Enterolactone Concentrations Research Articles

Urinary lignans and inflammatory markers in the US National Health and Nutrition Examination Survey (NHANES) 1999–2004 and 2005–2008

Chronic inflammation has been implicated in the etiology of various chronic diseases. We previously found that certain urinary isoflavones are associated with markers of inflammation. In the present study, we examined the associations of serum C-reactive protein (CRP) and white blood cell (WBC) count with lignans, which are more frequent in the Western diet than isoflavones. Our analysis included 2,028 participants of NHANES 2005-2008 and 2,628 participants of NHANES 1999-2004 aged 18 years and older. The exposures of interest were urinary mammalian lignans (enterodiol and enterolactone). Outcome variables were two inflammatory markers (CRP [≤10 mg/L] and WBC [≥3.0 and ≤11.7 (1,000 cells/μL)]). Log-transformed CRP concentration and WBC count by log-transformed creatinine-standardized concentrations of mammalian lignans were used for linear regression. Statistically significant inverse associations of urinary lignan, enterodiol, and enterolactone concentrations with circulating CRP and WBC counts were observed in the multivariate-adjusted models: In NHANES 2005-2008, per one-percent increase in lignan concentrations in the urine, CRP concentrations and WBC counts decreased by 8.1 % (95 % CI -11.5, -4.5) and 1.9 % (95 % CI -2.7; -1.2), respectively. Per one-percent increase in enterodiol and enterolactone, WBC counts decreased by 2.1 % (95 % CI -2.8, -1.3) and 1.3 % (95 % CI -1.9, -0.6), respectively. In NHANES 1999-2004, analogous results were 3.0 % (95 % CI -5.6, -0.3), 1.2 % (95 % CI -2.0; -0.4), 1.0 % (95 % CI -1.8, -0.2), and 0.8 % (95 % CI -1.4, 0.2). Mammalian lignans were inversely associated with markers of chronic inflammation. Due to the cross-sectional design, our findings require confirmation in prospective studies.

Low serum enterolactone concentration is associated with low colonicLactobacillus–Enterococcuscounts in men but is not affected by a synbiotic mixture in a randomised, placebo-controlled, double-blind, cross-over intervention study

The aims of the present study were to assess the possible differences in faecal microbiota between men with a low serum enterolactone concentration and those with a high concentration, and to investigate the impact of a synbiotic mixture on serum enterolactone concentration in men with a low concentration. We compared faecal microbiota between ten men with the lowest serum enterolactone concentration and ten men with the highest concentration at recruitment (n 84). Furthermore, we carried out a randomised, double-blind, placebo-controlled, cross-over intervention study (6-week intervention periods and 4-week washout period) to investigate the impact of a synbiotic mixture (two Lactobacillus strains, one Bifidobacterium strain, one Propionibacterium strain and galacto-oligosaccharides (32g/l)) on serum enterolactone concentration in fifty-two men who had a concentration <20nmol/l. Serum sensitive C-reactive protein (CRP) concentration was measured at the end of the first intervention period. Men with a low serum enterolactone concentration when compared with those with a high concentration had less faecal bacteria, especially those belonging to the Lactobacillus-Enterococcus group (median 8·2 (interquartile range 7·8-8·4)log10 colony-forming units/g v. median 8·8 (interquartile range 8·5-8·9)log10 colony-forming units/g, P=0·009). The synbiotic mixture that was used did not have a significant effect on serum enterolactone (synbiotic v. placebo ratio 0·96 (95% CI 0·76, 1·22), P=0·724) or serum sensitive CRP (synbiotic v. placebo ratio 0·99 (95% CI 0·74, 1·33), P=0·954) concentration. Men with a low serum enterolactone concentration harbour less colonic bacteria, especially those belonging to the Lactobacillus-Enterococcus group. A synbiotic mixture does not increase serum enterolactone concentration.

