A nationwide survey of adrenal incidentalomas in Japan: the first report of clinical and epidemiological features.

The aim of this study was to reveal clear epidemiologic and clinical characteristics of incidentally discovered adrenal masses, termed adrenal incidentalomas (AIs), and to establish appropriate managemental and therapeutic regimens in Japan. This study had been originally carried out as a project of a research proposed on behalf of the Japanese Ministry of Health, Labour and Welfare, from 1999 to 2004. This nationwide multicenter study on AIs included 3,672 cases with clinically diagnosed AIs, involving 1,874 males and 1,738 females, with mean age 58.1 ± 13.0 years (mean ± SD). In the present study, we focused on the investigation of the real prevalence of various adrenal disorders with AI. The mean nodule size of AI based on computed tomography was 3.0 ± 2.0 cm. Compared to non-functioning adenomas (NFAs), tumor diameters were significantly larger in adrenocortical carcinomas (ACCs), pheochromocytomas, cortisol-producing adenomas (CPAs), myelolipomas, metastatic tumors, cysts, and ganglioneuromas (p < 0.01). Endocrinological evaluations demonstrated that 50.8% of total AIs were non-functioning adenomas, while 10.5%, including 3.6% with subclinical Cushing's syndrome, were reported as CPAs, 8.5% as pheochromocytomas, and 5.1% as aldosterone-producing adenomas. ACCs were accounted for 1.4% (50 cases) among our series of AIs. In conclusion, while almost 50 % of AIs are non-functional adenomas, we must be particularly careful as AIs include pheochromocytomas or adrenal carcinomas, because they may be asymptomatic. To our knowledge, this is the first and the largest investigation of AI, thus providing basic information for the establishment of clinical guidelines for the management of AI.

Low serum albumin, aspartate aminotransferase, and body mass are risk factors for frailty in elderly people with diabetes-a cross-sectional study.

BACKGROUND: Frailty is broadly characterized by vulnerability and decline in physical, mental and social activities and is more common in elderly patients with type 2 diabetes mellitus (T2DM). Frailty is closely associated with nutrition, muscle strength, inflammation, and hormones etc. In hormones, dehydroepiandrosterone sulfate (DHEA-S) and cortisol are suggested to be such candidates affecting frailty. Little investigation has been performed using a wider range of measures of frailty to clarify risk factors for frailty including the above two hormones. METHODS: We performed a cross-sectional study to investigate the risk factors for frailty in elderly T2DM patients (n = 148; ≥65 years), using a broad assessment, the clinical frailty scale. We compared parameters between the non-frail and frail groups using the unpaired t and Mann-Whitney U tests. The Jonckheere-Therpstra test was used to identify relationships with the severity of frailty, and risk factors were identified using binary regression analysis. RESULTS: Simple regression analysis identified a number of significant risk factors for frailty, including DHEAS < 70 µg/dL and cortisol/DHEA-S ratio ≥ 0.2. Multiple regression analysis showed that low albumin (< 4.0 g/dl) (odds ratio [OR] = 5.79, p < 0.001), low aspartate aminotransferase (AST) activity (< 25 IU/L) (OR = 4.34, p = 0.009), and low body mass (BM) (< 53 kg) (OR = 3.85, p = 0.012) were independent risk factors for frailty. A significant decrease in DHEA-S and a significant increase in the cortisol/DHEA-S ratio occurred alongside increases in the severity of frailty. DHEA-S concentration positively correlated with both serum albumin and BM. CONCLUSIONS: Hypoalbuminemia, low AST, and low BM are independent risk factors for frailty in elderly T2DM patients, strongly implying relative malnutrition in these frail patients. DHEA-S may be important for the maintenance of liver function and BM. A decrease in DHEA-S and an increase in the cortisol/DHEAS ratio may be involved in the mechanism of the effect of malnutrition in elderly T2DM patients.

Frailty in elderly diabetes patients.

