Tina Skau Nielsen

Phyto-oestrogens in herbage and milk from cows grazing white clover, red clover, lucerne or chicory-rich pastures.

A grazing experiment was carried out to study the concentration of phyto-oestrogens in herbage for cattle and in milk during two periods (May and June). Forty-eight Danish Holstein cows were divided into four groups with four treatment diets; white clover, red clover, lucerne and chicory-rich pastures. Each experimental period lasted 15 days. Herbage samples from the first day and individual milk samples from the last day of the experimental period were analysed for phyto-oestrogens using LC-MS technique. The total concentration of phyto-oestrogens was 21 399 mg/kg dry matter (DM) for red clover and 238 to 466 mg/kg DM for the other three herbages mainly due to a much higher concentration of biochanin A, formononetin and glycitein in red clover. In the milk, the total concentration of phyto-oestrogens was 253 to 397 μg/l for red clover milk and 56 to 91 μg/l in the milk from the other three treatments. This was especially due to a higher concentration of equol, daidzein and formononetin in the red clover milk. The concentration of biochanin A was significantly higher in milk from the red clover treatment in May while no differences were observed in June. Enterodiol was similar across treatments while the concentration of enterolactone was significantly lower for red clover milk compared with the other treatments. Of the tested pastures, red clover appears to have the highest concentration and to be the best source of phyto-oestrogens, especially equol, in bovine milk.

Estrogenic activity of bovine milk high or low in equol using immature mouse uterotrophic responses and an estrogen receptor transactivation assay.

BACKGROUND Cows milk contain phytoestrogens especially equol depending on the composition of the feed ration. However, it is unknown whether milk differing in equol exhibits different estrogenicity in model systems and thereby potentially in humans as milk consumers. METHODS The estrogenicity of high and low equol milk (HEM and LEM, respectively) and purified equol was investigated in immature female mice including mRNA expression of six estrogen-sensitive genes in uterine tissue. Extracts of HEM and LEM were also tested for estrogenicity in vitro in an estrogen receptor (ER) reporter gene assay with MVLN cells. RESULTS The total content of phytoestrogens was approximately 10 times higher in HEM compared with LEM, but levels of endogenous milk estrone and 17beta-estradiol were similar in the two milk types (503-566 and 60-64.6pg/ml, respectively). There was no difference in uterine weight between mice receiving LEM and HEM, and no difference from controls. Equol (50 times the concentration in HEM) was not uterotrophic. The ERbeta mRNA expression was down-regulated in the uteri of HEM mice compared with LEM and controls, but there was no difference between milk types for any of the other genes. Extracts of HEM showed a higher estrogenicity than extracts of LEM in MVLN cells, and there was a dose-dependent increase in estrogenicity by equol. CONCLUSION The higher in vitro estrogenicity of HEM was not reflected as a higher uterine weight in vivo although the down-regulation of ERbeta in uterine tissue of HEM mice could suggest some estrogenic activity of HEM at the gene expression level.

Effects of Resistant Starch and Arabinoxylan on Parameters Related to Large Intestinal and Metabolic Health in Pigs Fed Fat-Rich Diets

This study compared the effects of a resistant starch (RS)-rich, arabinoxylan (AX)-rich, or low-DF Western-style control diet (all high-fat) on large intestinal gene expression, adiposity, and glycemic response parameters in pigs. Animals were slaughtered after 3 weeks of treatment. Plasma butyrate concentration was higher following the high-DF diets, whereas plasma glucose, insulin, and insulin resistance increased after 3 weeks irrespective of diet. The mRNA abundance in the large intestine of genes involved in nutrient transport, immune response, and intestinal permeability was affected by segment (cecum, proximal, mid or distal colon) and some genes also by diet. In contrast, there was no diet-induced effect on adipose mRNA abundance or adipocyte size. Overall, a high level of RS or AX did not demonstrate strong beneficial effects on large intestinal gene expression as indicators of colonic health or glycemic response parameters when included in a high-fat diet for pigs as a model of healthy humans.

Cell-based models to test the effects of milk-derived bioactives.