Plasma enterolactone and breast cancer risk in the Nurses’ Health Study II

Lignans are plant-based phytoestrogens with both estrogenic and anti-estrogenic properties that may be important for breast carcinogenesis. Retrospective studies have observed decreased breast cancer risk associated with highcirculating enterolactone concentrations, a biomarker of lignan intake, but results from prospective studies are conflicting. To prospectively examine this association, we measured plasma enterolactone levels in 802 breast cancer cases and 802 matched controls nested among predominantly premenopausal women in the Nurses' Health Study II cohort. We used conditional logistic regression and polytomous logistic regression models, adjusting for known breast cancer risk factors, to calculate relative risks (RR) and 95% confidence intervals (CI). Compared to women with enterolactone concentrations ≤4nmol/L, the multivariate-adjusted RRs for breast cancer were 1.18 (95% CI 0.86-1.62), 0.91 (95% CI 0.66-1.25), and 0.96 (95% CI 0.70-1.33) for women with enterolactone levels in the second to the fourth quartiles, respectively; P trend=0.60. Results were similar across tumors defined by estrogen and progesterone receptor status. Among premenopausal women with follicular estradiol levels below the median (<47pg/mL), women in the highest category of enterolactone levels had a 51% lower breast cancer risk compared to those in the lowest category (95% CI 0.27-0.91); P trend=0.02. No association was observed among women with high-follicular estradiol levels (≥47pg/mL), (comparable RR=1.39, 95% CI 0.73-2.65; P interaction=0.02). We did not observe an overall association between plasma enterolactone and breast cancer risk in a large nested case-control study of US women. However, a significant inverse association was observed among premenopausal women with low-follicular estradiol levels, suggesting that enterolactone may be important in a low-estrogen environment. This should be confirmed in future studies.

Sociodemographic and Lifestyle Variables Are Compound- and Class-Specific Correlates of Urine Phytoestrogen Concentrations in the U.S. Population

Isoflavones and lignans are plant-derived dietary compounds generally believed to be beneficial to human health. We investigated the extent to which sociodemographic (age, sex, race-ethnicity, education, and income) and lifestyle variables (smoking, alcohol consumption, BMI, physical activity, and dietary supplement use) were correlates of spot urine concentration for daidzein, genistein, O-desmethylangolensin (DMA), equol, enterodiol, and enterolactone in the U.S. population aged ≥20 y (NHANES 2003–2006). We performed correlation analyses with continuous variables and calculated stratified unadjusted geometric means for each sociodemographic and lifestyle variable. We used bivariate significance testing and covariate adjustment by use of multiple regression models to identify influential variables and used β coefficients to estimate relative effects. Urine creatinine was also included in our analyses because of its use in correcting for variable dilution in spot urine samples. We observed many significant (P < 0.05) associations with the sociodemographic and lifestyle variables that withstood covariate adjustment. Smoking was a significant correlate of urine DMA and enterolactone, with concentrations at least 25% lower in smokers vs. nonsmokers. Consumers of 1 daily alcoholic drink vs. none were estimated to have 18–21% lower urine equol and DMA concentrations. A 25% increase in BMI was associated with a 21% lower urine enterolactone concentration, and increasing physical activity was associated with a >6% higher urine enterolactone concentration. Dietary supplement use was not significantly associated with any of the urine phytoestrogens. Overall, we found that relationships between sociodemographic and lifestyle variables and urine phytoestrogen concentration were highly compound and class specific.

