Effects of in utero linuron exposure on rat Wolffian duct development

Linuron is an herbicide that displays weak androgen receptor antagonist activity. Male offspring exposed in utero to 50 mg/kg/day linuron often exhibit malformations in Wolffian duct derivatives (i.e. the epididymis and vas deferens). The objectives of this study were to determine the point during the perinatal period that linuron-induced epididymal lesions can be identified, to characterize linuron-mediated perinatal testicular and epididymal pathology, and to determine whether male rat fetuses exposed prenatally to linuron exhibit decreased intratesticular and serum testosterone (T) levels. Pregnant rats were administered corn oil vehicle or linuron by gavage at 0 or 50 mg/kg/day (n = 3 controls, 5-11 linuron-treated dams per time point) from gestation days (GD) 12 to 21 or to termination. Male fetuses or offspring were necropsied on GD 17, 19, and 21, and postnatal days (PND) 7 and 14. Epididymal malformations were not observed in fetuses from linuron-treated dams but were seen in linuron-exposed male offspring on PND 7 and 14. No testicular lesions were observed at any time point. The growth and development of linuron-exposed fetuses were altered, as evidenced by slight decreases in fetal weight and increased levels of immunoreactive proliferating cell nuclear antigen (PCNA) on GD 21. Intratesticular and serum T levels were not decreased in linuron-exposed male fetuses. These findings indicate that the adversely altered adult phenotype following in utero exposure to linuron is very similar to that produced by the antiandrogens di-n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP). However, the absence of testicular lesions or alterations in fetal testosterone levels would suggest that the effect of linuron on the developing Wolffian ducts is distinctly different from DBP or DEHP.     

Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate.

Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.     

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental defects in the rat vagina.

At puberty, female rats exposed in utero to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit a persistent thread of mesenchymal tissue surrounded by keratinized epithelium that partially occludes the vaginal opening. Our objective was to determine the earliest time during fetal development that morphological signs of this vaginal canal malformation could be detected and to obtain greater insight into mechanisms involved in this effect. Pregnant rats were administered a single dose of vehicle (control) or TCDD (1.0 microg/kg, po) on gestation day (GD) 15 and were sacrificed on GD 18, 19, 20, and 21 for histological evaluation of female. Gestational exposure to TCDD affected vaginal morphogenesis as early as GD 19, 4 days after exposure of pregnant dams. In exposed fetuses, the thickness of mesenchymal tissue between the caudal Mullerian ducts was increased, which resulted in a failure of the Mullerian ducts to fuse, a process normally completed prior to parturition. In addition, TCDD exposure appeared to inhibit the regression of Wolffian ducts. Thus, TCDD interferes with vaginal development by impairing regression of the Wolffian ducts, by increasing the size of interductal mesenchyme, and by preventing fusion of the Mullerian ducts. Taken together, these effects appear to cause the persistent vaginal thread defect observed in rats following in utero and lactational TCDD exposure.     

Effects of in utero exposure to DI(n-Butyl) phthalate on development of male reproductive tracts in Sprague-Dawley rats

The purpose of this study was to determine the effects of di(n-butyl) phthalate (DBP) administration on male reproductive organ development in F1 Sprague-Dawley rats following in utero exposure. During gestation days (GD) 10-19, pregnant rats were administered daily, orally, DBP at 250, 500, or 700 mg/kg or flutamide (1, 12.5, or 25 mg/kg/d) as a positive control. The male offspring were sacrificed at 31 d of age. DBP and flutamide dose-dependently significantly increased the incidence of hypospadias and cryptorchidism in F1 male offspring. The weights of testes and accessory sex organs (epididymides, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowper's glands) were significantly reduced in DBP-treated animals. Furthermore, cauda agenesis of epididymides and ventral prostate atrophy were observed in high-dose 700-mg/kg DBP males. Anogenital distance (AGD) and levels of dihydrotestosterone (DHT) and testosterone were significantly decreased in the DBP (700 mg/kg/d)-treated groups. In particular, the expression of androgen receptor (AR) and 5α-reductase type 2 in the proximal penis was markedly depressed following administration of DBP (700 mg/kg/d) or flutamide (25 mg/kg/d). The expression of sonic hedgehog (Shh) in the urethral epithelium of the proximal penis was significantly less in the DBP (700 mg/kg/d)- or flutamide (25 mg/kg/d)-treated groups. In addition, DBP dose-dependently significantly increased the expression of estrogen receptor (ER α) in the undescended testis. Data demonstrated that in utero exposure to DBP produced several abnormal responses in male reproductive organs, and these effects may be due to disruption of the stage-specific expression of genes related to androgen-dependent organs development.     

Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate.

Di(n-butyl) phthalate (DBP) alters male reproductive development by decreasing testicular testosterone (T) production when fetuses are exposed on gestation days (GD) 12-21. Previous studies have shown altered gene expression for enzymes in the T biosynthetic pathway following exposure to DBP. The objectives of this study were to develop a more detailed understanding of the effect of DBP on steroidogenesis, using a robust study design with increased numbers of dams and fetuses, compared with previous studies, and to explore messenger RNA (mRNA) expression for other critical genes involved in androgen biosynthesis and signaling. Additionally, immunohistochemical localization of protein expression for several key genes was performed to further confirm mRNA changes. Fetal Leydig cell lipid levels were also examined histochemically, using oil red O. Six to seven pregnant Crl:CD(SD)BR rats per group were gavaged with corn oil or DBP at 500 mg/kg/day on GD 12-19. Testicular RNA isolated from three randomly selected GD 19 fetuses per litter was used for real-time RT-PCR for the following genes: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, 17beta-hydroxysteroid dehydrogenase (17beta-HSD), androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), proliferating cell nuclear antigen (PCNA), and testosterone-repressed prostate message-2 (TRPM-2). mRNA expression was downregulated for SRB1, StAR, P450scc, 3beta-HSD, P450c17, and c-kit following DBP exposure, and TRPM-2 was upregulated. 17beta-HSD, AR, LHR, FSHR, and PCNA were not significantly changed. Immunohistochemical staining for c-kit was seen in fetal Leydig cells, which has not been previously reported. Downregulation of most of the genes in the T biosynthetic pathway confirms and extends previous findings. Diminished Leydig cell lipid content and alteration of cholesterol transport genes also support altered cholesterol metabolism and transport as a potential mechanism for decreased T synthesis following exposure to DBP.     

Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate

Adult male rats previously exposed on gestation days (GD) 12-21 to di(n-butyl) phthalate (DBP) have reproductive tract malformations, particularly agenesis of the epididymis, decreased sperm production, and Leydig cell hyperplasia and adenomas. Although similar effects are produced by the potent androgen receptor (AR) antagonist flutamide and are indicative of disruption of male sexual differentiation via an antiandrogenic mechanism, DBP is not an AR antagonist. The purpose of the study was to determine whether DBP causes pathologic changes and alterations in androgen status in the testis during the prenatal period of male reproductive tract differentiation. Pregnant CD rats were given corn oil, DBP (500 mg/kg/day), or flutamide (100 mg/kg/day) p.o. on GD 12-21. At GD 16-21, DBP caused hyperplasia of Leydig cells, many of which were 3beta-hydroxysteroid dehydrogenase- and/or AR-positive. Focal areas of hyperplasia had increased numbers of Leydig cells positive for proliferating cell nuclear antigen (PCNA). At GD 21, testis atrophy was apparent, seminiferous cords in DBP-exposed fetuses were enlarged and contained multinucleated gonocytes that, unlike controls, were PCNA-positive. DBP, but not flutamide, markedly decreased testicular testosterone levels at GD 18 and 21. Fewer epididymal ducts and reduced AR staining in some ducts were evident with DBP treatment, whereas decreased overall AR staining was seen with flutamide in the presence of mild Leydig cell hyperplasia. Leydig cell proliferation is likely a compensatory mechanism to increase testicular steroidogenesis triggered by testosterone insufficiency. The overall decrease in androgen concentration is not corrected and results in reproductive tract malformations. The multinuclearity and proliferation of gonocytes suggests an underlying Sertoli cell dysfunction.     

Dose-dependent alterations in gene expression and testosterone synthesis in the fetal testes of male rats exposed to di (n-butyl) phthalate.

