Administration Of Diethylstilbestrol Research Articles

An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells

The utilization of chicken oviductal epithelial cells (OECs) as a bioreactor to produce therapeutic proteins has shown promise, but the time taken to obtain transgenic offspring impedes efficient validation of protein production. To overcome this barrier, we focused on the immortalization of chicken OECs (cOECs) using retroviral vector-mediated c-MYC oncogene expression to establish an in vitro pre-validation system for chicken bioreactors. The resulting immortalized cOECs exhibited sustained proliferation, maintained a normal diploid chicken karyotype, and expressed key oviduct-specific genes (OVA, OVM, LYZ, AVD, and ESR1). Notably, hormonal administration of diethylstilbestrol (DES) or progesterone (P4) upregulated oviduct-specific genes in these cells. To enhance the utility of these immortalized cOECs as an in vitro validation system for chicken bioreactors, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was employed to knock-in (KI) an enhanced green fluorescence protein (EGFP) gene at the ovalbumin (OVA) locus. The resulting OVA EGFP KI immortalized cOECs secreted both EGFP and OVA proteins into the culture medium, with secretion enhanced under DES treatment. This successful integration of an exogenous gene into cOECs enhances their potential as a versatile in vitro validation system for chicken bioreactors. The established immortalized cOECs overcome previous challenges associated with long-term culture and maintenance, providing a reliable platform for efficient protein production validation. This study presents a comprehensive characterization of the immortalized cOECs, addressing critical limitations associated with in vivo systems and laying a foundation for the development of a streamlined and effective chicken bioreactor model.

Neonatal administration of synthetic estrogen, diethylstilbestrol to mice up-regulates inflammatory Cxclchemokines located in the 5qE1 region in the vaginal epithelium.

A synthetic estrogen, diethylstilbestrol (DES), is known to cause adult vaginal carcinoma by neonatal administration of DES to mice. However, the carcinogenic process remains unclear. By Cap Analysis of Gene Expression method, we found that neonatal DES exposure up-regulated inflammatory Cxcl chemokines 2, 3, 5, and 7 located in the 5qE1 region in the vaginal epithelium of mice 70 days after birth. When we examined the gene expressions of these genes much earlier stages, we found that neonatal DES exposure increased these Cxcl chemokine genes expression even after 17 days after birth. It implies the DES-mediated persistent activation of inflammatory genes. Intriguingly, we also detected DES-induced non-coding RNAs from a region approximately 100 kb far from the Cxcl5 gene. The non-coding RNA up-regulation by DES exposure was confirmed on the 17-day vagina and continued throughout life, which may responsible for the activation of Cxcl chemokines located in the same region, 5qE1. This study shows that neonatal administration of DES to mice causes long-lasting up-regulation of inflammatory Cxcl chemokines in the vaginal epithelium. DES-mediated inflammation may be associated with the carcinogenic process.

Antifibrotic Potential of Tetracarpidium conophorum (African Walnut) Leaves Extract on Diethylstilbestrol-Induced Rat Model of Uterine Fibroid

Background: The increased prevalence of uterine fibroid (UF) and its life-threatening impact among women of reproductive age led to the development of this study. The study investigated the antifibrotic potential of Tetracarpidium conophorum aqueous extract on UF-induced rats. Materials and Methods: Sixty-four female Wistar rats, with an average weight of 200 g, were used for the study. The rats were randomly divided into eight groups of eight animals each. UF was induced by oral administration of diethylstilbestrol (DES) and intramuscular injection of progesterone at dosages 1.35 and 1.0 mg/kg body weight, respectively. Group 1 was administered normal saline orally for 8 weeks. Groups 2 and 3 were treated with progesterone and a combination of DES and progesterone, respectively. Groups 4 and 5 were pretreated with 200 and 400 mg/kg T. conophorum extract, respectively, for 3 weeks before the administration of DES and progesterone for 5 weeks. Groups 6 and 7 were administered DES and progesterone for 5 weeks before being treated with 200 and 400 mg/kg T. conophorum extract, respectively, for 3 weeks. Group 8 was the self-recovery group-administered DES and progesterone for 5 weeks after which they were given normal saline orally for 3 weeks. Results: After the treatment period, the rats were euthanized, and blood was collected, while the uteruses were harvested. Co-administration of DES and progesterone produces UF conditions. However, pre- and post-treatment with 200 mg/kg of extract mitigated the effects that were induced by DES and progesterone, but no remarkable preventive and curative effects were observed with the higher dosage (400 mg/kg). There were a reduction of the serum prolactin level in the treatment groups and an increased serum progesterone level in the posttreatment group. Conclusion: The study has shown that T. conophorum has both preventive and curative effects on UF at low dosage (200 mg/kg).