Levels of urinary isoflavones and lignan polyphenols in Japanese women

High consumption of soybean products has been associated with a reduced risk of hormone-sensitive tumors. Soybean products contain phytoestrogens, such as daidzein, and sesame seeds contain secoisolariciresinol. These compounds are further metabolized to equol, enterodiol, and enterolactone by intestinal bacteria. However, individual differences in the metabolizing potential remain unclear. The aim of this study was to evaluate the urinary daidzein, equol, enterodiol, and enterolactone concentrations in women from several different regions of Japan according to age group. Five hundred urine samples collected from Japanese women living in Sapporo, Sendai, Kyoto, Kochi, and Naha were analyzed for daidzein, equol, enterodiol, and enterolactone concentration by gas chromatography-mass spectrometry. The urinary isoflavone and lignan polyphenol levels did not differ significantly among the sampling sites, except for daidzein, which was highest in urine collected at Naha. The prevalence of equol producers was 39 % in the total study cohort. In equol producers, a positive correlation was observed between the urinary daidzein and equol levels (r = 0.399, p < 0.001). However, there was no significant difference between daidzein concentrations in equol producers and non-producers. Moreover, the levels of enterodiol and enterolactone were higher in equol producers than in equol non-producers. In the multivariate logistic analyses, two factors, Sendai dwelling and current smoking, were found to be significant [equol producers to non-producers: odds ratio 2.15 (95 % confidence interval: 1.17-4.02) and odds ratio 0.32 (0.15-0.63), respectively]. Our data suggest that geographic factors and smoking status should be considered during the evaluation of equol in urine samples and that the same pathway may be responsible for the metabolism of both isoflavones and lignan polyphenols.

Abstract 115: Enterolactone levels and prognosis after invasive postmenopausal breast cancer: Potential effect modifiers.

Abstract Background: We previously reported that high levels of serum enterolactone, a metabolite of the phytoestrogen group of lignans, are associated with lower mortality in postmenopausal breast cancer patients (Buck et al. JCO 2011). Two subsequent studies confirmed this finding. In order to provide more insight into this association, we investigated whether enterolactone concentrations are affected by other lifestyle factors and tumor characteristics and whether the association might be restricted to subgroups defined by these factors. Methods: To increase study power, we additionally measured enterolactone concentration in patients of the second study center of the two-center MARIEplus study. The 1,990 postmenopausal breast cancer patients, aged 50-74 years and diagnosed between 2001 and 2005 in Germany, were prospectively followed up until end of 2009. Vital status was assessed via local population registries, deaths were verified by death certificates. Information on the clinical course and treatment was collected and verified by clinical records. Hazard ratios (HR) and 95% confidence intervals (CI) for post-diagnostic enterolactone levels in relation to overall survival (OS) were estimated using delayed entry Cox proportional hazards models stratified by age at diagnosis and study center and adjusted for prognostic factors. Potential differential effects by hormone receptor status, HER2 status, tumor characteristics, and lifestyle factors such as BMI, physical activity and smoking, were assessed. Results: Overall 257 patients had died. Median follow-up time from recruitment to death or censoring was 5.4 years. Median enterolactone levels for deceased and non-deceased patients were 17.3 and 20.8 nmol/L, respectively. Patients with high enterolactone levels were more likely to have tumors of smaller size, lower grade, hormone receptor expression positive and screen-detected, and to be HRT users, of normal weight and non-/former smokers and had not received chemotherapy. High enterolactone levels were associated with better OS (highest, Q4, vs. lowest quartile, Q1, HR 0.60, 95% CI 0.41-0.90). Per 10 nmol/L increment, HR was 0.94 (95% CI 0.90-0.98). In subgroup analysis, we found a stronger inverse association with estrogen receptor (ER) negative tumors (Q4 vs. Q1, HR 0.39, 95% CI 0.16-0.93) than with ER positive tumors (HR 0.76, 0.46-1.25). Stronger associations were also observed in normal weight women (Q4 vs. Q1: HR 0.56, 0.35-0.90) than overweight women (HR 0.80, 0.36-1.75), in more active (HR 0.54, 0.33-0.90) than less active women (HR 0.78, 0.39-1.58) and in current smokers (HR 0.35, 0.14-0.89) than non-smokers/former smokers (HR 0.72, 0.46-1.13). However, subgroup differences were not statistically significant. Conclusions: The inverse association of high enterolactone levels with overall mortality may be modified by hormone receptor status and lifestyle factors. Citation Format: Petra Seibold, Katharina Buck, Alina Vrieling, Theron Johnson, Rudolf Kaaks, Jakob Linseisen, Judith Heinz, Nadia Obi, Dieter Flesch-Janys, Jenny Chang-Claude. Enterolactone levels and prognosis after invasive postmenopausal breast cancer: Potential effect modifiers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 115. doi:10.1158/1538-7445.AM2013-115

Abstract 4823: Isoflavones from phytoestrogens and colorectal cancer risk: A nested case-control study within the Korean Multicenter Cancer Cohort.

