Inhibition of Canonical Transient Receptor Potential Channels 4/5 with Highly Selective and Potent Small-Molecule HC-070 Alleviates Mechanical Hypersensitivity in Rat Models of Visceral and Neuropathic Pain.

Transient receptor potential channels C4/C5 are widely expressed in the pain pathway. Here, we studied the putative analgesic efficacy of the highly selective and potent TRPC4/C5 antagonist HC-070 in rats. Inhibitory potency on human TRPC4 was assessed by using the whole-cell manual patch-clamp technique. Visceral pain sensitivity was assessed by the colonic distension test after intra-colonic trinitrobenzene sulfonic acid injection and partial restraint stress. Mechanical pain sensitivity was assessed by the paw pressure test in the chronic constriction injury (CCI) neuropathic pain model. We confirm that HC-070 is a low nanomolar antagonist. Following single oral doses (3-30 mg/kg in male or female rats), colonic hypersensitivity was significantly and dose-dependently attenuated, even fully reversed to baseline. HC-070 also had a significant anti-hypersensitivity effect in the established phase of the CCI model. HC-070 did not have an effect on the mechanical withdrawal threshold of the non-injured paw, whereas the reference compound morphine significantly increased it. Analgesic effects are observed at unbound brain concentrations near the 50% inhibitory concentration (IC50) recorded in vitro. This suggests that analgesic effects reported here are brought about by TRPC4/C5 blocking in vivo. The results strengthen the idea that TRPC4/C5 antagonism is a novel, safe non-opioid treatment for chronic pain.

Dietary phytoestrogen, serum enterolactone and risk of prostate cancer: the cancer prostate Sweden study (Sweden).

OBJECTIVE: Based on evidence that phytoestrogens may protect against prostate cancer, we evaluated the associations between serum enterolactone concentration or dietary phytoestrogen intake and risk of prostate cancer. METHODS: In our Swedish population-based case-control study, questionnaire-data were available for 1,499 prostate cancer cases and 1,130 controls, with serum enterolactone levels in a sub-group of 209 cases and 214 controls. Unconditional logistic regression was performed to estimate multivariate odds ratios (ORs) and 95% confidence intervals (CIs) for associations with risk of prostate cancer. RESULTS: High intake of food items rich in phytoestrogens was associated with a decreased risk of prostate cancer. The OR comparing the highest to the lowest quartile of intake was 0.74 (95% CI: 0.57-0.95; p-value for trend: 0.01). In contrast, we found no association between dietary intake of total or individual lignans or isoflavonoids and risk of prostate cancer. Intermediate serum levels of enterolactone were associated with a decreased risk of prostate cancer. The ORs comparing increasing quartiles of serum enterolactone concentration to the lowest quartile were, respectively, 0.28 (95% CI: 0.15-0.55), 0.63 (95% CI: 0.35-1.14) and 0.74 (95% CI: 0.41-1.32). CONCLUSIONS: Our results support the hypothesis that certain foods high in phytoestrogens are associated with a lower risk of prostate cancer.

Suitability of a batch in vitro fermentation model using human faecal microbiota for prediction of conversion of flaxseed lignans to enterolactone with reference to an in vivo rat model.

BACKGROUND: In vitro fermentation models have been used widely for studies of shortchain fatty acid (SCFA) formation from carbohydrates, whereas the suitability of these methods for enterolactone (ENL) formation has received less attention. AIM: The aim was to study the suitability of an in vitro fermentation model for prediction of bioconversion of lignans to ENL, to compare the approach with that of an in vivo rat model and to study the SCFA formation in both models. METHODS: Predigested samples of rye bran (R), flaxseed meal (F) alone, or in combination with rye bran (R&F) and a faecal control were incubated in an in vitro fermentation model using human faecal microbiota. In the in vivo experiment rats consumed a non-fibre control diet (C) or diets supplemented either with rye bran (R), flaxseed meal (F) alone, or with their combination (R&F) for four weeks. Enterodiol (END), ENL and SCFA concentrations were measured from in vitro faecal fermentation samples and from the intestinal contents of rats. Plasma ENL concentrations from rats were also measured. RESULTS: The highest ENL production was found in vitro with the F supplement (areas under curve: 740 +/- 4, 7,500 +/- 400, 2,600 +/- 500 and 1,520 +/- 70 nmol x h for the R, F, R&F supplements and faecal control, respectively). In vivo, the concentration of ENL in caecal digesta from flaxseed meal was significantly (P < 0.05) enhanced by the presence of rye bran (medians 261, 407 and 24 nmol/g in the F, R&F and C groups, respectively). No correlation was found between the models regarding ENL production, possibly due to different responses to the presence of rye bran matrix, differences in microbiota or application of a batch in the in vitro fermentation model. Rye bran supplementation enhanced butyrate production both in vitro and in vivo. CONCLUSION: In vitro fermentation and the in vivo rat models responded differently to the presence of rye bran and no correlation with regard to the ENL formation from flaxseed lignans was observed.