Exposure to di (n-butyl) phthalate (DBP) in utero impairs the development of the male rat reproductive tract. The adverse effects are due in part to a coordinated decrease in expression of genes involved in cholesterol transport and steroidogenesis with a resultant reduction in testosterone production in the fetal testis. To determine the dose-response relationship for the effect of DBP on steroidogenesis in fetal rat testes, pregnant Sprague-Dawley rats received corn oil (vehicle control) or DBP (0.1, 1.0, 10, 50, 100, or 500 mg/kg/day) by gavage daily from gestation day (GD) 12 to 19. Testes were isolated on GD 19, and changes in gene and protein expression were quantified by RT-PCR and Western analysis. Fetal testicular testosterone concentration was determined by radioimmunoassay. DBP exposure resulted in significant dose-dependent reductions in mRNA and protein concentration of scavenger receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450c17. Testicular testosterone was reduced at doses of 50 mg/kg/day and above. Whole-testis expression of peripheral benzodiazepine receptor (PBR) mRNA, which functions with StAR to transport cholesterol across the mitochondrial membrane, was upregulated following exposure to DBP at 500 mg/kg/day. By immunocytochemistry, however, PBR protein was reduced in interstitial cells and also expressed but not reduced in gonocytes. Our results demonstrate a coordinate, dose-dependent reduction in the expression of key genes and proteins involved in cholesterol transport and steroidogenesis and a corresponding reduction in testosterone in fetal testes following maternal exposure to DBP, at dose levels below which adverse effects are detected in the developing male reproductive tract. Alterations in gene and protein expression and testosterone synthesis may serve as sensitive indicators of testicular response to DBP.     

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits luminal cell differentiation and androgen responsiveness of the ventral prostate without inhibiting prostatic 5alpha-dihydrotestosterone formation or testicular androgen production in rat offspring.

In utero and lactational exposure to a single maternal dose of 1 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg causes some overt toxicity and impairs prostate growth in male offspring. As similar effects on the ventral prostate can be caused by decreased testosterone production during perinatal development, we determined whether intratesticular testosterone content, testicular responsiveness to gonadotropin stimulation, or plasma testosterone concentrations were reduced in fetal and newborn rats. Because these endpoints were not affected, the ability of TCDD exposure to inhibit synthesis of the proximal androgen in prostate development, 5alpha-dihydrotestosterone (DHT), from the circulating precursor testosterone and 5alpha-androstane-3alpha,17ss-diol (3alpha-Diol), was studied on postnatal days (PNDs) 14, 21, and 32. The ability of the ventral prostate to form DHT from 3alpha-Diol was slightly impaired on PND 14, but this transient effect was not statistically significant, and recovery was evident by PND 21. Subsequent experiments used organ culture to study the effects of in vivo TCDD exposure on androgen metabolism, androgen responsiveness, androgen receptor expression, and luminal epithelial cell differentiation after in vitro exposure to graded androgen concentrations. In utero and lactational TCDD exposure had no effect on DHT formation in organ culture, but transiently reduced the androgen -induced expression of prostatic-binding protein subunit C3, decreased ventral prostate epithelial cell androgen receptor expression, and inhibited the formation of androgen responsive luminal epithelial cells. These results suggest that TCDD exposure impairs prostate growth and androgen responsiveness by inhibiting prostatic epithelial cell differentiation.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in the C57BL/6J mouse prostate: lobe-specific effects on branching morphogenesis.