Abstract Background The association of soybean products related with colorectal cancer risk is inconsistent. This may be due to information bias from measuring food intake by questionnaires and different degrees of bias according to study design. To assess the association between soybean products and colorectal cancer, we directly measured phytoestrogen biomarkers in a nested case-control study in Korea. Methods Study population was composed of 101 cases and 398 matched controls matched for age within 5-year, sex, area and year at recruitment of cases within the Korean Multicenter Cancer Cohort. The concentrations of four biomarkers in the plasma were measured by Liquid Chromatography-Mass Spectrometry. Conditional logistic regression models were used to calculate the odds ratio and 95% confidence intervals (CI). Results Median plasma concentrations of genistein, daidzein, glycitein and enterolactone were not statistically significant between cases and controls. However, there was a significant trend of decreasing risk according to an increase of genistein (p=0.03). This protective effect of genistein was more prominent in women. There was no association between other plasma concentrations of isoflavones or lignans and colorectal cancer. Conclusions High serum concentrations of isoflavones, i.e, genistein, were associated with a decreased trend of colorectal cancer, especially in women. Citation Format: Yohwan Yeo, Kwang-Pil Ko, Seung-Hyun Ma, Jae Jeong Yang, Aesun Shin, Sue K. Park, Soung-Hoon Chang, Hai-Rim Shin, Daehee Kang, Keun-Young Yoo. Isoflavones from phytoestrogens and colorectal cancer risk: A nested case-control study within the Korean Multicenter Cancer Cohort. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4823. doi:10.1158/1538-7445.AM2013-4823

Flaxseed-Derived Enterolactone Is Inversely Associated with Tumor Cell Proliferation in Men with Localized Prostate Cancer

Enterolactone and enterodiol, mammalian lignans derived from dietary sources such as flaxseed, sesame seeds, kale, broccoli, and apricots, may impede tumor proliferation by inhibiting activation of nuclear factor kappa B (NFκB) and vascular endothelial growth factor (VEGF). We examined the associations between urinary enterolactone and enterodiol with prostatic tumor expression of NFκB, VEGF, and Ki67 among 147 patients with prostate cancer who participated in a presurgical trial of flaxseed supplementation (30 g/day) for ~30 days. Urinary enterolignans and tissue biomarkers were determined by high-performance liquid chromatography and immunohistochemistry, respectively. After supplementation, we observed significant correlations between intakes of plant lignan and urinary concentrations of total enterolignans (ρ=0.677, P<.0001), enterolactone (ρ=0.676, P<.0001), and enterodiol (ρ=0.628, P<.0001). Importantly, we observed that total urinary enterolignans and enterolactone were significantly and inversely correlated with Ki67 in the tumor tissue (ρ=-0.217, P=.011, and ρ=-0.230, P=.007, respectively), and a near-significant inverse association was observed for enterodiol (ρ=-0.159, P=.064). An inverse association was observed between enterolactone and VEGF (ρ=-0.143, P=.141), although this did not reach statistical significance. We did not observe an association between enterolignans and NFκB. In conclusion, flaxseed-derived enterolignans may hinder cancer cell proliferation via VEGF-associated pathways.

