1. AbstractWe have previously reported that human primary cultured bone cells (hObs) respond to estradiol-17β (E2) and different phytoestrogens and vitamin D analogs by modulating cell proliferation. In the present study hObs derived from biopsies of pre- or post- menopausal females or males were cultured and treated with different estrogens and with the non-calcemic vitamin D JK-1624F2-2 (JKF) and assayed for the expression of 12LO and 15LO mRNA as well as for the synthesis of 12 and 15HETEs and the formation of ROS. All cells express 12LO and 15LO and produce 12 and 15 HETEs. Treatment with all estrogens: E2, daidzein (D) and femarelle (F) as well as JKF modulated the expression of both 12 and 15LO mRNA in pre- or post-menopausal female hObs and to some extent in male hObs. 12 and 15HETEs production was also modulated by these compounds in these cells. ROS formation was increased by all hormones in pre- and post- but not in male hObs. ROS formation was also increased by 12 and 15HETEs in pre- and post- but not in male hObs. In conclusion, we provide herein evidence for a novel mechanism of hormonal-induced human bone biology operating through LO enzymes and ROS formation, but the exact role of LO/HETE/ROS pathway is not clear yet. We also provide a mechanism of new phytoestrogen which is not behaving like E2 and can react as both estrogenic compound and a disruptor of estrogen regulation in a mechanism which has still to be further studied.

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

BACKGROUND:We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17β (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 incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) βmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17β (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERβ-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.

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.

Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol‐17β (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. J. Cell. Biochem. 103: 1826–1832, 2007. © 2007 Wiley‐Liss, Inc.

Introduction: Targeting of therapeutics and contrast media to tumors is limited by unfavorable biodistribution. We previously reported the exclusion of intravenously administered biotin-BSA-GdDTPA to stroma tracks in ovarian tumor xenografts (1, 2). Within the stroma tracks, biotin-BSA-GdDTPA was effectively internalized by the myofibroblasts by caveolae mediated uptake, which could be suppressed by treatment with nystatin (1). Upon internalization, the material is sequestered within small intracellular vacuoles leading to suppressed MRI relaxivity, which is regained only after the material is redistributed in the cells with cell division (1, 2). The contrast material does not partition to the interstitial space within the tumor nodules (1). We aimed to alter the distribution of biotin-BSA-GdDTPA, and to enhance the partition of the contrast material to the tumor by addition of daidzein as a targeting ligand, recently reported to show high affinity to the ovarian carcinoma cells (3). The use of daidzein-BSA-GdDTPA/CyTE-777 (or FAM) as a bifunctional targeted contrast media revealed a novel mechanism regulating endocytosis by ovarian cancer cells, involving competition between receptor mediated internalization through binding of daidzein and caveolae mediated internalization via binding of albumin. Materials and methods: MRI measurements: MRI experiments were performed on a horizontal 4.7 T Bruker Biospec spectrometer using an actively radio-frequency decoupled 1.5 cm surface coil embedded in a Perspex board (for in-vitro) and a birdcage transmission coil (for in-vivo). In-vitro: R1 measurements spin echo images were acquired at 8 different repetition times ranging between 2000 and 100 ms; 2 averages, field of view 4X4 cm, slice thickness 1 mm, matrix 128 X 128). In-vivo: T1 weighted 3D gradient-echo (GE) images, with pulse flip angles of 50,150,300, 500 and 700 were acquired to determined the R1 values. The acquisition parameters: TR 10ms; TE 3.561ms; 2 averages; field of view 4X4X2 cm; 128X128X128 pixels. MRI in-vivo model: The tumor bearing mice were injected with BSA-GdDTPA or daidzein-BSA-GdDTPA. R1 was measured 24h, 48h and 72h after injection of the contrast material. Fluorescence microscopy: MLS cells were incubated with Daidzein-BSA-FAM or BSA-FAM (200 μg/ml) or combination of both (Daidzein-BSA-FAM with BSAROX) in the presence or absence of a blocking dose of nystatin (50 μg/ml). The excess of fluorescent material was washed 3 times with PBS, and the cells were fixed with 4% PFA, then washed, and stained with DAPI and mounted. The images were monitored by two-photon and by confocal microscopy (2PM; Zeiss LSM 510 META NLO). Results: Uptake of daidzein-BSA-FAM by ovarian carcinoma cells: Binding and uptake of BSA-FAM and daidzein-BSA-FAM by MLS human epithelial ovarian carcinoma cells could be detected by two-photon and by confocal fluorescence microscopy, showing internalization of the fluorescent probes into intracellular vesicles. Suppression of caveolae mediated uptake by treatment of the cells with nystatin, resulted in slightly reduced uptake of BSA-FAM and in significantly enhanced internalization of daidzein-BSA-FAM by MLS cells. The distribution of contrast material inside the tumor was visualized by fluorescence microscopy in tumors isolated 24 after injection with daidzein-BSA-FAM or BSA-FAM. Daidzein-BSA-FAM was localized in the tumor cells areas, while BSA-FAM that was localized to tumor blood vessels. In vivo MRI detection of targeted delivery of daidzein-BSA-CyTE-777 to ovarian carcinoma: Analog contrast media was synthesized to allow detection of targeted tumor delivery by MRI, by conjugation of GdDTPA to daidzein-BSA.MRI data acquired from MLS tumor bearing mice 24h after administration of daidzein-BSAGdDTPA or BSA-GdDTPA (12 mg/200 μl), showed significantly higher accumulation of daidzein-BSA-GdDTPA in the tumor site as compared to vehicle-injected mice or mice injected with BSA-GdDTPA (Figure 1 B,C). Time course experiments showed specific localization and retention of targeted contrast agent (daidzeinBSA-GdDTPA) in the tumor site 24, 48 and 72h after injection as compared to controls or mice injected with non targeted contrast agent (BSA-GdDTPA). Statistically significant elevation of R1 relaxation was visualized in tumors injected with daidzein-BSA-GdDTPA as compared to non injected animals or mice injected with BSAGdDTPA. Conclusions: We show presence of two competing endocytic pathways for uptake of targeted bifunctional daidzein-BSA MRI/NIR contrast media to human ovarian carcinoma cells. The ability to manipulate between this pathways by nystatin or BSA, augments both in-vivo and in-vitro the distribution and endocytosis of the contrast material to the cancer cells.

