An alternative for extracorporeal photopheresis: 8-methoxypsoralen and UVA-treated leucocytes from allogeneic donors improve graft-versus-host disease in mice.

BACKGROUND AND OBJECTIVE: Extracorporeal photopheresis (ECP) is an important immune tolerance inducing therapy for graft-versus-host disease (GvHD). However, a sufficient number of ECP cycles cannot be performed in patients with severe GvHD and contraindications for apheresis. Allogeneic sources of leucocytes for use as ECP treatment would be of great benefit. Therefore, this study aimed to test the therapeutic potential of novel sources of leucocytes for ECP. MATERIALS AND METHODS: Graft-versus-host disease mice were treated with ECP using therapeutic cells from different allogeneic sources. Splenocytes were incubated with 8-methoxypsoralen (8-MOP), irradiated with UVA light and injected into GvHD mice as a model for ECP. RESULTS: The therapy with 8-MOP/UVA-treated cells from healthy mice of the bone marrow transplantation (BMT) donor strain reduced the GvHD symptoms, at least in a model of chronic GvHD. In the acute GvHD model, 8-MOP/UVA-treated cells from the BMT donor or recipient strain did not show significant improvements in GvHD symptoms or survival time. Pre-activation of cells by mixed lymphocyte reactions before 8-MOP/UVA treatment also failed to result in significant differences in survival time or GvHD score. In contrast, ECP with third-party 8-MOP/UVA-treated cells from a HLA-mismatched donor resulted in a mean survival time of 37 days compared to 21 days in the control group. CONCLUSION: In our analysis of novel allogeneic leucocyte sources for ECP, we could demonstrate that the source of the 8-MOP/UVA-treated cells is crucial. The underlying immunologic effect of allogeneic 8-MOP/UVA-treated cells needs to be investigated in future studies.

Synaptopodin is regulated by aromatase activity.

Locally synthesized estradiol plays an important role in synaptic plasticity in the hippocampus. We have previously shown that in hippocampal neurons, activity of the enzyme aromatase, which converts testosterone into estradiol, is reduced via Ca2+ -dependent phosphorylation. Synaptopodin is a highly estrogen responsive protein, and it has been shown that it is an important regulator of synaptic plasticity, mediated by its close association with internal calcium stores. In this study, we show that the expression of synaptopodin is stronger in the hippocampus of female animals than in that of male animals. Phosphorylation of aromatase, using letrozole, however, down-regulates synaptopodin immunohistochemistry in the hippocampus of both male and females. Similarly, in aromatase knock-out mice synaptopodin expression in the hippocampus is reduced sex independently. Using primary-dissociated hippocampal neurons, we found that evoked release of Ca2+ from internal stores down-regulates aromatase activity, which is paralleled by reduced expression of synaptopodin. Opposite effects were achieved after inhibition of the release. Calcium-dependent regulation of synaptopodin expression was abolished when the control of aromatase activity by the Ca2+ transients was disrupted. Our data suggest that the regulation of aromatase activity by Ca2+ transients in neurons contributes to synaptic plasticity in the hippocampus of male and female animals as an on-site regulatory mechanism.

Lack of anti-androgenic effects of equol on reproductive neuroendocrine function in the adult male rat.

