Theodore Fotsis

Soybean phytoestrogen intake and cancer risk.

Because many Western diseases are hormone-dependent cancers, we have postulated that the Western diet, compared with a vegetarian or semi-vegetarian diet, may alter hormone production, metabolism or action at the cellular level. Recently, our interest has been focused on the cancer-protective role of some hormone-like diphenolic phytoestrogens of dietary origin, the lignans and isoflavonoids. The precursors of the biologically active compounds originate in soybean products (mainly isoflavonoids but also lignans), as well as whole grain cereals, seeds, probably berries and nuts (mainly lignans). The plant lignan and isoflavonoid glycosides are converted by intestinal bacteria to hormone-like compounds with weak estrogenic and antioxidative activity; they have now been shown to influence not only sex hormone metabolism and biological activity but also intracellular enzymes, protein synthesis, growth factor action, malignant cell proliferation, differentiation and angiogenesis, making them strong candidates for a role as natural cancer protective compounds. Epidemiological investigations support this hypothesis, because the highest levels of these compounds are found in countries or regions with low cancer incidence. This report is a review of results that suggest that the diphenolic isoflavonoids and lignans are natural cancer-protective compounds.

Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG)

A brief account of our present knowledge on the enterohepatic metabolism of estrogens and on the origin, metabolism and biological effects of mammalian lignans and phytoestrogens is undertaken. Furthermore, recently published results on the effects of dietary fiber, fat and carbohydrates on estrogen metabolism are reviewed. New preliminary results are presented on quantitative assays of lignans and phytoestrogens in urine of women belonging to various dietary and population groups and in a group of chimpanzees. The highest values of lignans and phytoestrogens were found in the non-human primates, and in macrobiotic, lactovegetarian and Japanese women, all groups considered having a low risk for the development of breast and other hormone-dependent cancer. New results on correlations between intake of various fibers, lignan and phytoestrogen excretion and plasma levels of estrogens, free testosterone and SHBG in women are presented. There is a significant positive correlation between the intake of fiber and urinary excretion of lignans and phytoestrogens, and the concentration of plasma SHBG. Fiber intake and urinary excretion of lignans and equol correlated negatively with plasma percentage free estradiol. Enterolactone excretion correlated negatively with plasma free testosterone. It is concluded that dietary macro- and micronutrients seem to play an important role in estrogen metabolism.

Determination of urinary lignans and phytoestrogen metabolites, potential antiestrogens and anticarcinogens, in urine of women on various habitual diets

Recently two groups of compounds with diphenolic structure, the lignans and the isoflavonic phytoestrogens, were detected and identified in human urine and other biological fluids. These compounds are of great biological interest because they exhibit both in vitro and in vivo weak estrogenic and sometimes also antiestrogenic activities and many plant lignans have been shown to have anticarcinogenic, antiviral, antifungal and other interesting biological effects. The compounds found in relatively large amounts (10-1000 times more than estrogens) in urine are modified by intestinal bacteria from plant lignans and phytoestrogens, which are present in fiber-rich food such as grain and beans. They bind with low affinity to estrogen receptors and preliminary results suggest that they may induce production of sex hormone binding globulin (SHBG) in the liver and in this way may influence sex hormone metabolism and biological effects. Five compounds, the lignans enterolactone (Enl), enterodiol (End) and the isoflavonic phytoestrogen metabolites daidzein (Da), equol (Eq) and O-desmethylangolensin (O-Dma) were measured in urine by gas chromatography-mass spectrometry (selected ion monitoring) using deuterated internal standards in 5 groups of women (total number 53). The members of three dietary groups (omnivores, lactovegetarians and macrobiotics) were living in Boston and of two groups in Helsinki (omnivores and lactovegetarians). Until now measurements have been carried out in 94 72-h samples. The highest mean excretion of the most abundant compound, enterolactone, was found in the macrobiotic group and the lowest in the omnivoric groups. Total mean 24-h excretion of enterolactone was 17,680 nmol in the macrobiotics, 4,170 nmol in the Boston lactovegetarians, 3,650 nmol in the Helsinki lactovegetarians, 2,460 nmol in the Helsinki omnivores and 2,050 nmol in the Boston omnivores. The other diphenols followed approximately the same pattern. In an earlier study the lowest excretion of enterolactone (1,040 nmol/24 h) was found in a group of postmenopausal apparently healthy breast cancer patients living in Boston. It is concluded that further studies are necessary to elucidate the possible role of these compounds in cancer and other diseases. However, the evidence obtained until now seems to justify the conclusion that these compounds may be among the dietary factors affording protection against hormone-dependent cancers in vegetarians and semivegetarians.