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photo-induced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3-48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll-serine (Bchl-Ser), which is water soluble, highly toxic upon illumination in the near-infrared (lambda max 765-780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76-212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin-spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA-enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl-Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and raloxifene (Ral), and male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. We also found that pre-treatment with the less-calcemic analog of Vitamin D, JK 1624 F(2)-2 (JKF), upregulated the response of CK to E(2) and Ral. In this study, we analyzed the response of human bone cells from pre- and post-menopausal females and males, to phytoestrogens. Bone cells derived from pre-menopausal women showed greater stimulation of CK than cells from post-menopausal women, after treatment with E(2) (30 nM), daidzein (D, 3000 nM), genistein (G, 3000 nM) and Ral (3000 nM); whereas the responses to biochainin A (BA 3000 nM), quecertin (Qu 3000 nM) or the carboxy derivative of G (cG 300 nM) were not age-dependent. Male-derived cells did not respond to phytoestrogens. When cells derived from female bones at both age groups were pre-treated with JKF, by daily addition of 1nM, for 3 days, there was an upregulation of the response to E(2), Ral, G and D but not to BA or Qu. Nuclear binding of (3)[H] E(2) was characteristic of the different phytoestrogens, with increase of the specific binding after pre-treatment with JKF. In contrast, the membranal binding of E(2)-Ov-Eu, which was specific for the estrogenic compounds except Ral, was inhibited by pre-treatment with JKF except for ICI 161480 (ICI). Male bone cells did not bind E(2)-Ov-Eu but bound T-BSA-Eu; this binding was abolished by pre-treatment with JKF. Pre-treatment with JKF increased mRNA for ERalpha and decreased mRNA for ERbeta in bone cells from both age groups of females and from males, all of which expressed both ERs, with a ratio of ERalpha to ERbeta of 121:1 in pre- and 78:1 in post-menopausal and 105:1 in male bone cells. This study raises the possibility of combined Vitamin D analog and phytoestrogen for hormonal replacement therapy to prevent post-menopausal osteoporosis, which is a subject of ongoing research in animal models.

One of the technologies for performing rapid analysis is based on commercially available automated multi-channel surface plasmon resonance (SPR) biosensors (Biacore) in which analysis can be performed within a few minutes. The application of these instruments in food control was evaluated for the detection of sulfonamides in chicken serum. Different antibodies (from specific to generic) were tested using biosensor chips with different coatings. The most robust assays were obtained using biosensor chips coated with low molecular weight ligands (sulfonamide-derivatives) and these chips could be used for hundreds of cycles. For the multi-sulfonamide detection in chicken serum three different assays were evaluated of which one, based on a mutant antibody, detected all tested sulfonamides (n=17) below the 100 ng/ml level.

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.

Three different group-specific anti-sulfonamide antibodies were compared in inhibition assay formats in an optical biosensor (BIACORE 3000) using CM5 sensor chips coated with three different sulfonamide derivatives. The antibodies used were an anti-sulfamethazine monoclonal antibody (Mab) 21C7, the sulfonamide binding protein (SBP) in the Qflex Kit Sulfonamides and a recently developed mutant antibody (M.3.4). Each of these antibodies showed interactions with all 17 sulfonamides tested and one (Mab 21C7) was sensitive for the N4-acetyl metabolites also. The limits of detection of the different sulfonamides in chicken serum varied between 7 and >1000 ng ml−1 (Mab 21C7), 15 and 340 ng ml−1 (Qflex) and 4 and 82 ng ml−1 (Mutant M.3.4). The mutant M.3.4 based assay was found to be the most sensitive towards most of the sulfonamides whereas the Qflex Kit Sulfonamides detected the five sulfonamides registered for application in poultry in The Netherlands within the narrowest measurement range.

In cultured human vascular smooth muscle cells (VSMC), estradiol-17β (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2–BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERα and ERβ. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERα mRNA (100–200%) but decreased ERβ mRNA (30–40%) expression as measured by real time PCR. ERα protein expression assessed by Western blot analysis increased (25–50%) in parallel, whereas ERβ protein expression declines (25–55%). Using ovalbumin bound to E2 (Ov–E2) linked to Eu (Eu–Ov–E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70–80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60–100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERα/ERβ as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.

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.