cancer

The proinflammatory cytokines IL-1α and IL-1β promote tumor angiogenesis that might be counteracted by the IL-1 receptor antagonist (IL-1Ra), anakinra, a clinically approved agent. A diet with high amounts of phytoestrogens, such as flaxseed (Flax), genistein (GEN), and the mammalian lignan enterolactone (ENL), may affect breast cancer progression in a similar fashion as the antiestrogen tamoxifen. Both cancer cells and tumor stroma may be targets for cancer therapy. By using microdialysis in a model of human breast cancers in nude mice, we could perform species-specific analyses of released proteins in the microenvironment. We show that tumors treated with tamoxifen and fed Flax or ENL exhibited decreased in vivo release of IL-1β derived from the murine stroma and decreased microvessel density whereas dietary GEN had no effects. Cancer cell-released IL-1Ra were approximately 5 times higher than stroma-derived IL-1Ra. Tamoxifen, Flax, and ENL increased IL-1Ra levels significantly whereas GEN did not. The tumor stroma contained macrophages, which expressed the estrogen receptor. In vitro, estradiol decreased IL-1Ra released from breast cancer cells and from cultured macrophages. IL-1Ra decreased endothelial cell proliferation significantly in vitro whereas breast cancer cell proliferation was unaffected in presence of estradiol. Finally, IL-1Ra therapy of tumor-bearing mice opposed estrogen-dependent breast cancer growth and decreased angiogenesis. We conclude that the release of IL-1s both by cancer cells and the stroma, where macrophages are a key component, may offer feasible targets for antiestrogen therapy and dietary interventions against breast cancer.

Abstract P1-02-06: Tamoxifen, Flaxseed and the Lignan Enterolactone Increase Stroma and Cancer Cell Derived IL-1Ra, Decrease Tumor Growth and Angiogenesis in Estrogen Dependent Breast Cancer Explants

Abstract The proinflammatory cytokines IL-1α and IL-1β have been shown to promote tumor angiogenesis, which may be counteracted by the IL-1 receptor antagonist (IL-1Ra). A diet with high amounts of phytoestrogens such as flaxseed (Flax), genistein (GEN), and the mammalian lignan enterolactone (ENL) may affect breast cancer progression possibly in a similar fashion as the anti-estrogen tamoxifen. Both cancer cells and the tumor stroma may be targets for cancer therapy. Therefore, we set up a model with species-specific components of the microenvironment by establishing human breast cancers in nude mice and sampled released proteins by microdialysis. We show that tumors of mice treated with tamoxifen, fed Flax or ENL exhibited decreased in vivo release of IL-1β derived from the murine stroma and decreased microvessel density whereas dietary GEN had no effects compared to controls. Cancer cells released IL-1Ra were approximately five times higher compared with stroma derived and tamoxifen, Flax, and ENL increased these levels significantly whereas GEN did not. In vitro, estradiol decreased released IL-1Ra of breast cancer cells. ENL completely reversed this whereas tamoxifen further increased IL-1Ra levels. IL-1Ra had no effect on breast cancer cell proliferation in presence of E2 in vitro whereas endothelial cell proliferation was significantly decreased by IL-1Ra in a dose dependent manner. Finally, IL-1Ra therapy of tumor bearing mice opposed estrogen dependent breast cancer growth and decreased tumor angiogenesis. These data may provide insights into tumor-stroma mechanisms involved in estrogen effects on breast cancer progression and tamoxifen and dietary interventions against this disease. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-02-06.

The interaction of monensin and flaxseed hulls on ruminal and milk concentration of the mammalian lignan enterolactone in late-lactating dairy cows

Four ruminally fistulated multiparous Holstein cows were assigned to a 4x4 Latin square design with a 2x2 factorial arrangement of treatments to study the effects of dietary supplementation of monensin and flaxseed hulls on ruminal and milk concentration of the mammalian lignan enterolactone (EL) and ruminal and faecal activity of beta-glucuronidase. The hypothesis was that monensin supplementation has no effect on the incorporation of EL into milk when cows are fed flaxseed hulls. Treatments were: 1) control, neither flaxseed hulls nor monensin (CO); 2) diet containing (dry matter basis) 20% flaxseed hulls (FH); 3) diet with monensin (16 mg/kg of dry matter; MO); 4) diet containing 20% (dry matter basis) flaxseed hulls and 16 mg/kg monensin (HM). Intake of dry matter was higher for CO and MO than for FH and HM and monensin had no effect. Milk production decreased in cows fed flaxseed hulls while monensin had no effect. Production of 4% fat-corrected milk and concentrations of milk fat, lactose, urea N, and total solids were similar among treatments. Although there was a decrease in ruminal activity of beta-glucuronidase when feeding flaxseed hulls, the metabolism of plant into mammalian lignans may be increased as shown by enhanced concentration of EL in the rumen and milk. Supplementation with flaxseed hulls then may contribute to favourably change milk composition for better human health by enhancing mammalian lignan EL concentration.