Diethylstilbestrol inhibits human and rat 11β-hydroxysteroid dehydrogenase 2.

Glucocorticoid hormone might cause intrauterine growth restriction. The glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) in the placenta eliminates excess levels of glucocorticoids during pregnancy. The aim of the current study was to define the effects of diethylstilbestrol (DES) on HSD11B2 activity in the mammalian placentas and identify its mode of action. Rat and human placental microsomal HSD11B2 were incubated with different concentrations of DES, and IC50 values were determined. The mode of action was analyzed by incubation of DES together with substrates, glucocorticoid and NAD+. DES suppressed rat and human HSD11B2 with IC50 values of 5.33 and 12.62 μM, respectively. DES was a competitive inhibitor of rat and human HSD11B2 when steroid substrates were added, while it was an uncompetitive inhibitor when cofactor NAD+ was exposed. Oral administration of DES (0.5 mg/kg) to the rat delayed the cortisol metabolism in adult female Sprague–Dawley rats, as indicated by the increases in cortisol’s elimination half-life, maximum concentration and area under the curve. In conclusion, DES is a potent HSD11B2 inhibitor, possibly contributing to the intrauterine growth restriction.

Transcriptional profiling of uterine leiomyoma rats treated by a traditional herb pair, Curcumae rhizoma and Sparganii rhizoma.

The aim of this study was to elucidate the concise effects of a traditional herb pair, Curcumae rhizoma-Sparganii rhizoma (CRSR), on uterine leiomyoma (UL) by analyzing transcriptional profiling. The UL rat model was made by intramuscular injection of progesterone and gavage administration of diethylstilbestrol. From 11 weeks of the establishment of the model, rats of the UL+CRSR group were gavaged daily with CRSR (6.67 g/kg). The serum concentrations of progesterone (P) and estradiol (E2) were determined by radioimmunoassay, the uterine index was measured by caliper measurement, and the pathological status was observed by hematoxylin and eosin stain. Gene expression profiling was checked by NimbleGen Rat Gene Expression Microarrays. The results indicated that the uterine mass of UL+CRSR rats was significantly shrunk and serum P and E2 levels significantly reduced compared to UL animals and nearly to the level of normal rats. Results of microarrays displayed the extensive inhibition of CRSR upon the expression of proliferation and deposition of extracellular matrix (ECM)-related genes, and significantly regulated a wide range of metabolism disorders. Furthermore, CRSR extensively regulated key pathways of the UL process, such as MAPK, PPAR, Notch, and TGF-β/Smad. Regulation of the crucial pathways for the UL process and ECM metabolism may be the underlying mechanisms of CRSR treatment. Further studies will provide clear clues for effectively treating UL with CRSR.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

228 - Oral Administration of Reduced Coenzyme Q10 Ameliorates the Endocrine-Disrupting Chemical-Induced Sperm Toxicity in Rats