Anti‐proliferative effects of physiological concentrations of enterolactone in models of prostate tumourigenesis

There is evidence that a mammalian lignan, enterolactone (ENL), decreases the proliferation rate of prostate cancer cells, although previous studies have used concentrations difficult to achieve through dietary modification. We have therefore investigated the anti-proliferative effects of ENL in an in vitro model of prostate tumourigenesis at concentrations reported to occur in a range of male populations. The effects of 0.1 and 1 μM ENL on three markers of viability and proliferation (metabolic activity, growth kinetics, and cell cycle progression) were assessed in the RWPE-1, WPE1-NA22, WPE1-NB14, WPE1-NB11, WPE1-NB26, LNCaP, and PC-3 cell lines over 72 h. Based on these data, we quantified the expression levels of 12 genes involved in the control of DNA replication initiation using TaqMan real-time PCR in the WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26 cell lines. ENL significantly inhibited the abnormal proliferation of the WPE1-NB14 and WPE1-NB11 cell lines and appears to be a consequence of decreased expression of abnormal chromatin licensing and DNA replication factor 1. In contrast to previous studies, concentrations of ENL that are reported after dietary intervention restrict the proliferation of early-stage tumourigenic prostate cell lines by inhibiting the abnormal formation of complexes that initiate DNA replication.

Plasma enterolactone and incidence of endometrial cancer in a case–cohort study of Danish women

The phyto-oestrogen enterolactone has been hypothesised to protect against hormone-dependent cancers, probably through its antioestrogenic potential. We investigated whether a higher level of plasma enterolactone was associated with a lower incidence of endometrial cancer in a case-cohort study in the ‘Diet, Cancer and Health’ cohort. The cohort study included 29 875 women aged 50–64 years enrolled between 1993 and 1997. Information on diet and lifestyle was provided by self-administrated questionnaires and blood was drawn from each participant. Time-resolved fluoroimmunoassay was used for biochemical determination of plasma enterolactone. A total of 173 cases and 149 randomly selected cohort members were included. We estimated incidence rate ratio (IRR) and 95% CI by a Cox proportional hazards model. A 20 nmol/l higher plasma concentration of enterolactone was associated with a non-significant lower risk of endometrial cancer (IRR 0.93, 95% CI 0.84, 1.04). When excluding women with low enterolactone concentrations (quartile 1) due to potential recent antibiotic use, the association became slightly stronger, but remained non-significant (IRR 0.90, 95% CI 0.79, 1.02). Menopausal status, hormone replacement therapy or BMI did not modify the association. In conclusion, we found some support for a possible inverse association between plasma enterolactone concentration and endometrial cancer incidence.

Validation of FFQ-based assessment of dietary lignans compared with serum enterolactone in Swedish women

The validity of using FFQ to assess dietary lignans is uncertain. We aimed to validate the use of FFQ for the assessment of dietary intake of lignans compared to the serum biomarker enterolactone, the main product of dietary lignans' metabolism in human subjects. A random sample of women, aged 55-75 years, from the Swedish Mammography Cohort was selected. Information from two FFQ, the FFQ-87 (sixty-seven food items) and the FFQ-97 (ninety-three food items), and blood samples were collected. Dietary intake of lignans (secoisolariciresinol, matairesinol, lariciresinol, pinoresinol, medioresinol and syringaresinol) was assessed by the FFQ. Serum concentrations of enterolactone were analysed by time-resolved fluoroimmunoassay. The correlation coefficient between energy-adjusted lignan intake and serum enterolactone was estimated in crude and multivariable-adjusted models, taking into account the factors potentially influencing the serum enterolactone. Among the 135 participants aged 55-75 years, with a mean BMI of 26·7 kg/m², the average energy-adjusted intake of total lignans was 1616 (sd 424) and 1516 (sd 409) μg/d according to the FFQ-87 (forty-five food items containing lignans) and the FFQ-97 (sixty-five food items containing lignans), respectively. The mean concentration of serum enterolactone was 23·2 (sd 15·4) nmol/l. The adjusted Pearson's correlation between dietary intake of lignans assessed by the FFQ-97 and serum enterolactone was statistically significant (r 0·22, P= 0·01). No significant correlation was observed for the FFQ-87 (r 0·09, P= 0·30). The present study indicates that the FFQ-97 might be better than the FFQ-87 for assessing dietary intake of lignans, although the correlation was low.