Frailty is a state of vulnerability and a consequence of cumulative decline in multiple physiological systems over a lifespan. The occurrence of frailty depends on deterioration in muscle and nerve function, declining cardiopulmonary reserve and loss of executive function. Diabetes mellitus (DM) often causes functional impairment in each of the above systems, thus leading to a loss of whole body homeostasis and deterioration in physical function. Inability of self-management in DM patients may also have considerable impact on the development of sarcopenia/frailty. Thus, there may be positive feedback between the progression of diabetic complications and frailty/sarcopenia. While various factors are involved in this process, insulin resistance or insulin depletion may be an important factor in the progression of frailty in diabetes patients since insulin is well known to be an anabolic hormone in muscle. Interestingly, in our study targeting elderly DM patients, low HbA1c was a significant and independent risk factor for frailty, as assessed using a broad sense frailty scale, the Clinical Frailty Scale (CSF), suggesting that reverse metabolism due to malnutrition in elderly type 2 DM patients might be involved. Therefore, an intervention that includes proper nutrition and exercise training may be essential for the prevention of frailty. The pathogenesis of frailty in DM patients is extensively discussed in this review.

Dehydroepiandrosterone-enhanced dual specificity protein phosphatase (DDSP) prevents diet-induced and genetic obesity.

Dehydroepiandrosterone (DHEA) exerts a wide variety of therapeutic effects against medical disorders, such as diabetes and obesity. However, the molecular basis of DHEA action remains to be clarified. Previously, we reported that DHEA-enhanced dual specificity protein phosphatase, designated DDSP, is one of the target molecules of DHEA. To examine the role of DDSP in DHEA signaling, we generated mice that carry a DDSP transgene in which expression is driven by the CAG promoter (DDSP-Tg). DDSP-Tg mice weighed significantly less than wild-type (WT) control mice when a high fat diet was supplied (p < 0.01). No difference in food-intake or locomotor activity was found between DDSP-Tg and WT mice. Oxygen consumption of DDSP-Tg mice was higher than that of WT mice (p < 0.01), which suggested an increase in basal metabolism in DDSP-Tg mice. To further investigate the role of DDSP in genetic obese mice, DDSP-Tg mice with a db/db background were generated (DDSP-Tg db/db). We observed cancellation of obesity by the db/db mutation and development of a cachexic phenotype in DDSP-Tg db/db mice. In conclusion, our study shows that expression of DDSP leads to prevention of diet-induced and genetic (db/db) obesity. Anti-obese effects of DHEA might be mediated through DDSP, which might be a therapeutic target for intervention of obesity.

The pituitary function of androgen receptor constitutes a glucocorticoid production circuit.

Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland.

Nuclear compartmentalization of N-CoR and its interactions with steroid receptors.

The repression mechanisms by the nuclear receptor corepressor (N-CoR) of steroid hormone receptor (SHR)-mediated transactivation were examined. Yellow fluorescent protein (YFP)-N-CoR was distributed as intranuclear discrete dots, while coexpression of androgen receptor (AR), glucocorticoid receptor alpha, and estrogen receptor alpha ligand-dependently triggered redistribution of YFP-N-CoR. In fluorescence recovery after photobleaching analysis, mobility of the N-CoR was reduced by 5alpha-dihydrotestosterone (DHT)-bound AR. The middle region of N-CoR mostly contributed to the interaction with agonist-bound SHRs and the suppression of their transactivation function. N-CoR impaired the DHT-induced N-C interaction of AR, and the impaired interaction was dose-dependently recovered by coexpression of SRC-1 and CBP. N-CoR also impaired the intranuclear complete (distinct) focus formation of SHRs. Coexpression of SRC-1 or CBP released YFP-N-CoR or endogenous N-CoR from incomplete foci and simultaneously recovered complete foci of AR-green fluorescent protein. These results indicate that the relative ratio of coactivators and corepressors determines the conformational equilibrium between transcriptionally active and inactive SHRs in the presence of agonists. The intranuclear foci formed by agonist-bound SHRs were completely destroyed by actinomycin D and alpha-amanitin, indicating that the focus formation does not precede the transcriptional activation. The focus formation may reflect the accumulation of SHR/coactivator complexes released from the transcriptionally active sites and thus be a mirror of transcriptionally active complex formation.

Selective androgen receptor modulator, S42 has anabolic and anti-catabolic effects on cultured myotubes.

