Lee Jane W Lu

Sex and long-term soy diets affect the metabolism and excretion of soy isoflavones in humans.

Soybean consumption may be protective against hormone-dependent cancers, possibly in part because of the isoflavones daidzein and genistein, which are weakly estrogenic. This paper reviews our studies of the metabolism and disposition of these phytoestrogens in humans. During 1 mo of daily soy ingestion in a metabolic unit [1.065 L (36 oz) soymilk, providing 80-210 mg of each isoflavone daily], women initially excreted more isoflavone conjugates in urine than did men. Recoveries of conjugates of genistein, daidzein, and equol were 24%, 66%, and 28% of the amounts ingested in women, respectively, and 15%, 47%, and 15%, respectively, of those in men. A progressive decrease in urinary excretion of genistein and daidzein was observed in women but not in men during the study. At least 10% of ingested daidzin was excreted in urine as equol conjugate in one man and one woman after the first soy ingestion. Three more women but no more men developed the ability to produce and excrete large amounts of equol. Absorption rate constants (k(e)) of the isoflavones were estimated to be 0.24-0.50 h(-1). The elimination rates (k(e)) for genistein, daidzein, and equol were 0.1, 0.16, and 0.08 h(-1), respectively, in women and 0.19, 0.25, and 0.13 h(-1), respectively, in men. Thus, the excretion half-life values of genistein were longer in women (7, 4, and 9 h, respectively) than in men (4, 3, and 5 h, respectively) after the first soy ingestion. The excretion half-life shortened progressively in women but lengthened progressively in men over the study period. Thus, isoflavone metabolism and disposition were affected by the duration of soy ingestion and by sex.

Altered kinetics and extent of urinary daidzein and Genistein excretion in women during chronic Soya exposure

Soybean consumption may be protective for breast cancer, possibly due in part to the presence of the isoflavones daidzein and genistein, which are weakly estrogenic. The metabolism and disposition of these phytoestrogens during chronic soya exposure were studied on a metabolic unit. Six healthy 22- to 29-year-old women consumed an unrestricted hospital diet for most of the study and ingested 12 oz of soymilk with each meal for one month. At two-week intervals, excretion of isoflavones in urine was studied, during which time the subjects consumed a constant basal diet for three to four days, ingested the full daily 36-oz portion of soymilk within 30 minutes each day for one to two days, and collected urine continuously. Urinary recovery of genistein [initially 23.9 +/- 17.3% (SD) of ingested genistin + genistein], daidzein (initially 66.2 +/- 23.5% of ingested daidzin + daidzein), and equol (initially 28% of the ingested precursors daidzin + daidzein in 1 subject and < 1% in 5 subjects) decreased progressively over four weeks of daily soya ingestion by 42% for genistein (p < 0.05) and 31% for daidzein (p < 0.01) but increased by 3- to 100-fold for equol (4 subjects, p < 0.05). Total amounts excreted and peak levels were similarly affected. The absorption half-lives (t 1/2) for genistein and daidzein were initially 2.7 +/- 0.8 and 1.6 +/- 0.5 hours, respectively, and during four weeks of soymilk ingestion decreased to 2.0 +/- 0.6 (p = 0.04) and 1.4 +/- 0.2 hours (p = 0.06), respectively, suggesting more rapid absorption. The appearance t 1/2 of equol can be estimated for only one subject initially (2.9 hrs), but during four weeks of soya ingestion it could be estimated for three more subjects (4.7 +/- 2.3 hrs). The excretion t 1/2 values for genistein and daidzein were initially 6.7 +/- 0.8 and 4.4 +/- 0.7 hours, respectively, and during four weeks of soymilk ingestion decreased to 4.2 +/- 1.2 (p = 0.005) and 3.2 +/- 1.1 hours (p = 0.005), respectively, suggesting more rapid excretion. For equol, the excretion t 1/2 was initially 9.1 hours (1 subject), and after two and four weeks of soymilk ingestion it was 13.4 +/- 9.7 and 5.5 +/- 1.6 hours (4 subjects, p = 0.046, 2 wks vs. 4 wks), respectively. These results indicate that metabolism and disposition of ingested isoflavones are altered during chronic soya ingestion in women, perhaps from increased metabolic degradation to formation of nonisoflavone metabolites. Increased production of the longer- and stronger-acting estrogenic equol in some women during chronic soymilk ingestion may alter the estrogenic potency of dietary soya isoflavones.