Branching morphogenesis is an essential component of prostate development. This study was conducted to test the hypothesis that in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure differentially inhibits branching morphogenesis and ductal canalization in the ventral, dorsal, lateral, and anterior mouse prostate. Pregnant C57BL/6J mice were given TCDD (5 microg/kg, orally) or vehicle on gestation day (GD) 13 and their pups examined at 1, 7, 14, 21, 35, and 90 days of age. Prostate lobes were microdissected after incubation in 0.5% collagenase and the numbers of ductal tips, main ducts, and ductal tips per main duct were determined by examining photographs of microdissected, whole-mount specimens. Ductal canalization was determined using histological sections of the dorsolateral and anterior prostate lobes. TCDD inhibited branching morphogenesis in all prostate lobes. The ventral prostate (VP) was extremely small throughout development and never developed any ductal structure. TCDD reduced the numbers of ductal tips and main ducts in the dorsal (DP) and lateral prostate (LP), but reductions in ductal tip numbers appeared to result entirely from reductions in the number of main ducts. Dorsolateral prostate (DLP) weights were slightly reduced by TCDD, but there was no effect on ductal canalization in the dorsal, lateral, or anterior lobes. TCDD had no effect on main duct number in the anterior prostate, but weight, ductal tip number, and the number of ductal tips per main duct was substantially reduced. These results demonstrate that the severe inhibition in ventral prostate development caused by in utero and lactational TCDD exposure is accompanied by a complete absence of branching morphogenesis. The impairment in dorsal, lateral, and anterior prostate (AP) development is associated with a lobe-specific inhibition of the various processes involved in duct formation.     

Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in fetal rat testis

Phthalates are widely used as plasticizers in various consumer products and building materials. Some of the phthalates are known to interfere with male reproductive development in rats, and di-n-butyl phthalate (DBP), diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) were recently banned for use in toys in the EU mainly due to their reproductive toxicity. Diisobutyl phthalate (DiBP) has similar structural and application properties as DBP, and is being used as a substitute for DBP. However, knowledge on male reproductive effects of DiBP in experimental animals is lacking. METHODS: In the current study, four groups of pregnant Wistar rats were exposed to either 0mg/kg bw/day or 600 mg/kg bw/day of DiBP from gestation day (GD) 7 to either GD 19 or GD 20/21. Male offspring was examined at GD 19 or GD 20/21 for effects on testicular testosterone production and testicular histopathology. Changes in anogenital distance (AGD) were evaluated as an indication of feminisation of males. RESULTS: Anogenital distance was statistically significantly reduced at GD 20/21 together with reductions in testicular testosterone production and testicular testosterone content. Histopathological effects (Leydig cell hyperplasia, Sertoli cell vacuolisation, central location of gonocytes and presence of multinuclear gonocytes) known for DBP and DEHP were observed in testes of DiBP-exposed animals at GD 20/21. Additionally, immunohistochemical expression of P450scc and StAR proteins in Leydig cells was reduced by DiBP. At GD 19, these effects on anogenital distance, testosterone levels and histopathology were less prominent. CONCLUSION: In this study, GD 20/21 rather than GD 19 appears to be the optimal time for investigating changes in anogenital distance, testosterone levels, and testicular histopathology. DiBP has similar testicular and developmental effects as DBP and DEHP, and although more developmental and especially postnatal studies are needed to clearly identify the reproductive effects of DiBP, this study indicates a reason for concern about the use of DiBP as a substitute for DBP.     

Prenatal Aroclor 1254 exposure and brain sexual differentiation: effect on the expression of testosterone metabolizing enzymes and androgen receptors in the hypothalamus of male and female rats

Polychlorinated biphenyls (PCBs) are industrial pollutants detected in human milk, serum and tissues. They readily cross the placenta to accumulate in fetal tissues, particularly the brain. These compounds affect normal brain sexual differentiation by mechanisms that are incompletely understood. The aim of this study was to verify whether a technical mixture of PCBs (Aroclor 1254) would interfere with the normal pattern of expression of hypothalamic aromatase and 5-alpha reductase(s), the two main enzymatic pathways involved in testosterone activation and of androgen receptor (AR). Aroclor 1254 was administered to pregnant rats at a daily dose of 25 mg/kg by gavage from days 15 to 19 of gestation (GD15–19). At GD20 the expression of aromatase, 5-alpha reductase types 1 and 2 and androgen receptor (AR) and aromatase activity were evaluated in the hypothalamus of male and female embryos. The direct effect of Aroclor was also evaluated on aromatase activity adding the PCB mixture to hypothalamic homogenates or to primary hypothalamic neuronal cultures. The data indicate that aromatase expression and activity is not altered by prenatal PCB treatment; 5-alpha reductase type 1 is similarly unaffected while 5-alpha reductase type 2 is markedly stimulated by the PCB exposure in females. Aroclor also decreases the expression of the AR in females. The observed in vivo effects are indicative of a possible adverse effect of PCBs on the important metabolic pathways by which testosterone produces its brain effects. In particular the changes of 5-alpha reductase type 2 and AR in females might be one of the mechanisms by which Aroclor exposure during fetal development affects adult sexual behavior in female rats.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disrupts early morphogenetic events that form the lower reproductive tract in female rat fetuses.