We previously identified a novel selective androgen receptor modulator, S42, that does not stimulate prostate growth but has a beneficial effect on lipid metabolism. S42 also increased muscle weight of the levator ani in orchiectomized Sprague-Dawley rats. These findings prompted us to investigate whether S42 has a direct effect on cultured C2C12 myotubes. S42 significantly lowered expression levels of the skeletal muscle ubiquitin ligase (muscle atrophy-related gene), atrogin1 and Muscle RING-Finger Protein 1(MuRF1) in C2C12 myotubes, as determined by real time PCR. Phosphorylation of p70 S6 kinase (p70S6K), an essential factor for promoting protein synthesis in skeletal muscle, was significantly increased by S42 to almost the same extent as by insulin, but this was significantly prevented by treatment with rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). However, phosphorylation of Akt, upstream regulator of mTORC1, was not changed by S42. S42 did not increase insulin-like growth factor 1 (Igf1) mRNA levels in C2C12 myotubes. These results suggest that S42 may have an anabolic effect through activation of mTORC1-p70S6K signaling, independent of IGF-1-Akt signaling and may exert an anti-catabolic effect through inhibition of the degradation pathway in cultured C2C12 myotubes.

A High Serum Cortisol/DHEA-S Ratio Is a Risk Factor for Sarcopenia in Elderly Diabetic Patients.

CONTEXT: Elderly patients with type 2 diabetes mellitus (T2DM) have a high prevalence of frailty and/or sarcopenia. Sarcopenia is thought to be related to discordant secretions of the adrenal hormones cortisol and dehydroepiandrosterone (DHEA), as well as the sulfate ester of DHEA (DHEA-S). The current study sought to evaluate the risk factors for sarcopenia in elderly patients with T2DM. DESIGN AND PATIENTS: We enrolled 108 consecutive elderly patients aged ≥65 years with T2DM (mean age, 76.2 ± 7.3 years; 43.5% males). Sarcopenia was assessed and diagnosed based on the Asian version of the diagnostic criteria regarding muscular strength, physical function, and muscle mass. We assessed various physical parameters, blood tests, and atherosclerosis markers and statistically determined the risk factors for sarcopenia. RESULTS: Multiple regression analysis showed that the independent risk factors for sarcopenia were a serum cortisol/DHEA-S ratio ≥0.2, diastolic blood pressure <70 mm Hg, Hb concentration <13 g/dL, and an ankle brachial index <1.0. The strongest risk factor for sarcopenia was a serum cortisol/DHEA-S ratio ≥0.2. An increase in the serum cortisol/DHEA-S ratio reflected higher cortisol values and lower DHEA-S values in patients with sarcopenia compared with those in nonsarcopenic patients. The concentrations of cortisol and DHEA-S, as well as the cortisol/DHEA-S ratio, changed in accordance with the severity of sarcopenia. CONCLUSIONS: A relative increase in cortisol may reflect the presence of stress and stimulate muscle catabolism, whereas a relative decrease in DHEA-S may cause a decrease in the anabolic action of DHEA on muscle; the combination of these factors may lead to sarcopenia.

Functional potentiation of leptin-signal transducer and activator of transcription 3 signaling by the androgen receptor.

Hypogonadism is associated with increased fat mass and dysregulation of metabolic homeostasis in men. Our previous study revealed that androgen receptor (AR)-null male mice (ARL-/Y) develop late-onset obesity and are leptin-resistant. The present study evaluated how hypothalamic AR contributes to central leptin-signal transducer and activator of transcription 3 (STAT3) signaling. We evaluated leptin action in wild-type and ARL-/Y mice, the anatomic co-relationship between AR and leptin signaling in the hypothalamus, and the effects of AR on leptin-mediated STAT3 transactivation and nuclear translocation. AR deletion in male mice results in a weaker leptin-induced suppression of food intake and body weight drop even before the onset of overt obesity. In wild-type male but not female mice, AR was highly expressed in various hypothalamic nuclei that also expressed the long-form leptin receptor (OBRB) and co-resided with OBRB directly in the arcuate neurons. In vitro, AR significantly enhanced STAT3-mediated transcription of leptin target genes including POMC and SOCS3. This effect relied on the AR N-terminal activation function-1 (AF-1) domain and was specific to AR in that none of the other sex steroid hormone receptors tested showed similar effects. AR enhanced the low concentrations of leptin-induced STAT3 nuclear translocation in vitro, and ARL-/Y mice receiving leptin had impaired STAT3 nuclear localization in the arcuate neurons. These findings indicate that AR in the hypothalamus functions as a regulator of central leptin-OBRB-STAT3 signaling and has a physiological role in energy homeostasis and metabolic regulation in male mice.