Altered time course of urinary daidzein and genistein excretion during chronic soya diet in healthy male subjects

Soybean consumption is associated with reduced rates of prostate and other cancers, possibly due in part to the presence of isoflavones. The metabolism and disposition of these soya-derived phytoestrogens after chronic soya exposure were studied on a metabolic unit in six healthy males (21-35 yrs of age) who consumed an unrestricted hospital diet and a 12-oz portion of soymilk with each meal for one month. The daily isoflavone intake was about 100 mg of daidzein (mostly as diadzin) and about 100 of mg of genistein (mostly as genistin). At two-week intervals, excretion of isoflavones in urine was studied, during which time the subjects consumed a constant basal diet for three to four days, ingested the full daily 36-oz portion of soymilk within 30 minutes each day for one to two days, and collected urine continuously. The urinary recovery of ingested diadzin plus daidzein (46.9 +/- 15.2%, mean +/- SD) and genistin plus genistein (14.6 +/- 9.2%) did not change with prolonged soya ingestion. The absorption half-lives (t1/2) for daidzein and genistein and the appearance t1/2 for equol (1 subject) were initially 1.5 +/- 0.4, 1.9 +/- 0.6, and 2.2 hours, respectively, and 2.5 +/- 1.1 (p = 0.06 compared with baseline) 1.4 +/- 0.9 (p = 0.03) compared with baseline), and 4.2 hours, respectively, during one month of soymilk ingestion. The excretion t1/2 for daidzein, genistein, and equol were initially 2.9 +/- 0.5, 3.8 +/- 0.7, and 5.2 hours, respectively, and 3.9 +/- 1.2 (p - 0.03), 5.5 +/- 1.6 (p = 0.02), and 9.7 hours, respectively, during one month of soymilk ingestion. These results indicate that chronic soya exposure did not induce significant changes in the metabolic pathways of isoflavones but altered the time courses of daidzein and genistein excretion. Thus chronic exposure to soya might prolong the tissue exposure to the presumed biologically active free and unconjugated forms of these isoflavones and thereby enhance their oncoprotective effects.

Genetic damage and the inhibition of 7,12-dimethylbenz[a]anthracene-induced genetic damage by the phytoestrogens, genistein and daidzein, in female ICR mice

Populations consuming soybeans have reduced rates of breast, colon and prostate cancer possibly due, in part, to the presence in soybeans of two estrogenic isoflavones, genistein and daidzein. This study investigated the genotoxicity of these soya isoflavones and their interactions with 7,12-dimethylbenz[a]anthracene (DMBA)-induced sister chromatid exchanges (SCE) in bone marrow cells and DNA adduct formations in liver and mammary glands of mice. Groups of female ICR mice were pretreated i.p. with daidzein and/or genistein (10-20 mg/kg per day for 6 days or 50 mg/kg per 12 h for 3 days) or with the solvent, dimethylsulfoxide (DMSO). The mice were implanted with bromodeoxyuridine (BrdU) tablets s.c., and treated with DMBA (50 mg/kg) i.p. and colchicine (4 mg/kg) i.p. 24, 23, and 2 h before sacrifice, respectively. In bone marrow cells. DMBA alone induced 11.73 +/- 1.42 SCE/cell compared to 4.35 +/- 0.83 SCE/cell in the DMSO treated controls (P = 0.001). DMBA induced 20% fewer SCE (P < 0.05) in mice pretreated with daidzein, genistein or a combination of genistein and daidzein (6 x 20 mg/kg per day for 6 days) when compared to mice that received no pretreatments. Genistein at 50 mg/kg per 12 h for 3 days also inhibited DMBA-induced SCE by 20%. However, treatment for 3 days with 50 mg/kg per 12 h of genistein or daidzein alone, or a combination of daidzein plus genistein (without DMBA treatment) also induced more SCE than treatment with only the solvent (DMSO, P < 0.05). Pretreatment with both the low and the high doses of daidzein plus genistein or the high dose of genistein reduced the replication index of bone marrow cells when compared to pretreatment with DMSO (P < 0.05). Pretreatment with genistein reduced DMBA-induced DNA adduct formation by 34%, but this was only marginally significant (P = 0.08) due to the large inter-individual variability in adduct levels. These results show that genistein and daidzein suppress SCE and possibly DNA adduct formation induced by the known carcinogen, DMBA. This response to a low dose isoflavone exposure may be partly responsible for the protective effect against endocrine cancers of soya consumption.

Drug effects on nucleic acid modification. I. A specific effect of 5-azacytidine on mammalian transfer RNA methylation invivo

Abstract Administration of the potent antineoplastic agent, 5-azacytidine (1-β-D-ribofuranosyl-4-amino- s -triazine-2 (1 H )-one), to mice causes a marked reduction of the 5-methylcytidine content of liver transfer RNA. This effect is dose-dependent and specific for the methylation of the 5-position of cytidine; the drug has no effect on 3-methylcytidine, 4-acetylcytidine, 5-methyluridine, dihydrouridine, pseudouridine, and modified purines. The mechanism of this effect has not yet been elucidated but probably involves the selective inhibition of 5-methylcytosine methyltransferase(s) by 5-azacytidine itself or its metabolites. The results presented show that the modified constituents of nucleic acids provide potential targets for chemotherapeutic agents.