Tissue Distribution and Cytochrome P450 Inhibition of Sesaminol and Its Tetrahydrofuranoid Metabolites

Sesame lignans such as sesamin, sesaminol, and sesamolin are major constituents of sesame oil, and all have a methylenedioxyphenyl group and multiple functions in vivo. It was previously reported that sesaminol, a tetrahydrofurofuran type lignin, was metabolized to mammalian lignans. The present study examined the tissue distribution of sesaminol in Sprague-Dawley (SD) rats. Changes in the concentration of sesaminol and its metabolites (sesaminol glucuronide/sulfate, hydroxymethylsesaminol-tetrahydrofuran, enterolactone, and enterodiol) were determined in tissues within a 24 h period after tube feeding (po 220 mg/kg) to SD rats. The concentrations of enterodiol and enterolactone were significantly higher than those of sesaminol and its tetrahydrofuranoid metabolites in the organs (liver, heart, brain, and kidney). This study demonstrates that sesaminol has potent inhibition of cytochrome P450 (CYPs), compared to tetrahydrofuranoid metabolites. The IC(50) values of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 for sesaminol were determined as 3.57, 3.93, 0.69, 1.33, and 0.86 μM, respectively. In addition, hydroxymethylsesaminol-tetrahydrofuran and enterodiol were weak inhibitors of CYP2C9 and CYP1A2, respectively.

Effects of feeding dairy cows different legume-grass silages on milk phytoestrogen concentration

Phytoestrogens are hormone-like substances in plants that can substantially influence human health (positively or negatively), and when fed to dairy cows are partly transferred to their milk. The aim of this study was to investigate the effects of varying the botanical composition and regrowth interval of legume-grass silage on phytoestrogen intake and milk phytoestrogen concentrations. In one experiment, 15 Swedish Red dairy cows were fed 2- or 3-cut red clover-grass silage, or 2-cut birdsfoot trefoil-grass silage. In a second experiment, 16 Norwegian Red dairy cows were fed short-term ley silage with red clover or long-term ley silage with white clover, and the effects of supplementation with α-tocopherol were also tested. High concentrations of formononetin and biochanin A were found in all silage mixtures with red clover. The milk concentration of equol was highest for cows on the 2-cut red clover-grass silage diet (1,494μg/kg of milk). Because of the metabolism of biochanin A, genistein, and prunetin, their concentrations in milk and the apparent recovery were low. Coumestrol was detected in only short-term and long-term ley silage mixtures, and its milk concentration was low. Concentrations of secoisolariciresinol and matairesinol were higher in 2-cut birdsfoot trefoil-grass and long-term ley silage mixtures, those with legume species other than red clover, and the highest grass proportions. The 2-cut birdsfoot trefoil-grass silage diet also resulted in higher enterolactone concentration than the other diets (226μg/kg of milk). Lengthening the regrowth interval increased the intake of secoisolariciresinol and decreased the recovery of lignans. Feeding long-term ley silage resulted in higher milk lignan concentrations but lower milk isoflavone concentrations than feeding short-term ley silage. The apparent recovery of all phytoestrogens except prunetin was highest on the 2-cut birdsfoot trefoil-grass silage diet. No effect of α-tocopherol supplementation was observed on milk concentrations of any of the measured phytoestrogens. Variations were observed in milk concentrations of phytoestrogens, especially of equol, among cows, which could not be explained by variations in diet composition or phytoestrogen intake. The results show that milk phytoestrogen concentration is strongly influenced by silage botanical composition, but questions regarding phytoestrogen metabolism remain to be answered.