The life science industries have a strong interest in screening for novel bioactives in complex mixtures like milk and dairy products. Food bioactives are not only important for public health in general, but also have potential therapeutic applications for the treatment of a number of diseases. To identify these novel bioactives, establishment of robust screening assays is essential. The use of in vitro cell-based models for screening and testing have the advantage that several concentrations of mixtures or specific compounds can be assayed at the same time in cells from specific tissues. Primary cell cultures from target organs or established cell lines can be used to identify the most sensitive cells. In addition, a large number of transfected cell lines with very specific sensitivities have been developed. Different endpoints inherent to basal or more sophisticated cellular functions can be investigated, such as cell viability, apoptosis, migration, intracellular signalling, regulation of gene expression, morphology and metabolic alterations. The gastrointestinal tract is an obvious target for bioactive molecules delivered through milk and dairy products, because it lies at the interface between dietary components in the lumen and the internal processes of the host. Identification of bioactive factors that affects proliferation or migration of epithelial cells may have potential applications in promoting gastrointestinal health in both humans and animals. The mammary gland is another target organ of considerable interest since it has been estimated that up to 50% of all newly diagnosed breast cancers may be related to dietary factors. A large number of gastrointestinal and mammary epithelial cell lines are commercially available, but in order to study some cellular functions, primary cultures of freshly isolated cells are often preferred, as established cell lines do not always express specialised properties in culture.

Effect of harvest time on fermentation profiles of maize ensiled in laboratory silos and determination of drying losses at 60°C

Abstract The objectives were to investigate the effect of premature ensiling of maize on alcohol fermentation in laboratory silos and the loss of fermentation products and glucose in silage following drying at 60°C for 48 h. During four consecutive weeks maize was harvested and ensiled for 60 days in vacuum-sealed laboratory silos. The content of DM in silage increased (p<0.001), whereas acetate and ethanol decreased (p<0.01) with later time of harvest. Concentrations of methanol and propanol were generally <1 g/kg DM and the content of 2-butanol <0.03 g/kg DM. The content of D-glucose was lower (p=0.03) in silage harvested the second week compared with the other harvest times (40.9 vs. 66–69 g/kg DM). Overall, silage pH was negatively correlated with L-lactate (r=−0.93). Drying increased pH, butyrate and valerate, although the numerical effect was small. A major portion of acetate was lost in drying, and the D-glucose content was reduced by approximately 45% after drying. Alcohols and esters were completely lost in drying. We conclude that ensiling of pre-mature maize does not lead to extensive alcohol fermentation in laboratory silos following 60 days of ensiling, and that dry matter correction based on fermentation products determined in dry samples may be erroneous due to inability to predict wet sample content from samples dried at 60°C.

Effects of Maternal Exposure to Cow's Milk High or Low in Isoflavones on Carcinogen-Induced Mammary Tumorigenesis among Rat Offspring

We investigated whether maternal exposure during pregnancy to cows milk containing endogenous estrogens and insulin-like growth factor 1 (IGF-1) and either high or low levels of isoflavones from dietary legumes (HIM and LIM, respectively) affected carcinogen-induced mammary carcinogenesis in female rat offspring. Pregnant Sprague-Dawley rats were given HIM, LIM, or tap water (control) from gestational day (GD) 11 until birth; hereafter all rats received tap water. Mammary tumorigenesis was induced by administrating 7,12-dimethylbenz[a]anthracene (DMBA) on postnatal day 50. No differences in maternal serum estradiol (P = 0.19) and IGF-1 levels (P = 0.15) at GD 19 or birth weight among the milk and water groups were seen, but estradiol, and IGF-1 levels and birth weight were numerically higher in the LIM group than in the HIM group. Puberty onset occurred earlier in the LIM offspring than in controls (P = 0.03). Although the high isoflavone content seemed to prevent the effect on circulating estradiol and IGF-1 levels and advanced puberty onset seen in the LIM group, HIM increased DMBA–DNA adducts in the mammary gland and tended to increase mammary tumorigenesis. In contrast, offspring exposed to LIM in utero, did not exhibit increased breast cancer risk, despite having higher estradiol and IGF-1 environment and consequently earlier puberty onset. These results indicate that the phytochemical content in the cows milk, consumed by a pregnant dam, determines how milk affects the offspring. Cancer Prev Res; 4(5); 694–701. ©2011 AACR.

Prepubertal exposure to cow's milk reduces susceptibility to carcinogen-induced mammary tumorigenesis in rats.