Adipose tissue-derived and bone marrow-derived mesenchymal cells develop into different lineage of steroidogenic cells by forced expression of steroidogenic factor 1.

Steroidogenic factor 1 (SF-1)/adrenal 4 binding protein is an essential nuclear receptor for steroidogenesis, as well as for adrenal and gonadal gland development. We have previously clarified that adenovirus-mediated forced expression of SF-1 can transform long-term cultured mouse bone marrow mesenchymal cells (BMCs) into ACTH-responsive steroidogenic cells. In the present study, we extended this work to adipose tissue-derived mesenchymal cells (AMCs) and compared its steroidogenic capacity with those of BMCs prepared from the identical mouse. Several cell surface markers, including potential mesenchymal cell markers, were identical in both cell types, and, as expected, forced expression of SF-1 caused AMCs to be transformed into ACTH-responsive steroidogenic cells. However, more elaborate studies revealed that the steroidogenic property of AMCs was rather different from that of BMCs, especially in steroidogenic lineage. In response to increased SF-1 expression and/or treatment with retinoic acid, AMCs were much more prone to produce adrenal steroid, corticosterone rather than gonadal steroid, testosterone, whereas the contrary was evident in BMCs. Such marked differences in steroidogenic profiles between AMCs and BMCs were also evident by the changes of steroidogenic enzymes. These novel results suggest a promising utility of AMCs for autologous cell regeneration therapy for patients with steroid insufficiency and also a necessity for appropriate tissue selection in preparing mesenchymal stem cells according to the aim. The different steroidogenic potency of AMCs or BMCs might provide a good model for the clarification of the mechanism of tissue- or cell-specific adrenal and gonadal steroidogenic cell differentiation.

Methylation of a conserved intronic CpG island of mouse SF-1 is associated with cell-specific expression of SF-1 in a culture system but not with tissue-specific expression.

The mechanism for the steroidogenic tissue or cell-specific expression of SF-1 has not been well clarified. We examined whether the methylation status of a large CpG island in the first intron of mouse SF-1 gene is associated with the expression level of SF-1 in cultured cells and in tissues. The island consists of three small islands (ICI-1, ICI-2, and ICI-3). In cultured adrenocortical Y-1 cells and in Leydig tumor cells, I-10, that both express high levels of SF-1, the upstream region of ICI-2, ICI-2-1, was clearly hypomethylated compared to cultured mouse bone marrow cells that do not express SF-1. However, this methylation status was not clearly associated with the tissue-specific expression of SF-1, in either adult or during development. These results suggest that methylation of ICI-2-1of SF-1 may partly determine the level of SF-1 expression at the cellular level, but may not be essential at the tissue level.

Frequent microsatellite instability in non-Hodgkin lymphomas irresponsive to chemotherapy.

Microsatellite instability (MSI) in haematopoietic malignancies has been controversial. Particularly in non-Hodgkin lymphoma, the data published to date lack unity. Using a unique fluorescent technique, we found MSI in eight (14%) tumours in a panel of 59 carefully selected non-Hodgkin lymphoma patients. Our fluorescent technique also reveals two qualitatively distinct modes of MSI, i.e. Type A and Type B. Based on our previous studies using DNA mismatch repair (MMR) gene-knock out animals, we have concluded that Type A MSI is a direct consequence of defective MMR. MSI observed in non-Hodgkin lymphomas was uniformly Type A, which implies that MMR deficiency occurs in this malignancy. Intriguingly, in non-Hodgkin lymphoma patients treated by CHOP/VEPA-based therapies, response to chemotherapy was significantly worse in those with microsatellite-unstable tumours (p=0.027). As a consequence, the patient outcomes at 1 year after treatment were significantly less favourable in this population (p=0.046), although the survival difference was not statistically confirmed in a longer term. These findings suggest that in some non-Hodgkin lymphomas MMR deficiency may lead to drug resistance in tumour cells and, consequently, to poor patient outcomes. In non-Hodgkin lymphoma, MSI may be a potential biomarker that predicts the tumour response against chemotherapy.

Androgens and metabolic syndrome: lessons from androgen receptor knock out (ARKO) mice.