The measurement of the isoflavone daidzein by time resolved fluorescent immunoassay : a method for assessment of dietary soya exposure

We report a novel method for the measurement of urinary daidzein that is suitable for assessment of dietary soya exposure. The method incorporates the following features: (i) a highly specific monoclonal antibody to daidzein (clone 4E4) raised through the 7 position of daidzein and (ii) a europium labeled ovalbumin daidzein conjugate. In the present format, dilute urine samples of subjects who ingested soy milk are hydrolyzed with beta-glucuronidase for 30 min on rabbit anti-mouse coated plates. Afterwards, the specific monoclonal antibody to daidzein, clone 4E4, and europium labeled ovalbumin daidzein conjugate are added. After 1 h incubation, the wall bound fluorescence of europium is measured by time resolved fluorescence and is inversely proportional to the concentration of daidzein over the range 0.1-10 ng daidzein/well. The method demonstrates good sensitivity, precision and comparability with the chemical method GC-FID. Unlike the chemical method, the present immunoassay technique for daidzein is applicable for the measurement of large amounts of samples in epidemiological studies for the assessment and monitoring of human exposure to soya food.

Phytoestrogens and healthy aging: gaps in knowledge. A workshop report.

There is an increasing public interest in foods and dietary supplements containing phytoestrogens for the maintenance of health. A workshop was convened to assess evidence for the potential benefits of phytoestrogen-containing foods or supplements on diseases or conditions affecting older populations. Preclinical, clinical, and epidemiologic data on the cardiovascular system, various cancers, bone diseases, and menopausal symptoms were the focus of the discussions. Research on the basis of consumer food choices as well as a presentation from the FDA regarding approval of the use of soy foods to reduce the risk of cardiovascular disease were also presented. Based on the information presented, isoflavone-containing soy foods may have favorable effects on the cardiovascular system, but major knowledge gaps still exist regarding effects of phytoestrogen supplements on bone diseases, various cancers, menopausal symptoms, and cognitive function.

DNA hypomethylation in Morris hepatomas

The 5-methylcytosine (m5C) content of DNAs from Morris hepatomas of varying growth rates and from normal liver was analyzed. DNA methylation in all hepatomas studied was found to be 20-45% less than in normal liver. This result was confirmed independently by restriction endonuclease (Hpa II and Msp I) analysis. While these results agreed with recent literature data suggesting hypomethylation of DNA from some neoplastic sources, no correlation was observed between the extent of DNA hypomethylation and the growth rates of the tumors.

Lean Body Mass, Not Estrogen or Progesterone, Predicts Peak Bone Mineral Density in Premenopausal Women

Estrogen and body fat content are important predictors of bone mineral density (BMD) in postmenopausal women, but their association with BMD in premenopausal women is less clear. Mounting evidence suggests that dietary fats can have detrimental effects on bone health. In a cross-sectional sample of healthy 30- to 40-y-old women (n = 242), we investigated the predictors of BMD at the hip and spine by multilevel multiple regression analyses. Predictor variables in the models included dietary intake of various fats, serum concentrations of sex steroids, blood chemistries and markers of metabolic syndrome, anthropometric variables, and ethnicity. Among these premenopausal women, lean body mass was the strongest independent predictor (P < 0.0001) and African-American ethnicity (P < 0.05) was another positive independent predictor of BMD at the hip and spine. Dietary fats were not independent predictors of BMD of hip and spine. Lean body mass and being African-American explained 33% of the variance in hip BMD. Lean body mass, African-American ethnicity, and serum concentrations of triglycerides (a negative predictor, P = 0.0001) explained 28% of the variance in spine BMD. In contrast, luteal phase serum concentrations of estradiol, progesterone, and testosterone were not predictors of BMD. It remains to be determined whether efforts to increase lean body mass in premenopausal women with normal levels of endogenous estrogen may be an effective preventive strategy to preserve bone health.

A Nonisotopic Enzyme-Based Immunoassay for Assessing Human Exposure to Genistein

Phytoestrogenic isoflavones that are abundant in soybeans may be an important group of natural products that could play a critical role in preventing several chronic human diseases. To facilitate studying the relationship of soya exposure and chronic diseases, we report a simple method for measuring an isoflavone, genistein, in human urine and plasma. The method is a competitive enzyme-linked immunoassay that utilizes a conjugate of horseradish peroxidase (HRP) and genistein as tracer and a monoclonal antibody to genistein (clone 10D8) generated through the 6-position of genistein. Genistein, in diluted hydrolyzed urine or plasma of subjects who ingested soy milk, competes with HRP-genistein conjugate for the binding sites of anti-genistein antibody on rabbit anti-mouse IgG-coated plates. After a one-hour incubation, the wall-bound genistein-HRP activity, after reaction with a chromogen, is measured colorimetrically at 450 nm and is inversely correlated with concentrations of genistein over the range of 0.1-32 ng/well. The sensitivity limit of the method is 0.5 ng of genistein per well or 0.5 ng per 10 microliters of urine and plasma. Urine and plasma levels of genistein measured by this immunoassay correlated well (R2 = 0.92 for urine and 0.77 for plasma) with those determined by chromatographic techniques. This method can be used to assess soya exposure in humans and could facilitate epidemiological studies of the relationship of soya diets and chronic diseases, including cancer.