Analysis of prostate cancer association with four single-nucleotide polymorphisms from genome-wide studies and serum phyto-estrogen concentrations

Both genetics and the environment are implicated as risk factors for prostate cancer (PCa). This population-based case-control study evaluated four single-nucleotide polymorphisms (SNPs) previously identified by genome-wide association studies to be associated with increased PCa susceptibility. Potential relationships between serum concentrations of phyto-estrogens and SNPs were also investigated. Four SNPs (rs10993994, rs2660753, rs1016343 and rs6983267) were genotyped in 247 PCa patients, 125 BPH patients and 274 control men recruited in Scotland. Serum concentrations of the phyto-estrogens enterolactone, equol, genistein and daidzein were measured by isotope dilution gas chromatography-mass spectrometry. Increased PCa risk was associated with TT genotype of rs10993994 compared with CC and CT genotypes combined (odds ratio (OR)=1.87; 95% confidence interval (CI), 1.26-2.77). TT homozygotes who had low serum enterolactone concentrations (below median) were more likely to have PCa (OR=2.90; 95% CI, 1.28-6.57) than individuals with CC/CT genotype and high serum enterolactone concentrations (above median). PCa was not associated with the other three SNPs tested. PCa susceptibility was associated with TT genotype of SNP rs10993994 in this cohort of Scottish men and the increased risk of PCa was modified by serum enterolactone concentrations.

105P Antiproliferative Effect of New Lignan from Abies Sibirica on Hormone-Dependent Breast Cancer Cells in Vitro

ABSTRACT Background Lignans is one of the main group phytoestrogens, natural compounds with estrogenic activity. Data of experimental studies suggest that lignans can exhibit tumor-protective properties and inhibit tumor growth. Here we studied the effect of lignan, secoisolariciresinol (Knotolan®) isolated from new nonfood vegetable raw material (Abies sibirica) on proliferation of hormone-dependent breast cancer cells in vitro. Material and methods Knotolan® was obtained from Abies sibirica wood. Commercial enterolaktone (Sigma) was as positive control. The final concentration of Knotolan and enterolactone in samples 1000-fold surpassed the concentration of 17-estradiol (E2) and was 1 µM, because previous studies showed that higher concentrations of enterolactone produced a growth-stimulating effect in vitro. The MCF7 cells were used as test object. The number of estrogens receptors was determined by a modified radioligand method. Cells were grown in RPMI 1640 medium without phenol red and 10% FCS containing estrogens. For the experiments cells were grown in the same conditions but without estrogens. Cell proliferation in samples was measured by MTT-test. Microscope examination was performed using Axiolmager invertal microscope. Results MCF7 cells revealed 6.1 ± 0.5 3H-E2 binding sites per cells. On day 5, incubation of cells with E2 in concentration 1nM increased proliferation of MCF7 cells by 41.7%. Incubation with Knotolan® or enterolactone in concentration 1 µM for 5 days had no effect on cell proliferation. Pre-incubation with Knotolan® or enterolactone for 2 days followed by incubation with E2 did not stimulate cell proliferation. The capacity of Knotolan® to abolish the growth stimulating effect of E2, i.e. to exhibit its antiestrogen properties was similar to the effect of enterolactone. We hypothesized that this effect can be realized in vivo and Knotolan® from Abies sibirica can serve as basis for the creation of new plant preparation with protective properties. Disclosure All authors have declared no conflicts of interest.

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.

Erratum for “Effects of a Breast-Health Herbal Formula Supplement on Estrogen Metabolism in Pre- and Post-Menopausal Women not Taking Hormonal Contraceptives or Supplements: A Randomized Controlled Trial”