In female rats, in uteroexposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during critical periods of organogenesis causes a permanent thread of tissue, consisting of a core of mesenchyme surrounded by keratinized epithelia, across the vaginal opening. The objective of the current study was to determine the earliest time after exposure to TCDD during fetal development that morphological changes in the development of the lower reproductive tract could be detected. In addition, the spatio-temporal expression of several growth factors within the developing reproductive tract was investigated to provide insight into the mechanism of action involved in TCDD-induced vaginal thread formation. Pregnant rats received a single oral dose of 1.0 microg TCDD/kg on gestation day (GD) 15. Dams were sacrificed on GD 17, 18, 19, and 21 and individual reproductive tracts were isolated from female fetuses. As early as GD 18, TCDD produced distinct abnormalities in the female reproductive tract. The width of mesenchyme separating the Mullerian ducts was significantly greater in TCDD-exposed female GD 18 and 19 fetuses and the zone of unfused Mullerian ducts was substantially increased on GD 19 and 21. TCDD induced alterations within the developing reproductive tract in the subcellular and temporal expression of transforming growth factor-beta3 (TGF-beta3) and epidermal growth factor receptor (EGFR). DNA array analysis suggested effects on several genes expressed on GD 18 and 19.     

Evidence that inhibited prostatic epithelial bud formation in 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed C57BL/6J fetal mice is not due to interruption of androgen signaling in the urogenital sinus.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits the androgen-dependent processes by which the urogenital sinus (UGS) of fetal mice forms prostatic epithelial buds. This inhibition is mediated by aryl hydrocarbon receptors in UGS mesenchyme and causes prostate lobes to develop abnormally. Experiments were conducted to test the hypothesis that TCDD inhibits prostatic budding in C57BL/6J mice by inhibiting androgen signaling. In utero TCDD exposure sufficient to inhibit budding (5 microg/kg maternal dose on gestation day [GD] 13) had no effect on testicular testosterone content on GD 16 or 18. Nor did it inhibit the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) by the UGS. Both hydroxyflutamide (OH-flutamide; a competitive androgen receptor antagonist) and TCDD inhibited prostatic epithelial budding by UGSs cultured in vitro with DHT. To determine if TCDD inhibits responsiveness to androgens, primary mesenchymal cells prepared from UGSs cultured for three days with DHT were transiently transfected with an androgen-responsive reporter plasmid (MMTV-luciferase). OH-flutamide prevented DHT from increasing luciferase activity in these cells but TCDD did not. The same results were obtained when the mesenchymal cells were isolated from UGSs cultured with both DHT and TCDD. The lack of effect of TCDD on androgen-dependent gene expression was not due to inability of transfected UGS mesenchymal cells to respond to TCDD, as shown by significant increases in luciferase activity after transfection with plasmids containing CYP1A1 and CYP1B1 promoters. Finally, while OH-flutamide prevented DHT from altering androgen receptor and 5alpha-reductase type II mRNA expression in UGS organ culture, TCDD had no such effects. Collectively, these results suggest that TCDD inhibits prostatic epithelial bud formation without impairing the androgen receptor signaling pathway.     

Fetal mouse phthalate exposure shows that Gonocyte multinucleation is not associated with decreased testicular testosterone.