Equol (EQ), a metabolite of the soy isoflavone daidzein, has well known estrogenic properties. Data from animal studies suggested that EQ may act also as an anti-androgen. However, data regarding how EQ may affect brain functions like the regulation of neuroendocrine activity and reproductive outcomes in adult male rats are still lacking. We therefore investigated the effects of EQ on sex-steroid regulated gene expression in the brain [medial preoptic area/anterior hypothalamus (MPOA/AH) and medial basal hypothalamus/median eminence (MBH/ME)], pituitary, and prostate as a reference androgen-dependent organ. Furthermore reproductive outcomes were evaluated. The anti-androgen flutamide (FLUT) served as reference compound. Male rats (n=12 per group) were treated by gavage for 5 days with either EQ (100 or 250 mg/kgBW/day), or FLUT 100 mg/kgBW/day. All vehicle- and EQ-treated males showed successful reproductive outcomes, whereas FLUT-exposed males had severe reproductive impairments resulted in infertility. FLUT decreased relative weights of prostate, seminal vesicles and epididymides, and increased serum levels of luteinizing hormone, follicle-stimulating hormone, testosterone and 5α-dihydrotestosterone without altering prolactin levels, whereas EQ exerted opposite effects. Both EQ and FLUT decreased gonadotropin releasing hormone (GnRH) expression in the MPOA/AH. Only FLUT upregulated levels of GnRH receptor expression both in the MBH/ME and pituitary. While EQ downregulated the hypothalamic ERα and ERß expressions, but FLUT did not. In the prostate, only FLUT upregulated both ERα and AR mRNA expression levels. Taken together, our findings are the first data that EQ did not induce anti-androgenic effects on brain, prostate and male reproductive parameters, however, estrogenic neuroendocrine and reproductive effects of EQ were observed.

Embryo implantation failure and other reproductive defects in Ube2q1-deficient female mice.

The ubiquitination process is indispensable for proteome regulation. Three classes of ubiquitin (Ub)-related proteins can be distinguished: E1, E2 and E3. Proteins from the E2 class are responsible for the transfer of Ubls from E1 to the target protein. For this activity, interaction with class E3 ligases is usually required. Ub-conjugating enzyme E2Q 1 (UBE2Q1) belongs to the E2 class of Ub-related enzymes and is demonstrated to be involved in the regulation of membrane B4GALT1 protein. Here, we demonstrate that human UBE2Q1 and mouse Ube2q1 are widely expressed and highly conserved genes. To elucidate the function of UBE2Q1 protein, we generated knockout mouse model. No overt phenotype was detected in UBE2Q1-deficient males, but in mutant females, pleiotropic reproductive defects were observed including altered oestrus cycle, abnormal sexual behaviour and reduced offspring care. Moreover, in the uterus of mutant females, significantly increased embryonic lethality and decreased implantation capacity of homozygous mutant embryos were noticed. We found that Ube2q1 is not expressed in the uterus of non-pregnant females but its expression is up-regulated during pregnancy. Taken together, Ube2q1 is involved in different aspects of female fertility.

Pre-infection physical exercise decreases mortality and stimulates neurogenesis in bacterial meningitis.

Physical exercise has been shown to increase neurogenesis, to decrease neuronal injury and to improve memory in animal models of stroke and head trauma. Therefore, we investigated the effect of voluntary wheel running on survival, neuronal damage and cell proliferation in a mouse model of pneumococcal meningitis. Mice were housed in cages equipped with voluntary running wheels or in standard cages before induction of bacterial meningitis by a subarachnoid injection of a Streptococcus pneumoniae type 3 strain. 24 hours later antibiotic treatment was initiated with ceftriaxone (100 mg/kg twice daily). Experiments were terminated either 30 hours or 4 days (short-term) or 7 weeks (long-term) after infection, and the survival time, inflammatory cytokines and corticosterone levels, neurogenesis in the dentate gyrus of the hippocampal formation and the cognitive function were evaluated in surviving mice. Survival time was significantly increased in running mice compared to control animals (p = 0.0087 in short-term and p = 0.016 in long-term experiments, log-rank test). At the end of the long-term experiment, mortality was lower in trained than in sedentary animals (p = 0.031, Fisher's Exact test). Hippocampal neurogenesis--assessed by the density of doublecortin-, TUC-4- and BrdU + NeuN-colabeled cells--was significantly increased in running mice in comparison to the sedentary group after meningitis. However, Morris water maze performance of both groups 6 weeks after bacterial meningitis did not reveal differences in learning ability. In conclusion, physical exercise prior to infection increased survival in a mouse model of bacterial meningitis and stimulated neurogenesis in the dentate gyrus of the hippocampal formation.