Introduction: Both indole-3-carbinol and dietary lignans have beneficial effects on estrogen metabolism and breast cancer risk. There is no published literature on the effects of a combination product. This study was designed to investigate the impact of a combination product on estrogen metabolism. The major trial objective was to determine whether a breast health supplement containing indole-3-carbinol and hydroxymatairesinol lignan would alter estrogen metabolism to favour C-2 hydroxylation and reduce C-16 hydroxylation. Higher concentrations of C-2 metabolites and lower concentrations of C-16 metabolites may reduce breast cancer risk and risk for other hormonally-related cancers. Methods: Forty-seven pre-menopausal and forty-nine post-menopausal women were recruited for this study, and were divided by random allocation into treatment and placebo group. The treatment supplement contained HMR lignan, indole-3-carbinol, calcium glucarate, milk thistle, Schisandra chinesis and stinging nettle, and each woman consumed either treatment or placebo for 28 days. At day 0 and day 28, blood samples were analysed for serum enterolactone concentrations, and first morning random urine samples were assessed for estrogen metabolites. Repeated measures ANOVA statistical testing was performed. Results: In pre-menopausal women, treatment supplementation resulted in a significant increase (P , 0.05) in urinary 2-OHE concentrations and in the 2:16α-OHE ratio. In post-menopausal women, treatment supplementation resulted in a significant increase in urinary 2-OHE concentrations. In pre- and post-menopausal women combined, treatment supplementation produced a significant increase in urinary 2-OHE concentration and a trend (P = 0.074) toward an increased 2:16α-OHE ratio. There were no significant increases in serum enterolactone concentrations in the treatment or placebo groups. Conclusions: Supplementation with a mixture of indole-3-carbinol and HMR lignan in women significantly increased estrogen C-2 hydroxylation. This may constitute a mechanism for the reduction of breast cancer risk as well as risk for other estrogen-related cancers. Further studies with higher numbers of subjects are indicated. Trial registration: ClinicalTrials.gov registration #NCT01089049.

Serum enterolactone levels and mortality outcome in women with early breast cancer: a retrospective cohort study

We previously demonstrated that high serum enterolactone levels are associated with a reduced incidence of breast cancer in healthy women. The present study was aimed at investigating whether a similar association might be found between serum enterolactone levels and the mortality of women with early breast cancer. The levels of enterolactone in cryopreserved serum aliquots obtained from 300 patients, operated on for breast cancer, were measured using a time-resolved fluoro-immunoassay. Levels were analyzed in respect to the risk of mortality following surgery. Cox proportional hazard regression models were used to check for prognostic features, to estimate hazard ratios for group comparisons and to test for the interaction on mortality hazards between the variables and enterolactone concentrations. The Fine and Gray competing risk proportional hazard regression model was used to predict the probabilities of breast cancer-related and breast cancer-unrelated mortalities. At a median follow-up time of 23 years (range 0.6-26.1), 180 patients died, 112 of whom died due to breast cancer-related events. An association between a decreased mortality risk and enterolactone levels ≥ 10 nmol/l was found in respect to both all-cause and breast cancer-specific mortality. The difference in mortality hazards was statistically significant, but it appeared to decrease and to lose significance after the first 10 years, though competing risk analysis showed that breast cancer-related mortality risk remained constantly lower in those patients with higher enterolactone levels. Our findings are consistent with those of most recent literature and provide further evidence that mammalian lignans might play an important role in reducing all-cause and cancer-specific mortality of the patients operated on for breast cancer.

Abstract B83: Isoflavones from phytoestrogens and cervical cancer risk: A nested case-control study

Abstract Objective: The effect of soybean products in cervical cancer risk has not been investigated yet. To examine the association between soybean product intake and cervical cancer risk, we measured phytoestrogen biological markers in cervical cancer patients and non-cancer control patients. Methods: Among patients visit Ewha womans university hospital between October 2007 and October 2010, 143 cases and 143 matched controls as same age distribution were enrolled. The concentrations of the four biomarkers in the plasma samples were measured using time-resolved fluoro-immunoassay. Dose-response relationship was analyzed with restricted cubic spline method. Conditional and unconditional logistic regression models were used to compute the odds ratio (OR) and 95% confidence intervals (CI). Results: There are linear relationships between two isoflavones concentrations (daidzein, equol) and cervical cancer risk. As daidzein concentration increased by 100 nmol/L, cervical cancer risk significantly increases by 41.4%. (P=0.028) Compared with the reference group, cervical cancer risk increased significantly in higher concentration of daidzein(P=0.04), genistein(3rd and 4th quantile, P=0.03), enterolactone(3rd quantile, P=0.003). Conclusions: Serum concentrations of daidzein, genistein, and enterolactone were siginificantly associated with a increased risk for cervical cancer. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B83.