The effects of native and synthetic estrogenic compounds as well as vitamin D less-calcemic analogs on adipocytes content in rat bone marrow.

BACKGROUND: We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17ß (E2) modulate bone morphology in rat female model. AIM: We now analyze the effects of phytoestrogens, E2, selective E2 re ceptor modulators, and the less-calcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). MATERIALS AND METHODS: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. RESULTS: In OVX young adults there is a decreased bone formation and a 10-fold increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or DT56a resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. CONCLUSIONS: Our results demonstrate that adipogenesis induced by OVX is a reversible process which can be corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone loss as well as a novel approach to diagnosis and treatment of postmenopausal osteoporosis.

The effects of peptides with estrogen-like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells.

Hormone replacement therapy (HRT) for post-menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post-menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen-mimetic peptide 1 (EMP-1) (VSWFFE) in comparison to estrogen (E(2)) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP-1-like E(2) inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP-1 did not affect E(2) stimulation of DNA, but inhibited E(2) inhibition of cell proliferation at high concentration. All effects by the combination of EMP-1 and E(2) were abolished at hg. A44-stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co-incubated with E(2) at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E(2) stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45-stimulated CK-specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP-1. EMP-1 and A44 similar to E(2)-stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP-1 provided it acts similarly in vivo can replace E(2) for treatment of post-menopausal women in hyperglycemia due to diabetes.

Daidzein but not other phytoestrogens preserves bone architecture in ovariectomized female rats in vivo.

Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol-17beta (E(2)) restored bone structure of ovariectomized (Ovx) female rats to values obtained in intact sham-operated female rats. E(2) also selectively stimulated creatine kinase (CK) specific activity a hormonal-genomic activity marker. In the present study, we compared the effects of E(2) and the phytoestrogens: daidzein (D), biochainin A (BA), genistein (G), carboxy-derivative of BA (cBA), and the SERM raloxifene (Ral) in Ovx, on both histological changes of bones and CK, when administered in multiple daily injections for 2.5 months. Bone from Ovx rats, showed significant disrupted architecture of the growth plate, with fewer proliferative cells and less chondroblasts. The metaphysis underneath the growth plate, contained less trabeculae but a significant increased number of adipocytes in the bone marrow. D like E(2) and Ral but not G, BA, or cBA, restored the morphology of the tibiae, similar to that of control sham-operated animals; the bony trabeculeae observed in the primary spongiosa was thicker, with almost no adipocytes in bone marrow. Ovariectomy resulted also in reduced CK, which in both epiphysis and diaphysis was stimulated by all estrogenic compounds tested. In summary, only D stimulated skeletal tissues growth and differentiation as effectively as E(2) or Ral, suggesting that under our experimental conditions, D is more effective in reversing menopausal changes than any of the other isolated phytoestrogens which cannot be considered as one entity.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A.

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

Role of putative membrane receptors in the effect of androgens on human vascular cell growth.

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.

Anti-idiotypic antibody as an oestrogen mimetic in vivo: stimulation of creatine kinase specific activity in rat animal models.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) significantly increased the specific activity of creatine kinase (CK) activity in the rat uterus, and in vitro in skeletal cells capable of responding to oestradiol (E2), suggesting that the antibody has oestrogenic-like activity. Moreover, the F(ab')2 dimer of clone 1D5 acted like an antagonist and completely inhibited the increase in CK specific activity by either E2 or clone 1D5 in these skeletal cells. In the present study, we examined the in vivo effects of clone 1D5 and its proteolytic fragment, the F(ab')2 dimer, E2 and dihydrotestosterone (DHT) on CK specific activity in the epiphyseal cartilage, diaphyseal bone, uterus, prostate, thymus and pituitary of immature or gonadectomized female and male rat animal models. In the intact immature animals, clone 1D5 caused an increase in CK in all organs of the female except in the pituitary. In the diaphyseal bone and prostate of male rats there was no stimulation by 1D5. The CK response in the uterus, epiphysis, and diaphysis of immature female rats was dose-dependent and was blocked by either the anti-oestrogen tamoxifen or the F(ab')2 dimer of clone 1D5. E2, DHT, as well as clone 1D5, stimulated CK specific activity in both the diaphysis and epiphysis of ovariectomized female and castrated male rats, whereas sex specificity in the CK response was observed also in the uterus and the prostate of gonadectomized animals. Collectively, these results suggest that, as in cell culture, an intact antibody is necessary for the observed stimulation of CK specific activity and the F(ab')2 dimer can act as an antagonist. Furthermore, the observed biological effects of clone 1D5 which are absolutely parallel to E2, imply that the anti-idiotypic antibody is able to penetrate the cell and reach the nuclear oestrogen receptor and transduces a signal to the nucleus, by as yet uncharacterized mechanisms.