Testosterone (T) is an important factor for determining body composition in males. Abdominal obesity is inversely correlated with serum T levels in men, leading to greater mortality. Pathologically hypogonadal men also have a significantly higher fat mass, which is reversed by T administration. However, the mechanism for such anti-obesity effect of androgen has not been well clarified. Androgen receptor (AR) null male mice revealed late-onset obesity. Male ARKO mice were euphagic compared to the wild-type male controls, but also less dynamic and less oxygen consuming. Transcript profiling indicated that male ARKO mice had lower transcripts for the thermogenetic uncoupling protein 1 (UCP1). We also found enhanced secretion of adiponectin, which is insulin-sensitizing, from adipose tissue in comparison to wild type, which might partly explain why the overall insulin sensitivity of male ARKO mice remained almost intact despite their apparent obesity. In addition, decreased lipolysis rather than increased lipid synthesis was observed, which might also account for the increased adiposity in male ARKO mice. The results revealed that AR plays important roles in male metabolism by affecting the energy balance, and is negative to both adiposity and insulin sensitivity.

Blocking of monocyte chemoattractant protein-1 (MCP-1) activity attenuates the severity of acute pancreatitis in rats.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) has been shown to affect the progression of various inflammatory disorders, including pancreatitis. To investigate the role of MCP-1 in acute pancreatitis and to seek possible therapeutic means, we evaluated the effect of a plasmid expression vector containing a dominant-negative mutant MCP-1 gene (mMCP-1). METHODS: Two rat models of acute pancreatitis were employed that used either cerulein (for mild pancreatitis) or a mixture of 5% taurocholic acid and trypsin (for severe pancreatitis). At 6 h after induction of acute pancreatitis with or without injection of mMCP-1, serum amylase levels and cytokine levels, as well as morphological evaluation of the pancreas, were determined. Survival rates were also evaluated. RESULTS: Severe pancreatitis was significantly reduced by mMCP-1 injection. mMCP-1 decreased serum levels of amylase, IL-6, IL-10, and LDH, and improved the survival rate 48 h after disease onset. Histopathological changes of pancreas and lungs were also improved by mMCP-1. CONCLUSIONS: MCP-1 appears to be involved in the progression of severe forms of acute pancreatitis. Our data suggested that MCP-1 is a candidate as a therapeutic target to treat acute pancreatitis.

[Steroid receptor superfamily and its mechanism of action].

Steroid receptors are evoluted from the common ancestor and have the same structure as steroid receptor superfamily. The nuclear receptors are characterized by a central DNA binding domain with specific DNA sequences known as hormone response element, ligand binding domain in the C terminal and N terminal domain with the tissue specificity. Nuclear receptor superfamily is divided into four classes based on the dimerization and DNA binding properties, homodimers as steroid hormone receptor, heterodimers with RXR, homodimers and monomers. Most of orphan receptors fall into the last 2 classes. The coregulators as coactivators and corepressor are determined. The mechanism of action of nuclear receptor on chromatin in the nucleus are clarified precisely from the stand point of the clasters of coregulators and the complex of nuclear receptor and coregulators. The crosstalk of nuclear receptor and peptide hormone signal in the cell is important in the autocrine and paracrine action. Non-genomic action of steroid hormone is also proposed.

Selective Androgen Receptor Modulator S42 Suppresses Prostate Cancer Cell Proliferation.

We previously identified the selective androgen receptor (AR) modulator S42, which does not stimulate prostate growth but has a beneficial effect on lipid metabolism. In the prostate cancer (PC) cell line LNCaP, S42 did not induce AR transactivation but antagonized 5α-dihydrotestosterone (DHT)‒induced AR activation. Next, we investigated whether S42 suppresses the growth of PC cell lines. Basal growth of LNCaP cells was significantly suppressed by treatment with S42 compared with vehicle, as determined by cell counting and 5-bromo-2'-deoxyuridine assays. The suppressive effect of S42 on cell growth was evident in the AR-positive PC cells LNCaP and 22Rv1 and was slightly observed even in the AR-negative PC-3 cells. However, S42 did not induce apoptosis as determined by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. S42 had an even greater suppressive effect on DHT-dependent LNCaP cell proliferation than on basal proliferation (P < 0.05). DHT treatment increased the expression of phosphorylated extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK), a major signaling molecule for PC proliferation, and this was significantly inhibited by S42. DHT also significantly upregulated AR, insulinlike growth factor-1 receptor (IGF-1R), and insulin receptor (IR)-ß protein levels, which were similarly reduced by S42 treatment. Importantly, S42 administration to mice attenuated the growth of LNCaP tumors and reduced tumor expression of the prostate-specific antigen, P504S, Ki67, and phosphorylated ERK-MAPK. These data suggest that S42 attenuates LNCaP tumor growth not by inducing apoptosis but by inhibiting the expression of proliferation-related receptors, including IGF-1R, IR, and AR, and by suppressing ERK-MAPK activation. S42 may thus be a feasible candidate for PC treatment.