Cows milk contains high levels of estrogens, progesterone and insulin‐like growth factor 1 (IGF‐1), all of which are associated with breast cancer. We investigated whether prepubertal milk exposure affects mammary gland development and carcinogenesis in rats. Sprague–Dawley rats were given either whole milk or tap water to drink from postnatal day (PND) 14 to PND 35, and thereafter normal tap water. Mammary tumorigenesis was induced by administering 7,12‐dimethylbenz[a]anthracene on PND 50. Milk exposure increased circulating E2 levels on PND 25 by 10‐fold (p < 0.001) and accelerated vaginal opening, which marks puberty onset, by 2.5 days (p < 0.001). However, rats exposed to milk before puberty exhibited reduced carcinogen‐induced mammary carcinogenesis; that is, their tumor latency was longer (p < 0.03) and incidence was lower (p < 0.05) than in the controls. On PND 25 and 50, mammary glands of the milk‐exposed rats had significantly less terminal end buds (TEBs) than the tap water‐exposed controls (p < 0.019). ER‐α protein levels were elevated in the TEBs and lobules of milk rats, compared to rats given tap water (p < 0.019), but no changes in cyclin D1 expression, cell proliferation or apoptosis were seen. IGF‐1 mRNA levels were reduced on PND 50 in the mammary glands of rats exposed to milk at puberty. Our results suggest that drinking milk before puberty reduces later risk of developing mammary cancer in rats. This might be mediated by a reduction in the number of TEBs and lower expression of IGF‐1 mRNA in the mammary glands of milk‐exposed animals.

The Influence of Bovine Milk High or Low in Isoflavones on Hepatic Gene Expression in Mice

Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17β-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array qPCR kit. It was revealed that Tween 80 and 17β-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones can regulate the transcription of especially phase II liver enzymes which potentially could give rise to an increase in reactive oxygen metabolites that may contribute to the development of cancer.

Proliferative effect of whey from cows' milk obtained at two different stages of pregnancy measured in MCF-7 cells

Dietary estrogens may play a role in the etiology of hormone-dependent cancers like breast cancer. Cows milk contains various endogenous estrogens and feed derived phytoestrogens that potentially contribute to an estrogenic effect of milk in consumers, and therefore we evaluated the effect of milk (whey) in a proliferation assay with estrogen-sensitive MCF-7 human breast cancer cells. Milk samples were obtained from 22 cows representing different stages of pregnancy (first and second half) and whey was produced from the milk. 0·1, 0·25 or 0·5% whey was included in the cell culture medium and after 6 days of treatment cell proliferation was assessed by a colorimetric method with a fluorometer. Whey induced significant (P<0·05) proliferative effects compared with control cells with no added whey at all concentrations tested. There was no difference in the proliferative effect of whey depending on the stage of pregnancy from which the milk was obtained. We did not observe anti-proliferative effects when whey was tested in the presence of 10 pm estradiol in the medium. In conclusion, these results indicate that whey, irrespective of the pregnancy stage from which the milk was obtained induced a significant proliferative response in MCF-7 cells and no anti-proliferative effect, which may be caused, at least in part, by estrogens present in milk. The implications of our findings in relation to for example breast cancer will have to be studied further in other model systems preferentially in vivo.

Impact of Diet-Modulated Butyrate Production on Intestinal Barrier Function and Inflammation.

A major challenge in affluent societies is the increase in disorders related to gut and metabolic health. Chronic over nutrition by unhealthy foods high in energy, fat, and sugar, and low in dietary fibre is a key environmental factor responsible for this development, which may cause local and systemic inflammation. A low intake of dietary fibre is a limiting factor for maintaining a viable and diverse microbiota and production of short-chain fatty acids in the gut. A suppressed production of butyrate is crucial, as this short-chain fatty acid (SCFA) can play a key role not only in colonic health and function but also at the systemic level. At both sites, the mode of action is through mediation of signalling pathways involving nuclear NF-κB and inhibition of histone deacetylase. The intake and composition of dietary fibre modulate production of butyrate in the large intestine. While butyrate production is easily adjustable it is more variable how it influences gut barrier function and inflammatory markers in the gut and periphery. The effect of butyrate seems generally to be more consistent and positive on inflammatory markers related to the gut than on inflammatory markers in the peripheral tissue. This discrepancy may be explained by differences in butyrate concentrations in the gut compared with the much lower concentration at more remote sites.