EXCRETION OF THE LIGNANS ENTEROLACTONE AND ENTERODIOL AND OF EQUOL IN OMNIVOROUS AND VEGETARIAN POSTMENOPAUSAL WOMEN AND IN WOMEN WITH BREAST CANCER

Dietary studies and assays of urinary lignans in postmenopausal women showed that lignan excretion is significantly lower in urine of women with breast cancer than in normal omnivorous and vegetarian women and confirmed that there is a significant correlation between fibre intake and lignan excretion. It is suggested that the precursors of the human lignans enterolactone and enterodiol formed by the intestinal microflora are to be found in fibre-rich foods such as grains, nuts, and legumes. Excretion of equol, which has antioestrogenic properties, was similar in all groups studied and did not correlate with fibre intake, but occasional high values were found in some subjects.

Lignan and isoflavonoid conjugates in human urine.

Lignans and isoflavonoids are two groups of diphenolic phytoestrogens of plant origin which have gained increasing interest because of their possible cancer protective properties. High excretion of these compounds occur in populations at low risk of breast, prostate and colon cancer consuming either high amounts of whole-grain (lignans and some isoflavonoids) or soy products (isoflavonoids and some lignans). We determined the pattern of conjugation of the phytoestrogens in four urine samples from vegetarian or semivegetarian women and in two samples from men. Seven compounds were investigated: enterodiol, enterolactone, matairesinol, diadzein, equol, genistein and O-desmethylangolensin. The fractions quantified are the free fraction, mono- and disulfate, as well as the mono-, di- and sulfoglucuronide fractions. For the fractionation and purification we used ion-exchange chromatography and the determination of the concentrations of each compound in all fractions was done by isotope dilution gas chromatography-mass spectrometry (GLC-MS) using deuterated internal standards of all diphenols. More than 60% of all compounds determined, occurred in the monoglucuronide fraction. Daidzein, enterodiol and equol are excreted to a relatively high extent as sulfoglucuronides and genistein as diglucuronide. We conclude that the general pattern of lignan and isoflavonoid conjugates in urine is similar to that of endogenous estrogens.

Isotope dilution gas chromatographic-mass spectrometric method for the determination of lignans and isoflavonoids in human urine, including identification of genistein

We describe an isotope dilution gas chromatographic-mass spectrometric method for the quantitative determination of the lignans enterolactone, enterodiol and matairesinol and the isoflavonoids daidzein, equol, O-desmethylangolensin and genistein in urine. Furthermore we present the gas chromatographic/mass spectrometer identification of genistein. Urine samples were extracted on Sep-Pak cartridges, conjugated fractions were isolated by chromatography on the acetate form of DEAE-Sephadex and deuterated internal standards of all seven compounds were added to the samples before hydrolysis. The hydrolysate was extracted on a Sep-Pak cartridge and following chromatography on the acetate form of QAE-Sephadex two fractions were obtained: Fraction 1 contained equol, enterolactone, enterodiol, matairesinol and all estrogens and fraction 2 contained O-desmethylangolensin, daidzein and genistein. The latter was ready for gas chromatography/mass spectrometry, but the first one was further purified to eliminate the estrogens by chromatography on the carbonate form of QAE-Sephadex. Following silylation, the samples were analyzed by combined capillary column gas chromatography/mass spectrometry in the selective ion monitoring mode. The within-assay imprecision varied from 0.8-15.2% (mean 8.7%) and the between-assay imprecision from 4.1-13.9% (mean 9.3%), depending on compound and concentration level. The mean recovery of authentic standards added to urine extracts before hydrolysis varied from 96.6 to 105.5%. Values obtained from 10 Finnish omnivorous men are presented. Individual values for matairesinol (excretion range 3.3-59.9 nmol/24 h) and genistein (range 21.8-1180 nmol/24 h) in human urine have never been published before.