Low glycated hemoglobin level is associated with severity of frailty in Japanese elderly diabetes patients.

AIMS/INTRODUCTION: Previously, a study using a narrowly defined (physical base) frailty scale reported that both good and bad (U-shaped curve) glycated hemoglobin (HbA1c) levels were frailty risk factors in patients with type 2 diabetes mellitus. However, no such studies in Japan have shown this. We aimed to evaluate the frailty risk factors including HbA1c in elderly Japanese patients with type 2 diabetes mellitus using a broadly defined (both physical and psychosocial base) frailty scale, the Clinical Frailty Scale (CFS). MATERIALS AND METHODS: We randomly enrolled 132 elderly patients with type 2 diabetes mellitus (aged ≥65 years) and categorized the patients into nine stages of frailty using CFS. Because no patient had CFS 9, patients with a CFS score of 1-4 and 5-8 were defined as non-frail and frail, respectively. We attempted to identify the risk factors of frailty by investigating the association between CFS stage and various patient factors. RESULTS: Multiple regression analysis showed that an increase in age, low levels of albumin, high-density lipoprotein cholesterol, systolic blood pressure, HbA1c, total cholesterol, and bodyweight were statistically significant and strong independent risk factors for frailty, suggesting that reverse metabolism owing to malnutrition in elderly type 2 diabetes mellitus patients might be involved. CONCLUSIONS: HbA1c level was not a U-shaped risk for frailty, suggesting that relatively good glycemic control might be more important for frailty than poor control in elderly type 2 diabetes mellitus patients.

Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans.

BACKGROUND: Atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP. METHODS: We compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1's role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1-dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII. RESULTS: Atrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this "orphan" receptor. CONCLUSION: The current findings are consistent with atrazine's endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans.

Long-term inhibition of RhoA attenuates vascular contractility by enhancing endothelial NO production in an intact rabbit mesenteric artery.

RhoA plays a critical role in regulating NO production in cultured endothelial cells. To determine its role in in situ endothelial cells, we investigated the effects of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors and a RhoA-binding domain of Rho-kinase (RB) on vascular contractility in the isolated rabbit mesenteric artery. Ex vivo treatment of the strips with 3x10(-5) mol/L simvastatin and fluvastatin for approximately 24 to 30 hours significantly attenuated the contractile response to phenylephrine and high K+ in the presence of endothelium. The addition of N(omega)-nitro-L-arginine methyl ester and the removal of endothelium abolished the attenuation of the contractile response. The cotreatment with geranylgeranyl pyrophosphate prevented the statin-induced attenuation of the contractile response, whereas geranylgeranyl transferase inhibitor mimicked the effect of simvastatin. Treatment with simvastatin enhanced the bradykinin-induced endothelium-dependent relaxation in the mesenteric artery, whereas it had no effect on the bradykinin-induced [Ca2+]i elevation in endothelial cells of the aortic valves. Introduction of RB to the strips using a cell-penetrating peptide of Tat protein (TATHA-RB) attenuated the contractile responses in a NO-dependent manner. However, a Rac1/Cdc42-binding fragment of p21-activated protein kinase, RB without Tat peptide or TATHA-protein A had no effect. The in vivo treatment of rabbit with simvastatin and TATHA-RB attenuated the contractility in a NO-dependent manner. Simvastatin and TATHA-RB significantly upregulated eNOS in the rabbit mesenteric artery. The present study provides the first evidence that RhoA plays a physiological role in suppressing NO production in in situ endothelial cells.