Pharmacological effects of the continuous administration of an LHRH agonist in the ageing male rat: comparison with orchidectomy.

The age-related changes in tissue response to chronic treatment for 1 month with a potent LHRH agonist were investigated in the ageing male rat, and the observed pharmacological effects were compared with orchidectomy. In both young (4 months) and old (22 months) rats, treatment resulted in a significant decrease in the weights of prostates and testes, a decrease in plasma LH and testosterone levels, a loss of LH receptors in the testes and in a complete depletion of prostatic nuclear androgen receptors, reaching levels observed after castration. In young rats, treatment with an LHRH agonist or orchidectomy induced a three- or sixfold increase in prostatic creatine kinase (CK) activity which may have been induced by the local stimulatory effect of oestradiol arising from the conversion of precursor steroids secreted by the adrenal. On the other hand, in old rats, 7 days after orchidectomy or after treatment with an LHRH agonist a twofold increase or no change was induced in prostatic CK activity respectively. SDS gel electrophoresis patterns of cytosolic prostatic proteins of young rats treated with an LHRH agonist or young rats orchidectomized 7 days previously revealed the presence of several intensified proteins, two of them having apparent molecular weight of 67 kDa and 43 kDa, whereas in the old rats treated with LHRH agonist or old rats castrated 7 days previously, these two proteins were not intensified. The results of this study confirmed that continuous treatment with an LHRH agonist to young and old rats induces medical castration since the pharmacological effects observed were the same as those induced with surgical castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxy derivatives of isoflavones as affinity carriers for cytotoxic drug targeting in adrenocortical H295R carcinoma cells.

Carboxy derivatives of isoflavones that exhibit oestrogenic/anti-oestrogenic properties were used as carriers for affinity drug targeting to H295R adrenocortical carcinoma cells that express transcripts of oestrogen receptor (ER) alpha and beta. These derivatives were prepared by introducing a carboxymethyl group at the 6-position of genistein and of biochanin A, yielding 6CG and 6CB respectively. In transactivation assays, 6CG displayed mixed agonist/antagonist activity for ERalpha, whereas 6CB displayed only weak antagonist activity. Low concentrations of oestrogen, 6CG and 6CB were capable of inducing proliferation in H295R cells and of stimulating creatine kinase (CK) specific activity, suggesting that these cells were sensitive to oestrogenic compounds. In in vivo experiments, both 6CG and 6CB were capable of inhibiting oestrogen-induced CK specific activity in rat tIssues. For affinity drug targeting, the cytotoxic drug daunomycin was coupled to 6CB and 6CG, yielding 6CB-Dau and 6CG-Dau respectively. These conjugates were tested for their antiproliferative ability to inhibit DNA synthesis as assessed by incorporation of [(3)H]thymidine in H295R cells. A dose-dependent cytoxicity was observed with both conjugates. At 0.3-3 nM, both conjugates were 10 to 30 times more potent than daunomycin. At 30 nM these conjugates were two to three times more potent than daunomycin. At concentrations ranging between 300 and 3000 nM, no difference in cytotoxicity was observed between the conjugates and daunomycin. When the cells were treated over a wide range of concentrations with a combination of 6CG plus daunomycin, the observed cytotoxicity was less than with daunomycin alone. When non-transformed rat enterocytes, which do not express ER, were treated with 6CG-Dau or daunomycin, the antiproliferative effect of 6CG-Dau was the same as that of daunomycin over the concentration range tested. These pilot studies suggest that the ready availability of oestrogenic binding sites in H295R cells can be exploited for site-directed chemotherapy.