Long-term treatment of ovariectomized mice with estradiol or phytoestrogens as a new model to study the role of estrogenic substances in the heart.

Epidemiological data reveal that the overall risk for heart disease is lower for premenopausal women compared to age-matched men. However, the beneficial effect for the female sex is lost upon menopause. Thus, it has been suggested that estrogens convey the protective effect for the female sex against heart disease. Numerous natural plant products, i.e., phytoestrogens (PE), interfere with or alter the development or function of the endocrine system. Although PEs have been studied intensively with regard to the effects on the reproductive organs, such as the uterus or mammary gland, surprisingly little data are available about the effects of PEs on the heart. Here, we conducted a long-term study with ovariectomized mice to examine putative estrogenic effects of the PEs genistein (GEN), resveratrol (RES), and equol (EQ), using estradiol (E2) as a reference compound on heart size, morphology, and cardiac gene expression. We report for the first time significant changes in these parameters by GEN and E2. Changes in the size of cardiomyocytes were observed by GEN and E2. In line with these observations, cardiac expression of insulin-like growth factor 1 ( IGF1) was significantly induced by both GEN and E2. Thus, we speculate that endocrine active compounds, like the isoflavone GEN, which is used as a food additive or as a drug for the treatment of menopausal symptoms, may directly affect heart function.

Estrogen synthesis in the hippocampus.

Estradiol plays essential roles in the modulation of synaptic plasticity and neuroprotection in males as well as in females, as has been shown particularly in the hippocampus. Although it has long been known that aromatase, the final enzyme in estrogen synthesis, is expressed in the hippocampus, a new paradigm emerged when it was shown that estradiol is actually synthesized de novo in this part of the brain. Increasing evidence indicates that hippocampus-derived estradiol plays a role in synaptic plasticity and neuroptrotection, rather than estradiol originating from the gonads. In recent years, a number of in vivo and in vitro studies have shown that hippocampus-derived estradiol substantially contributes to hippocampal function, in particular to structural synaptic plasticity.

Plant-derived alternative treatments for the aging male: facts and myths.

Soy- or red clover- derived products containing isoflavones have been amply studied in climacteric and postmenopausal women, and confusing contradicting results have been published. The beneficial effects on climacteric complaints, cholesterol and the development of osteoporosis are marginally at best and there are no uterine and mammary safety studies. In males, however, isoflavones may protect the prostate to make them less prone to develop cancer. Cell biological and animal experimental data support this notion. Clinical data about possible beneficial effects on cholesterol or in the bone are largely missing. Hence, soy or red clover products containing the mild estrogenic isoflavones with a slightly higher affinity to the estrogen receptor of the beta in comparison to the alpha subtype may prove to have some beneficial effects in males.

Effect of dihydrotestosterone, 17-ß-estrogen, genistein and equol on remodeling and morphology of bone in osteoporotic male rats during bone healing.

INTRODUCTION: The aim of this study was to investigate the effect of dihydrotestosterone (DHT), 17-ß-estrogen (E2), genistein (GEN) and equol (EQ) on bone remodeling and bone morphology during healing of osteoporotic male rat tibiae. MATERIALS AND METHODS: 180 Sprague-Dawley male rats were divided in 5 groups of 36 animals. After orchidectomy (ORX) and development of osteoporosis, trepanation of the tibia was performed. Until the time of trepanation all groups received soya free food (SF), then food change occurred and treatment started. At day 95, 102 and 151, samples were taken and histomorphometry was performed to analyze changes in bone structure under treatment. At day 33 and 70 all animals received calcein respective alizarin for polychrome bone labeling. RESULTS: The cortical bone was particularly affected. Treatment with DHT and E2 led to a significant long-term expansion of the thickness of the diaphyseal cortical bone, while the phytoestrogens EQ and GEN only had a positive short-term effect in this area. Only E2 preserved the trabecular bone for a limited time. In all groups, periosteal and endosteal bone areas showed the highest bone formation activity. The osteoporotic male injured bone shows a shift in mineral apposition rate (MAR) from periosteal to endosteal bone in the SF, DHT and E2 groups but not in the GEN and EQ phytohormones groups. An MAR decrease in trabecular bone formation was observed at day 70 in all groups except the E2 group. CONCLUSION: We conclude from our results that healing of cortical bone defects in a rat model of male osteoporosis are mainly influenced by the estrogen pathway. Nevertheless, effects via purely androgenic mechanisms can also be demonstrated. The role of a phytohormone therapy is only marginal and if only useful for a short-term supportive approach. The role of the periosteal to endosteal shift during male osteoporotic bone healing needs to be further examined.