Weekly excretion of the mammalian lignan enterolactone in milk of dairy cows fed flaxseed meal

Flaxseed meal (FM) is rich in the plant lignan secoisolariciresinol diglucoside (SDG) which is converted to the mammalian lignans enterodiol and enterolactone (EL) by ruminal microbiota. Feeding FM to dairy cows increases linearly EL concentration in milk but enterodiol is not detected. The objectives of the study were to determine the length of time to obtain peak EL concentration in the milk of dairy cows fed 20% FM and the length of time to return to EL baseline level in milk when cows are switched from high to low intake of flax SDG. A total of 12 multiparous lactating Holstein cows were assigned randomly to one of two feeding regimens: the control (CO) diet was fed for 6 weeks or the FM diet was fed from week 0 to 3 inclusive and then cows were switched to the control diet from week 3 to 6 inclusive. Milk samples were taken weekly for EL analysis. There was a significant interaction between feeding regimen and week for milk concentration of EL as a result of higher concentration of EL from week 1 to 3 for cows on the FM regimen compared with those on the CO regimen. Concentrations of milk EL on the FM regimen maintained uniform high levels from week 1 to 3 and they decreased significantly from week 3 to 4 when the CO diet was reintroduced in week 3. This study suggests that the conversion of SDG to the mammalian lignan EL and the transfer of EL to the mammary gland are well established after one week of feeding 20% FM in the diet of dairy cows and that milk concentration of EL returns to baseline level after one week of FM deprivation.

Antioxidants and dairy production: the example of flax

This manuscript reports on the main problems decreasing productivity of dairy cows (e.g. fatty liver syndrome and poor fertility) and how antioxidants could enhance it. High producing dairy cows are prone to oxidative stress, and the situation can be exacerbated under certain environmental, physiological, and dietary conditions. Antioxidants have important effects on the expression of genes involved in the antioxidant status, which may enhance animal health and reproduction. Moreover, antioxidants may contribute to decrease the incidence of spontaneous oxidized flavour in milk enriched in polyunsaturated fatty acids. Plant lignans are strong antioxidants and flax is the richest source of plant lignans. Flax lignans are converted in the mammalian lignans enterolactone and enterodiol. The main mammalian lignan in milk is enterolactone and flax lignans are converted in enterolactone mainly under the action of ruminal microbiota. Therefore, ruminal microbiota may be the most important flora to target for plant lignan metabolism in order to increase concentration of mammalian lignan antioxidants in milk of dairy cows. However, more research is required to improve our knowledge on metabolism of other antioxidants in dairy cows and how they can contribute in decreasing milk oxidation.

Milk concentrations of the mammalian lignans enterolactone and enterodiol, milk production, and whole tract digestibility of dairy cows fed diets containing different concentrations of flaxseed meal

A total of 32 lactating Holstein cows averaging 634 ± 27 kg of body weight were allotted at week 33 of lactation to eight groups of four cows blocked for similar days in milk. The objective of the experiment was to determine the effect of feeding four dietary concentrations (0, 50, 100 or 150 g/kg of the dry matter (DM)) of flaxseed meal (FM), which contains the plant lignan precursor secoisolariciresinol diglucoside, on concentrations of two mammalian lignans enterodiol (ED) and enterolactone (EL) in milk. The effects of the four diets on feed intake, milk production, milk composition, and digestion were also studied. Cows within each block were assigned to one of the four isonitrogenous and isoenergetic total mixed diets and the experiment was carried out from week 33 to 37 of lactation. Diets were fed for ad libitum intake. The mammalian lignan ED was not detected in the milk of cows. Concentration of EL in milk increased linearly with the level of incorporation of FM in the diet. Feed intake, milk yield, and milk composition were similar among diets, thus suggesting that FM is a good protein source for diets of mid-lactating dairy cows up to 150 g/kg of the DM and that FM is a feed ingredient enhancing concentration of the mammalian lignan EL in milk.

Ruminal metabolism of flaxseed (Linum usitatissimum) lignans to the mammalian lignan enterolactone and its concentration in ruminal fluid, plasma, urine and milk of dairy cows

Secoisolariciresinol diglucoside is the main flax (Linum usitatissimum) lignan that is converted to the mammalian lignans enterodiol (ED) and enterolactone (EL) by gastrointestinal microbiota. The objectives of the present study were to investigate the role of ruminal microbiota and the effects of flax oil on in vivo metabolism of flax lignans and concentration of EL in biological fluids. Four rumen-cannulated dairy cows were used in a 4 x 4 Latin square design. There were four periods of 21 d each and four treatments utilising flax hulls (1800 g/d) and oil (400 g/d) supplements. The treatments were: (1) oil and hulls administered in the rumen and abomasal infusion of water; (2) oil and hulls administered in the abomasum; (3) oil infused in the abomasum and hulls placed in the rumen; (4) oil placed in the rumen and hulls administered in the abomasum. Samples were collected during the last week of each period and subjected to chemical analysis. The site of supplementation of oil and hulls had no effect on ruminal EL concentration. Supplementing flax oil in the rumen and the abomasum led to similar EL concentrations in urine, plasma and milk. Concentrations of EL were higher in the urine, plasma and milk of cows supplemented with hulls in the rumen than in those placed with hulls in the abomasum. The present study demonstrated that ruminal microbiota play an important role in the metabolism of flax lignans.