Anti-idiotypic antibody as an oestrogen mimetic: removal of Fc fragment converts agonist to antagonist.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) caused an increase in uterine creatine kinase (CK) activity when administered in vivo to immature female rats, indicating that the antibody has oestrogenic-like activity. It was, therefore, of interest to investigate the structural requirements of clone 1D5 to act as an oestrogen mimetic in an in vitro model system. In the present study, the effect of clone 1D5 and its proteolytic fragments, F(ab')2, Fab' and Fc on CK activity was examined in cultured skeletal cells having functional oestrogen receptor (ER). Incubation of female-derived calvaria cells or epiphyseal cartilage cells with clone 1D5 (8.33 nM) or oestradiol (E2) (30 nM) for 24 h caused a significant increase in CK activity, indicating that clone 1D5 acted as an agonist. On the other hand, incubation of male-derived calvaria cells devoid of a functional ER with clone 1D5 or E2 did not have any effect on CK activity. Incubation of female-derived calvaria cells with clone 1D5 and E2 did not result in any further increase in CK activity, whereas dihydrotestosterone (DHT) did not alter the response to clone 1D5. The CK response to clone 1D5, in female-derived calvaria cells was time- and dose-dependent and could be inhibited in a dose-dependent manner by the oestrogen antagonist tamoxifen. In contrast, the proteolytic fragments of clone 1D5, the F(ab')2 dimer (12 nM) and the Fab' monomer (24 nM), and the Fc fragment (28 nM) did not have E2-like activity in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Carboxymethyl genistein: a novel selective oestrogen receptor modulator (SERM) with unique, differential effects on the vasculature, bone and uterus.

The novel genistein (G) derivative, 6-carboxymethyl genistein (CG) was evaluated for its biological properties in comparison with G. Both compounds showed oestrogenic activity in vitro and in vivo. On the other hand G and CG differed in the following parameters: (i) only CG displayed mixed agonist-antagonist activity for oestrogen receptor (ER) alpha in transactivation assays and (ii) only CG was capable of attenuating oestrogen (E(2))-induced proliferation in vascular smooth muscle cells and of inhibiting oestrogen-induced creatine kinase (CK) specific activity in rat tissues. On the other hand only G enhanced the stimulatory effect on CK specific activity in the uterus. In comparison to the selective oestrogen receptor modulator (SERM) raloxifene (RAL), CG showed the same selectivity profile as RAL in blocking the CK response to E(2) in tissues derived from both immature and ovariectomized female rats. Molecular modelling of CG bound to the ligand binding domain (LBD) of ERbeta predicts that the 6-carboxymethyl group of CG almost fits the binding cavity. On the other hand, molecular modelling of CG bound to the LBD of ERalpha suggests that the carboxyl group of CG may perturb the end of Helix 11, eliciting a severe backbone change for Leu 525, and consequently induces a conformational change which could position Helix 12 in an antagonist conformation. This model supports the experimental findings that CG can act as a mixed agonist-antagonist when E(2) is bound to its receptors. Collectively, our findings suggest that CG can be considered a novel SERM with unique effects on the vasculature, bone and uterus.

Characterization of a prolactin-daunomycin ligand as a probe for drug targeting.

Conjugates of ovine prolactin and daunomycin were prepared for use as affinity-labelled drug carriers in cancer cells carrying the prolactin receptor. The binding affinity of the conjugates to prolactin receptors in rat liver membrane preparations and in viable granulosa cells derived from estradiol- and pregnant mare serum gonadotropin (PMSG)-treated immature female rats was less than an order of magnitude lower than prolactin. The toxicity of the conjugate in cultured granulosa cells was dependent upon the concentration of the daunomycin present in the culture. The cytotoxic effect of the ligand was abolished by the addition of free prolactin or NH4Cl to the granulosa cell cultures. These conjugates may be useful probes in drug targeting against hormone-sensitive cancer.

The measurement of oestrone-3-glucuronide in urine by non-competitive idiometric assay.