Cholesterol-promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus.

Cholesterol of glial origin promotes synaptogenesis (Mauch et al., (2001) Science 294:1354-1357). Because in the hippocampus local estradiol synthesis is essential for synaptogenesis, we addressed the question of whether cholesterol-promoted synapse formation results from the function of cholesterol as a precursor of estradiol synthesis in this brain area. To this end, we treated hippocampal cultures with cholesterol, estradiol, or with letrozole, a potent aromatase inhibitor. Cholesterol increased neuronal estradiol release into the medium, the number of spine synapses in hippocampal slice cultures, and immunoreactivity of synaptic proteins in dispersed cultures. Simultaneous application of cholesterol and letrozole or blockade of estrogen receptors by ICI 182 780 abolished cholesterol-induced synapse formation. As a further approach, we inhibited the access of cholesterol to the first enzyme of steroidogenesis by knock-down of steroidogenic acute regulatory protein, the rate-limiting step in steroidogenesis. A rescue of reduced synaptic protein expression in transfected cells was achieved by estradiol but not by cholesterol. Our data indicate that in the hippocampus cholesterol-promoted synapse formation requires the conversion of cholesterol to estradiol.

Estrus cyclicity of spinogenesis: underlying mechanisms.

Hippocampal spine density varies with the estrus cycle. The cyclic change in estradiol levels in serum was hypothesized to underlie this phenomenon, since treatment of ovariectomized animals with estradiol induced an increase in spine density in hippocampal dendrites of rats, as compared to ovariectomized controls. In contrast, application of estradiol to hippocampal slice cultures did not promote spinogenesis. In addressing this discrepancy, we found that hippocampal neurons themselves are capable of synthesizing estradiol de novo. Estradiol synthesis can be suppressed by aromatase inhibitors and by knock-down of Steroid Acute Regulatory Protein (StAR) and enhanced by substrates of steroidogenesis. Expression of estrogen receptors (ERs) and synaptic proteins, synaptogenesis, and long-term potentiation (LTP) correlated positively with aromatase activity in hippocampal cultures without any difference between genders. All effects due to inhibition of aromatase activity were rescued by application of estradiol to the cultures. Most importantly, gonadotropin-releasing hormone (GnRH) increased estradiol synthesis dose-dependently via an aromatase-mediated mechanism and consistently increased spine synapse density and spinophilin expression. As a consequence, our data suggest that cyclic fluctuations in spine synapse density result from pulsative release of GnRH from the hypothalamus and its effect on hippocampal estradiol synthesis, rather than from varying levels of serum estradiol. This hypothesis is further supported by higher GnRH receptor (GnRH-R) density in the hippocampus than in the cortex and hypothalamus and the specificity of estrus cyclicity of spinogenesis in the hippocampus, as compared to the cortex.

Leupaxin, a novel coactivator of the androgen receptor, is expressed in prostate cancer and plays a role in adhesion and invasion of prostate carcinoma cells.