Luteolin Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis; Inhibition of Endothelial Cell Survival and Proliferation by Targeting Phosphatidylinositol 3′-Kinase Activity

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC50 of about 5 μmol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3′-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G1 progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound.

Inhibition of angiogenesis, tumour growth and metastasis by the NO‐releasing vasodilators, isosorbide mononitrate and dinitrate

1 The effect of the nitric oxide (NO)‐producing nitrovasodilators isosorbide mononitrate (ISMN) and isosorbide dinitrate (ISDN) were assessed on (a) the in vivo model of angiogenesis of the chick chorioallantoic membrane (CAM) and (b) on the growth and metastatic properties of the Lewis Lung carcinoma (LLC) in mice 2 Isosorbide 5‐mononitrate (ISMN) and isosorbide dinitrate (ISDN), inhibited angiogenesis in the CAM dose‐dependently. ISMN was more potent in inhibiting this process. Both compounds were capable of completely reversing the angiogenic effect of α‐thrombin. These effects of ISMN and ISDN on angiogenesis were comparable to those previously observed with sodium nitroprusside which generates NO non‐enzymatically 3 Mice, implanted intramuscularly with LLC, received daily i.p. injections of ISMN for 14 days resulting in a significant decrease in the size of the primary tumour and a reduction in the number and size of metastatic foci in the lungs. ISDN had a similar but less pronounced effect than that observed with ISMN 4 Addition of ISMN or ISDN to cultures of bovine, rabbit and human endothelial cells and to cultures of LLC cells had no effect on their growth characteristics 5 These results indicate that ISMN and ISDN inhibit angiogenesis and tumour growth and metastasis in an animal tumour model. The possibility should therefore be considered that these nitrovasodilators which are widely used therapeutically and have well characterized pharmacological profiles, may also possess antitumour properties in the clinic.

The multicomponent analysis of estrogens in urine by ion exchange chromatography and GC-MS—I. Quantitation of estrogens after initial hydrolysis of conjugates

A method for the multicomponent analysis of estrogens in urine after initial hydrolysis of the conjugates is described. Following protection of the carbonyl functions by ethoximation, estrogen conjugates were extracted on Sep-Pak C18 cartridges and purified on the acetate form of DEAE-Sephadex. The samples were subsequently hydrolysed by Helix pomatia juice and the hydrolysate was purified on the acetate form of QAE-Sephadex. Estrogens with vicinal cis-hydroxyls and diphenolic compounds were fractionated on the borate and bicarbonate form of QAE-Sephadex, respectively. Neutral steroids were removed by the free base form of DEAE-Sephadex after which estrogens were separated into two groups using Lipidex 5000 in a straight phase system. Following trimethylsilyl ether derivatization estrogens were analysed by selected ion monitoring (SIM). The method allows the quantitation of all the important estrogen metabolites including catechol estrogens. It is precise, accurate and sensitive permitting the quantitation of estrogens in urine of males and non-pregnant females.

Identification of lignans and phytoestrogens in urine of chimpanzees.

It was recently observed that the urinary excretion of animal lignans is low in postmenopausal breast cancer patients compared to normal omnivorous and vegetarian women. In addition, the mean excretion of the isoflavonic phytoestrogen equol tended to be lower. Because nonhuman primates appear to be remarkably resistant to the carcinogenic effect of estrogens, we investigated the possible occurrence of lignans and phytoestrogens in the urine of chimpanzees on their regular diet. Five major diphenols were isolated and identified by capillary gas chromatography and mass spectrometry by comparison with synthesized authentic reference compounds. Three of these compounds, the phytoestrogen equol and its precursor daidzein, the lignan enterolactone, were according to preliminary assays excreted in very large amounts. In addition, the lignan enterodiol and the daidzein metabolite O-desmethylangolensin were identified. It is concluded that the chimpanzee excretes both isoflavonic phytoestrogens and lignans in urine, apparently in high concentrations. It is suggested that these compounds may play a role in the maintenance of the resistance against carcinogenic effects of estrogens, which nonhuman primates possess, because both equol and enterolactone have been shown to have antiestrogenic properties in animals. However, much further work is necessary before the possible biological role of these compounds may be established.