The rat has been explored in detail for its in utero susceptibility to male reproductive tract malformation following phthalate exposure. Few other species have been studied in detail, and it is important for both mechanistic and risk assessment purposes to understand the species specificity of this response. We investigated the response of the fetal mouse testis to phthalate exposure and compared these results with those previously obtained from the rat. Initial experiments using a variety of phthalate congeners (monobutyl phthalate, di-(n-butyl) phthalate, or mono (2-ethylhexyl) phthalate) and exposure paradigms did not reduce fetal mouse testis testosterone levels. Pharmacokinetic data after a single 500 mg/kg di-(n-butyl)-phthalate (DBP) exposure on mouse gestation day (gd) 18 demonstrated that the concentrations and kinetics of the active metabolite monobutyl phthalate (MBP) in fetal and maternal plasma were similar to the rat. After a single 500 mg/kg or multiple day 250 mg/kg fetal mouse DBP exposure, rapid and dynamic changes in testis gene expression were observed, including induction of immediate early genes. Unlike the rat, expression of genes involved in cholesterol homeostasis and steroidogenesis were not decreased and were increased in a few cases. Similar to the rat, however, a 250- or 500-mg DBP/kg/day mouse exposure from gd 16 through 18 significantly increased seminiferous cord diameter, the number of multinucleated gonocytes per cord, and the number of nuclei per multinucleated gonocyte. Together, these results demonstrate that fetal mouse and rat phthalate exposure both induce immediate early gene expression and disrupt seminiferous cord and gonocyte development. This response in the mouse occurs without a measurable decrease in testicular testosterone, suggesting that altered seminiferous cord formation and gonocyte multinucleation may not be mechanistically linked to lowered testosterone.     

Altered mammary gland development in male rats exposed to genistein and methoxychlor.

Genistein (GE) is a prevalent phytoestrogen whose presence in human and animal foods may affect biological actions of synthetic endocrine active compounds. We have previously reported that in utero and lactational exposure to high doses of GE or the endocrine active pesticide methoxychlor (MXC) caused mammary epithelial proliferation in 21-day-old male rats. Combined exposure to GE and MXC resulted in significant feminization of the male mammary glands. The goals of the current study were to evaluate mammary responses to GE and MXC at the adult stage and investigate relevant mechanisms. Following in utero, lactational exposure (through maternal diet), and direct dietary exposure, the inguinal mammary gland of male rats (90 days of age) was found to exhibit significant morphological alterations in the groups treated with GE and/or MXC compared to the control. GE exposure (at 300 and 800 ppm concentrations) caused lobular enlargement and epithelial proliferation, whereas MXC exposure (800 ppm) led to ductal elongation and lobular enlargement. Combining the two treatments caused prominent proliferation of both ducts and alveoli; secretory material was seen in readily recognizable alveolar lumens, which are absent in untreated male mammary. We also surveyed gene expression in the mammary tissue using a cDNA microarray and evaluated relevant protein factors. The results indicated that the treatment effects are likely due to interactions between steroid hormone receptor-mediated signals and growth factor-driven cellular pathways. The distinctive responses associated with the GE+MXC combination were likely linked to enhanced actions of insulin-like growth factor 1 and related downstream pathways.     

Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats.

Antiandrogenic chemicals alter sex differentiation by several different mechanisms. Some, like flutamide, procymidone, or vinclozolin compete with androgens for the androgen receptor (AR), inhibit AR-DNA binding, and alter androgen-dependent gene expression in vivo and in vitro. Finasteride and some phthalate esters demasculinize male rats by inhibiting fetal androgen synthesis. Linuron, which is a weak competitive inhibitor of AR binding (reported Ki of 100 microM), alters sexual differentiation in an antiandrogenic manner. However, the pattern of malformations more closely resembles that produced by the phthalate esters than by vinclozolin treatment. The present study was designed to determine if linuron acted as an AR antagonist in vitro and in vivo. In vitro, we (1) confirmed the affinity of linuron for the rat AR, and found (2) that linuron binds human AR (hAR), and (3) acts as an hAR antagonist. Linuron competed with an androgen for rat prostatic AR (EC(50) = 100-300 microM) and human AR (hAR) in a COS cell-binding assay (EC(50) = 20 microM). Linuron inhibited dihydrotestosterone (DHT)-hAR induced gene expression in CV-1 and MDA-MB-453-KB2 cells (EC(50) = 10 microM) at concentrations that were not cytotoxic. In short-term in vivo studies, linuron treatment reduced testosterone- and DHT-dependent tissue weights in the Hershberger assay (oral 100 mg/kg/d for 7 days, using castrate-immature-testosterone propionate-treated male rats; an assay used for decades to screen for AR agonists and antagonists) and altered the expression of androgen-regulated ventral prostate genes (oral 100 mg/kg/d for 4 days). Histological effects of in utero exposure to linuron (100 mg/kg/d, day 14-18) or DBP (500 mg/kg/d, day 14 to postnatal day 3) on the testes and epididymides also are shown here. Taken together, these results support the hypothesis that linuron is an AR antagonist both in vivo and in vitro, but it remains to be determined if linuron alters sexual differentiation by additional mechanisms of action.     