In the present study, we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared with normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the PCa tissue sections analyzed, and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines, and at the subcellular level, leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different green fluorescent protein-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNA interference in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis, and a reduction of prostate-specific antigen secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 cells induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of PCa and may represent a novel coactivator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced PCa.

Effects of estradiol benzoate, raloxifen and an ethanolic extract of Cimicifuga racemosa in nonclassical estrogen regulated organs of ovariectomized rats.

The special extract of Cimicifuga racemosa (CR) BNO 1055 was shown to have bone protective effects without exerting estrogenic effects in the uterus or mammary gland. Whether the effects of CR BNO 1055 would be exerted in other organs that also express estrogen receptors (ERs) but in which the effects of estrogens and of the selective estrogen receptor modulator raloxifen (Ral) were not thoroughly studied was therefore investigated in the present contribution. Rats were ovariectomized (ovx) and their food immediately substituted with estradiol benzoate (EB), Ral or 2 doses of CR BNO 1055 for 3 months. Expressions of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and of insulin-like growth factor-1 (IGF-1) genes were determined in the vagina, liver, thyroid gland, lung, spleen, colon and kidney by means of quantitative RT-PCRs. Body weights in all treatment groups were significantly reduced and uterine weights in the EB treated animals were largely and in the Ral treated animals slightly but significantly increased. CR BNO 1055 was without effects in the uterus. We tested 3 genes: ERalpha gene expression was significantly reduced in the vagina, liver and kidney and remained unaffected in all other organs with the exception of the thyroid gland where ERalpha gene expression was stimulated by EB, Ral had--if any--similar effects in these organs. The CR extract BNO 1055 was devoid of any effect on ERalpha gene expression. ERbeta gene expression was suppressed in the vagina and colon by EB and this effect was shared by Ral in the colon. In the thyroid, EB and Ral stimulated ERbeta gene expression. Expression of IGF-1 gene was stimulated by EB and CR BNO 1055 in the vagina and kidney and inhibited by EB and Ral in the liver. No effects were observed by CR BNO 1055 in these organs. The effects of Ral, if occurring, were similar to those of EB while CR BNO 1055 was ineffective in all organs but the vagina. In the colon, reduced ERbeta gene activity may augment ERalpha mediated effects. In all other organs the effects of ER await further investigation. The CR BNO 1055 did not show any activity pattern which would be similar to the pattern observed under EB or Ral. Therefore the observed effects of CR BNO 1055 in these organs are most likely not estrogenic in nature.

Effects of a 5-day treatment with vinclozolin on the hypothalamo-pituitary-gonadal axis in male rats.

Vinclozolin (VZ), a potent antiandrogenic fungicide, is known to interfere with male reproductive function. Little data are currently available regarding possible impacts of VZ on brain function, particularly neuroendocrine activity and regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Therefore, we examined the effects of VZ on gene expression in the brain (MBH/ME, MPOA/AH, striatum, hippocampus), pituitary, prostate, seminal vesicles, and epididymis of 4-month-old male rats treated daily by gavage for 5 days with VZ (150 mg/kg body weight/day). Alterations in levels of serum hormones and gene expression were determined by RIA and qRT-PCR, respectively. Our results revealed that (i) VZ decreases epididymis weights, increases serum levels of LH and T, and decreases serum TSH and total T(4) levels; (ii) VZ affects the hypothalamic expression of both estrogen receptor (ERs) subtypes, ERalpha and ERbeta; (iii) in the extrahypothalamic brain areas, VZ alters expression of ERs and androgen receptor (AR); (iv) in the pituitary, VZ up-regulates expression of the GnRH receptor, LHbeta, alpha-subunit, and TERP-1/-2; (v) in the ventral prostate, VZ increases and decreases levels of AR and ERbeta mRNA, respectively; (vi) in the seminal vesicles, VZ increases levels of AR and ERalpha mRNA expressions; (vii) in the epididymis, VZ up-regulates AR and ERbeta mRNA expression. These results indicate that in vivo VZ is not a 'pure' antiandrogen, since it exerts mixed AR antagonistic/ERs agonistic actions observed at the levels of mRNA expression of selected androgen- and estrogen-regulated genes in the CNS, pituitary, and male accessory sex organs.