Effects of a flaxseed mixture and plant oils rich in alpha-linolenic acid on the adenoma formation in multiple intestinal neoplasia (Min) mice.

Flaxseed is a dietary source of possible chemopreventive compounds such as lignans and alpha-linolenic acid (ALA). To study the effects of a flaxseed mixture on adenoma formation in multiple intestinal neoplasia mice, the mice were fed a diet containing 2.7 % flaxseed, 4.5 % fibre and 3.7 % ALA. To elucidate the effect of oils of the mixture we also composed a diet without flaxseed but with the same oil composition. The median number of adenomas in the small intestine was fifty-four for the control group, and thirty-seven (P=0.023) and forty-two (P=0.095) for flaxseed and oil groups, respectively. Compared with controls (1.2 mm), the adenoma size was smaller in the flaxseed (0.9 mm; P=0.002) and oil (1.0 mm; P=0.012) groups. Both diets changed the proportions of n-3 and n-6 fatty acids in the colonic mucosa. Membrane beta-catenin and protein kinase C (PKC)-zeta levels were reduced in the adenoma v. mucosa (P<0.05), and an inverse association was found between the membrane PKC-zeta in the mucosa and the adenoma number (r -0.460, P=0.008, n 32). Only the flaxseed diet increased lignan levels in the caecum (P=0.002) and in plasma (P=0.002) but they were not associated with tumour formation. The results suggest that the preventive effect of flaxseed on colon carcinogenesis may be due to the oil part of flaxseed, and the loss of beta-catenin and PKC-zeta from the membranes of the mucosal tissue may play a permissive role in intestinal tumour development.

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects.

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.

Studies of the in vitro intestinal metabolism of isoflavones aid in the identification of their urinary metabolites.

Soy isoflavones have recently gained considerable interest due to their possible health benefits. However, detailed studies on the metabolism of isoflavones are lacking. The aims of the investigation presented here were (1) to study the in vitro intestinal metabolism of isoflavones and their hydroxylated analogues 3'-OH-daidzein, 6-OH-daidzein, 8-OH-daidzein, and 3'-OH-genistein and (2) to characterize the structures of some earlier identified urinary metabolites of soy isoflavones, for which no authentic reference compounds have been available. Isoflavone standards (1-2 mg) were fermented with human fecal flora (16.7%) for 24 h. Metabolites formed during the fermentation were tentatively identified by interpretation of the mass spectra of trimethylsilylated compounds obtained by GC-MS. Compounds having hydroxyl groups at 5-position (i.e., genistein and 3'-OH-genistein) were completely converted to metabolites that could not be detected by the methods used in this study. The metabolism of daidzein and its hydroxylated analogues, 3'-OH-daidzein, 6-OH-daidzein, and 8-OH-daidzein, occurred to a much lesser extent. Minor amounts of reduced metabolites (i.e., isoflavanones and alpha-methyldeoxybenzoins) of these compounds were tentatively identified in fermentation extracts. The retention times and the mass spectra of reduced isoflavone metabolites, obtained from in vitro fermentations of pure compounds, were utilized to identify unknown urinary metabolites of soy isoflavones. Four novel isoflavone metabolites were identified in human urine collected after soy supplementation: 3' '-OH-O-desmethylangolensin, 3',4',7-trihydroxyisoflavanone, 4',7,8-trihydroxyisoflavanone, and 4',6,7-trihydroxyisoflavanone.

Dietary lignins are precursors of mammalian lignans in rats.