Prenatal exposure to di-n-butyl phthalate (DBP) differentially alters androgen cascade in undeformed versus hypospadiac male rat offspring

This study was to compare the alterations of androgen cascades in di-n-butyl phthalate (DBP)-exposed male offspring without hypospadias (undeformed) versus those with hypospadias. To induce hypospadias in male offspring, pregnant rats received DBP via oral gavage at a dose of 750 mg/kg BW/day during gestational days 14–18. The mRNA expression levels of genes downstream of the androgen signaling pathway, such as androgen receptor (AR) and Srd5a2, in testes of undeformed rat pups were similar to those in controls; in hypospadiac rat pups these levels were significantly lower than those of control pups. In contrast, both undeformed and hypospadiac rats had decreased serum testosterone levels, reduced mRNA expression of key enzymes in the androgen synthetic pathway in the testes, and ablated genes of developmental pathways, such as Shh, Bmp4, Fgf8, Fgf10 and Fgfr2, in the genital tubercle (GT) as compared to those in DBP-unexposed controls, albeit hypospadiac rats had a more severe decrement than those of undeformed rats. Although other possibilities cannot be excluded, our findings suggest that the relatively normal levels of testosterone-AR-Srd5a2 may contribute to the resistance to DBP toxicity in undeformed rats. In conclusion, our results showed a potential correlation between decreased testosterone levels, reduced mRNA expression of AR and Srd5a2 and the occurrence of hypospadias in male rat offspring prenatally exposed to DBP.     

In Uteroand Lactational Exposure of the Male Rat to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Impairs Prostate Development: 2. Effects on Growth and Cytodifferentiation

In the male Holtzman rat,in uteroand lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases prostate weight without inhibiting testicular androgen production or decreasing circulating androgen concentrations. Therefore, the present study sought to characterize effects of TCDD exposure on prostate development, from very early outgrowth from the urogenital sinus (Gestation Day [GD] 20) until rapid growth and differentiation are essentially complete (Postnatal Day [PND] 32). Pregnant Holtzman rats were administered a single dose of TCDD (1.0 μg/kg po) or vehicle on GD 15 and offspring were exposed via placental transfer (GD 20 euthanasia) or placental and subsequent lactational transfer until euthanasia (if before PND 21) or weaning. Results show that the prostatic epithelial budding process was impaired byin uteroTCDD exposure, as evidenced by significant decreases in the number of buds emerging from dorsal, lateral, and ventral aspects of the GD 20 urogenital sinus. Ventral prostate cell proliferation index was significantly decreased on PND 1 but was similar to or higher than control at later times, whereas apoptosis was an extremely rare event in ventral prostates from both control and TCDD-exposed animals. Delays were noted in the differentiation of pericordal smooth muscle cells and luminal epithelial cells. In addition, ventral prostates from approximately 40% of TCDD-exposed animals examined on PNDs 21 and 32 exhibited alterations in the histological arrangement of cell types that could not be explained by a developmental delay. Compared to controls, these ventral prostates exhibited a disorganized, hyperplastic epithelium containing fewer luminal epithelial cells and an increased density or continuous layer of basal epithelial cells, as well as thicker periductal smooth muscle sheaths. In addition, in ventral prostates from TCDD-exposed animals, the intensity of androgen receptor staining was relatively low in the central and distal epithelium, and the number of androgen receptor-positive cells was relatively high in the periductal stroma. These data suggest thatin uteroand lactational TCDD exposure interferes with prostate development by decreasing very early epithelial growth, delaying cytodifferentiation, and, in the most severely affected animals, producing alterations in epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression that may be of permanent consequence.