We report a novel non-competitive idiometric assay for the measurement of oestrone-3-glucuronide (EG) in diluted urine. The method is based on the use of two types of anti-idiotypic antibody, the beta-type and alpha-type, that recognize different epitopes within the hypervariable region of the primary anti-EG antibody (Ab1). The beta-type anti-idiotypic antibody is analyte sensitive and competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the alpha-type is analyte insensitive, but does not bind the Ab1 in the presence of the beta-type due to epitope proximity. In the present format, reaction mixtures containing the europium labelled Ab1 are reacted sequentially with EG standards or diluted urine samples, with the beta-type anti-idiotypic antibody and biotinylated alpha-type anti-idiotypic antibody on immobilized streptavidin coated microtiter plates. After 1 h incubation, the fluorescence of europium is measured by a time-resolved fluorescence and is proportional to the concentration of EG over a range of 0-10 nmol/l. The method demonstrates good sensitivity, precision and comparability with an alternative competitive fluorescent immunoassay. The idiometric assay for EG may be applied for the monitoring of ovarian function in women and is suitable for dipstick technology.

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.

A monoclonal antibody to oestradiol potentiates the stimulation of the specific activity of the brain type creatine kinase by oestrogen in vivo and in vitro.

We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E2 15 plus oestrogen (E2) potentiated the specific activity of the brain type creatine kinase (CK) response to E2 in the rat tissues or skeletal cells. The enhancing CK response of anti E2 15 plus E2 was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E2 15 blocked the stimulatory CK response to E2. Two other high affinity anti-E2 antibodies, clones 8D9 and 11B6, had no effect in augmenting the response of CK to E2 in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E2 15 plus E2 since the intact anti-E2 in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E2 15 abolished the CK response to E2 in rat tissues and not to anti-E2 15 plus E2 whereas tamoxifen completely blocked the CK response to anti E2 plus E2. Anti E2 15 may therefore serve as a specific carrier in delivering E2 to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E2 effects in vivo and in vitro.

The measurement of progesterone in serum by a non-competitive idiometric assay.

A novel non-competitive idiometric time-resolved fluoroimmunoassay for the determination of serum progesterone was developed, based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary antiprogesterone antibody. The first anti-idiotype, the betatype, competes with progesterone for an epitope of the primary antiprogesterone antibody at the binding site. The second anti-idiotype, the alphatype, binds to the antiprogesterone antibody in the presence of progesterone, but does not bind to the betatype antiprogesterone complex due to epitope proximity. In the present configuration, the biotinylated alphatype was captured onto anti-biotin IgG which was immobilized on microtiter wells. Reaction mixtures containing europium-labeled antiprogesterone antibody complexed sequentially with progesterone in standards or serum samples and with the betatype anti-idiotypic antibody were then reacted with the immobilized alphatype anti-idiotypic antibody. After 30 min of incubation, the fluorescence of europium is measured by time-resolved fluorescence and is proportional to the concentration of progesterone over the range 0-320 nmol/mL. The method demonstrates good sensitivity, precision, and comparability with a direct competitive radioimmunoassay. The idiometric assay for progesterone is suitable for dipstick technology and biosensors.

Inhibitors of protein tyrosine phosphorylation: preliminary assessment of activity by time-resolved fluorescence.

Epidermal growth factor (EGF) receptor (ErbB1)-associated tyrosine kinase inhibitors may act as potential chemotherapeutic agents. In order to assess the inhibitory activity of these compounds, we developed a simple and sensitive assay based on time-resolved fluorescence. In this technique, crude cell lysates bearing the ErbB1 receptor were captured in microtitre plates immobilized with monoclonal anti-ErbB1 antibody SG 565. Subsequently, the phosphotyrosine content of the cell lysates was quantified by a europium-labelled anti-phosphotyrosine antibody. Thus, genistein, a tyrosine kinase inhibitor, was capable of reducing by half the tyrosine phosphorylation caused by the binding of EGF to A431 cells, whereas 6-carboxymethyl genistein did not inhibit protein tyrosine phosphorylation. This assay is simple to perform, does not use radioactive substrates, and can be useful for screening EGF receptor tyrosine kinase inhibitors from natural products or synthetic compounds. Moreover, the assay has a high signal:noise ratio and is suitable for large-scale screening.

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.