The mammalian lignans enterolactone (ENL) and enterodiol, commonly found in human plasma and urine, are phytoestrogens that may contribute to the prevention of breast cancer and coronary heart disease. They are formed by the conversion of dietary precursors such as secoisolariciresinol and matairesinol lignans by the colonic microflora. The identification of lignins, cell-wall polymers structurally related to lignans, as precursors of mammalian lignans is reported here for the first time. In study 1, rats were fed rye or wheat bran (15% diet) for 5 d. Untreated brans and brans extracted with solvents to remove lignans were compared. ENL was estimated in urine samples collected for 24 h by time-resolved fluoroimmunoassay. ENL urinary excretion was reduced from 18.6 to 5.3 nmol/d (n=8; P<0.001) when lignans were removed from rye bran and from 30.5 to 6.2 nmol/d (P<0.001) when they were removed from wheat bran. These results suggest that lignins, embedded in the cell wall and retained in the bran during solvent extraction, account for 26-32% of the ENL formed from cereal brans. In study 2, rats were fed a deuterated synthetic lignin (0.2% diet) together with wheat bran (15%) for 3 d. The detection of deuterated ENL by LC-tandem MS in urine (20 nmol/d) clearly confirms the conversion of lignin into mammalian lignans. More research is warranted to determine the bioavailability of lignins in the human diet.

Phytoestrogens, cancer and coronary heart disease.

Recent results obtained in collaboration with many other groups with regard to phytoestrogens (isoflavones and lignans) and breast cancer, prostate cancer and cardiovascular disease are presented and discussed in light of new developments in the field. Both isoflavones and lignans may be protective with regard to these diseases, but we do not yet understand some of the controversial results obtained. In this short communication the possible mechanisms of disease prevention were not discussed.

Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton.

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography-mass spectrometry (GC-MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.

Rye bread in the diet of pigs enhances the formation of enterolactone and increases its levels in plasma, urine and feces.

To obtain new insight into the quantitative and qualitative metabolism of rye and wheat lignans, we performed three series of experiments with catheterized pigs. Two diets with similar levels of dietary fiber and macronutrients but with contrasting levels of plant lignans (isolariciresinol, lariciresinol, matairesinol, pinoresinol, secoisolariciresinol and syringaresinol) were prepared from rye (high in lignans) and wheat (low in lignans) soft and crisp breads. In two series of experiments we quantified the uptake from the gut of enterolactone in four pigs fitted with catheters in the portal vein and mesenteric artery and with an ultrasonic flow probe attached to the portal vein to monitor the blood flow. In a third study with six pigs, we quantified the bioavailability of the plant lignans that can be converted to enterolactone (lariciresinol, matairesinol, pinoresinol, secoisolariciresinol and syringaresinol) and the concentration in the peripheral blood. Plant and mammalian lignans in diets and stool were analyzed by isotope dilution gas chromatography-mass spectrometry and enterolactone in plasma and urine determined by time-resolved fluoroimmunoassay. There was a significantly higher formation of enterolactone in pigs fed the rye diet, and higher fecal and urinary excretion and circulating levels of mammalian lignans than in pigs fed the wheat diet. The conversion of mammalian lignan precursors to enterolactone was 48% with the wheat diet and 60% with the rye diet. Mammalian lignans are absorbed by passive diffusion from the large intestine and a substantial fraction of the absorbed mammalian lignans undergoes enterohepatic circulation, resulting in low diurnal variation in plasma levels of enterolactone.

Process-induced changes on bioactive compounds in whole grain rye.

Manufacturing of healthy wholegrain foods demands knowledge of process-induced changes in macro-, micro- and non-nutrients. The high content of dietary fibre is a challenge in relation to good product texture and sensory quality. The stability and bioavailability of bioactive compounds have a marked influence on the health effects of cereal foods. It was confirmed that sterols, folates, tocopherols and tocotrienols, alkylresorcinols, lignans, phenolic acids and total phenolics are concentrated in the bran layers of the rye grain, and are only present at low levels in the flour endosperm. The levels of folate and easily-extractable phenolic compounds increase in germination and sourdough baking, but there are negligible changes in the levels of sterols, lignans and alk(en)ylresorcinols. The levels of tocopherols and tocotrienols are reduced during the sourdough fermentation. In conclusion, many of the bioactive compounds in wholegrain rye are stable during food processing, and their levels can even be increased with suitable processing.