Pharmacokinetics and metabolism of benzophenone 2 in the rat.

Twelve derivatives of benzophenone (BP1-BP12) are widely used as UV-screens to protect industrial products from light induced damage. There is growing public concern about industrially produced chemicals that might interfere with hormonal signalling pathways, thus having potential adverse effects on human health. The derivative 2,2',4,4'-tetrahydroxybenzophenone (BP2) which is used in cosmetic products and in packaging materials, was previously shown to be an estrogenic endocrine active chemical (EAC). While the metabolisation of BP3 has been analyzed in vivo, according to our knowledge little is known about the pharmacokinetics of BP2. Therefore we performed a dose-response experiment with 5 dosages of BP2 which was applied per gavage to adult ovariectomised (ovx) rats for 5 days. Serum samples were analyzed via HPLC. Metabolites were further identified by Helix pomatia glucuronidase treatment and subsequent ion-trap-mass spectrometry. Additionally we analyzed the time dependent metabolisation and excretion of BP2 in a kinetic study. The parent compound BP2 is metabolised to glucuronide - and sulfate-conjugates. In the serum maximum levels of BP2, BP2-glucuronide and BP2-sulfate were observed already 30 min after BP2 application while highest concentrations of BP2 and its metabolites in urine were measured 120 min after treatment. It is suggested that this biotransformation occurs via a first-pass effect in the gut wall or the liver. Despite this rapid metabolisation and excretion, the amount of unconjugated BP2 was sufficient to induce a dose dependent estrogenic effect in the uterotrophic assay.

Phytoestrogens: endocrine disrupters or replacement for hormone replacement therapy?

OBJECTIVES: This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed. METHODS: Review of selected publications since 1980 and summary of unpublished own results of the authors. RESULTS: Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect. CONCLUSION: If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.

Dietary quercetin does not affect pituitary lutenizing hormone (LH) expression and has no uterotropic effects in ovariectomized Sprague-Dawley rats.

INTRODUCTION: The aim of this study was to investigate the potency of LH suppression and the uterotrophic effects of quercetin, a flavonoid widely present in our diet which in vitro has been shown to posses estrogenic properties. METHODS: Fifty-nine female Sprague-Dawley (SD) rats were ovariectomized (ovx) and fed with soy-free rodent chow with the addition of quercetin or estradiol-3 benzoate (E2B). Quercetin was added to the rodent chow at the dose of 200mg/kg (n=12) and 1000 mg/kg (n=11) which on average corresponded to 3.55 mg and 18.42 mg per animal per day, respectively, and E2B at the dose of 4.3mg/kg (n=12) or 17.3mg/kg (n=12) which corresponded to 0.07 mg and 0.20 mg per animal per day, respectively. The control group (n=12) received soy-free chow only. After three months of treatment, animals were sacrificed and using real time RT-PCR, pituitary LHbeta and uterine insulin like growth factor (IGF)-1, progesterone receptor (PR) and complement 3 protein (C3) mRNA levels were measured. Additionally, the in vitro binding capacity of quercetin with a porcine cytosolic ER preparation was evaluated. RESULTS: In contrast to E2B, dietary quercetin did not decrease pituitary LH expression, had no effects on uterine weight and uterine expression of estrogen regulated genes. The binding capacity of quercetin with the ERs was also 35000-fold lower compared with 17beta-estradiol (E2). CONCLUSION: Our study shows that quercetin does not show any estrogenic effects in the pituitary and the uterus of the ovx SD rats.

Testosterone metabolites inhibit proliferation of castration- and therapy-resistant prostate cancer.

Novel treatments for castration-resistant prostate cancer (CRPC) such as abiraterone acetate (AA) or enzalutamide effectively target the androgen pathway to arrest aberrant signalling and cell proliferation. Testosterone is able to inhibit tumour cell growth in CRPC. Estrogen receptor-beta (ERß) binds the testosterone-metabolites 3ß-androstanediol and 3α-androstanediol in parallel to the canonical estradiol. In the prostate it is widely accepted that ERß regulates estrogen signalling, mediating anti-proliferative effects. We used the prostate cancer cell lines LNCaP, PC-3, VCaP, and the non-neoplastic BPH-1. VCaP cells were treated with 1 nmol/L testosterone over 20 passages, yielding the cell line VCaPrev, sensitive to hormone therapies. In contrast, LNCaP cells were grown for more than 100 passages yielding a high passage therapy resistant cell line (hiPLNCaP). VCaP and hiPLNCaP cell lines were treated with 5 µmol/L AA for more than 20 passages, respectively, generating the AA-tolerant-subtypes VCaPAA and hiPLNCaPAA. Cell lines were treated with testosterone, dihydrotestosterone (DHT), R1881, and the androgen-metabolites 3ß-androstanediol and 3α-androstanediol. 3ß-androstanediol or 3α-androstanediol significantly reduced proliferation in all cell lines except the BPH-1 and androgen receptor-negative PC-3 and markedly downregulated AR and estrogen receptor alpha (ERα). Whereas ERß expression was increased in all cell lines except BPH-1 or PC-3. In summary, 3ß-adiol or 3α-adiol, as well as DHT and R1881, significantly reduced tumour cell growth in CRPC cells. Thus, these compounds represent novel potential therapeutic approaches to overcome drug-resistance in CRPC, especially with regard to AR-V7 function in therapy resistance. Furthermore, these data confirm the tumour suppressor properties of ERß in CRPC.

The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase.

Endocrine disrupting chemicals (EDCs), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone (TH) axis. Here, we examined whether selected EDCs inhibit the key reactions of TH biosynthesis catalyzed by thyroid peroxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human follicular thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates), and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half-maximal inhibition in the guaiacol assay occurred at 450 nmol/liter BP2, a concentration 20- and 200-fold lower than those required in case of the TPO-inhibiting antithyroid drugs methimazole and propylthiouracil, respectively. BP2 at 300 nmol/liter combined with the TPO substrate H(2)O(2) (10 mumol/liter) inactivated hrTPO; this was, however, prevented by micromolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T(4) was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDCs, most potently BP2, may disturb TH homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world.

Isoflavones--safe food additives or dangerous drugs?

The sales volume of products containing isoflavone has increased since the publication of the Women's Health Initiative. The many apparently contradictory results published on the effects of isoflavones on a variety of estrogen-regulated organs point to both beneficial as well as adverse effects on human health. It is of particular importance that psychovegetative climacteric complaints such as hot flushes are, if at all, only slightly influenced by isoflavones. The substances appear to have weak anti-osteoporotic effect. Their anti-atherosclerotic action is debatable, as not all authors find any beneficial effect on lipids. Most importantly, there is dispute as to whether isoflavones derived from soy or red clover have negative, positive or any effect at all on the mammary gland or endometrium. It is beyond any doubt that soy products may have cancer preventing properties in a variety of organs including the mammary gland. However, these properties may only be exerted if the developing organ was under the influence of isoflavones during childhood and puberty. This may also explain the often quoted "Japanese Phenomenon", the fact that breast cancer occurs to a lesser extent in Japanese women. When administered to isoflavone "inexperienced" women at the time of menopause, the phytoestrogens appear to share the same effects as estrogen used in classical preparations for hormone replacement therapy, i.e. they may stimulate the proliferation of endometrial and mammary gland tissue with at present unknown and unpredictable risk to these organs. Therefore, the following question arises for the clinician: Why should soy or red clover products containing isoflavone be recommended, if the positive effects are only negligible but the adverse effects serious?