Aim Food additives and agricultural chemicals consumed in daily life can be endocrine-disrupting chemicals (EDCs) for humans. EDCs have been reported to influence our genital function, learning ability and recognition. We have reported that the production of reactive oxygen species (ROS) precedes the decrease in sperm motility and/or numbers (FRR 2010), suggesting oxidative stress plays a key role. Therefore, we have studied the effects of a strong antioxidant, coenzyme Q 10 (CoQ10) on the rat sperm toxicity induced by diethylstilbestrol (DES), one of EDCs. Methods Drinking water containing DES (0.1 mg/L) ± CoQ10 (300 or 600 mg/kg/day; Kaneka Co.) were given to Wistar rats (13 weeks old) for 8 weeks. The numbers and the motility of sperm, and lipid peroxidation products in the testicle were measured. Result Reduced CoQ10 was absorbed and transferred to rat blood and liver, but only a little to the testicle. Administration of DES decreased the number of sperm in the cauda epididymidis, and motility functions (curve line speed, and the head amplitude). Supplementation of reduced CoQ10 did not rescue the decline of sperm numbers. However, both the curved line speed and the head amplitude of the sperm which are important functions of fertilization were improved significantly by reduced CoQ10. HNE (4-hydroxy-2-nonenal) modified proteins (28 and 53 kDa) in the testicle were increased significantly by DES administration. Reduced CoQ10 significantly decreased HNE-modified proteins. Conclusion Although the reduced CoQ10 supplementation to rats did not rescue the decline of total sperm numbers induced by DES administration, it significantly reduced oxidative stress in the testis and improved sperm function. These results suggested that the systemic absorption of reduced CoQ10 might inhibit oxidative stress of the testicle and dysfunction of the sperm.

Diethylstilbestrol exposure in neonatal mice induces changes in the adulthood in the immune response to taenia crassiceps without modifications of parasite loads.

Industrial growth has increased the exposition to endocrine disruptor compounds (EDC's), which are exogenous agents with agonist or antagonist action of endogenous steroid hormones that may affect the course of parasite infections. We wanted to determine if the exposure to diethylstilbestrol (DES), an estrogen agonist, to both male and female mice affected the immune response and their susceptibility to T. crassiceps cysticercosis. In all infected groups, females showed higher parasite loads than males, and neonatal DES administration did not modify this pattern. In the spleen, noninfected mice showed sex-related differences in the percentage of the CD8+ subpopulation, but DES decreased the percentage of CD3+, CD19+, and CD8+ subpopulations in infected mice. In the mesenteric lymphatic node (MNL), DES showed a dimorphic effect in the percentage of CD19+ cells. Regarding estrogen receptor alpha (ER-α) expression, DES treatment induced a reduction in the expression of this receptor in both noninfected female and male mice in the spleen, which was decreased only in males in CD3+ and CD8+ lymphocytes in MNL cell subpopulations. Our study is the first one to demonstrate that DES neonatal treatment in male and female mice affects the immune cell percentage, without effect on the susceptibility to T. crassiceps cysticercosis.

The effects of diethylstilbestrol administration on rat kidney. Ultrastructural study.

To assess the histological and ultrastructural changes that can be induced by diethylstilbestrol (DES) on renal tissues using histological, immunohistochemical, and ultrastructural methods. Thirty adult male Wistar rats were divided into 3 groups (10 rats each): Group 1 - control; Group 2 - received DES at a dose of 60 ug/kg/day, dissolved in 0.1 ml corn oil for 20 days; and Group 3 - received the same dose of DES for 50 days by oral gavage. The renal tissues were studied histologically, immunohistochemically (using an anti-BCL2-associated X protein [BAX protein] antibody), and ultrastructurally. This study was carried out at the Anatomy Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia between December 2011 and December 2012. The DES administration for 50 days caused noticeable degeneration, and alteration of the morphology of the renal tissues in the form of damaged renal tubules with loss of the brush border of the proximal convoluted tubules and increased cellularity of the glomeruli. In addition, there was a significant increase in BAX protein expression based on immunoreactivity, and in renal tubules, as well as glomerular cells. These changes were less obvious after 20 days of treatment. Non-steroidal, synthetic estrogens showed harmful effects on the renal tissues and altered their morphology with an increased number of apoptotic cells, and these changes were duration dependent.

Inhibition of XO or NOX attenuates diethylstilbestrol-induced endothelial nitric oxide deficiency without affecting its effects on LNCaP cell invasion and apoptosis

Oestrogen protects cardiovascular health partially via an up-regulation of NO• (NO radical) production. Its synthetic analogue DES (diethylstilbestrol), used as a potent androgen deprivation therapy for patients with prostate cancer, is however associated with high incidence of thromboembolic events. Exposure of BAECs (bovine aortic endothelial cells) to pharmacologically relevant dosage (12.5 μmol/l, 24 h) of DES resulted in a marked reduction in endothelial NO• bioavailability determined by ESR (electron spin resonance), while 17β-oestradiol instead increased NO• production as expected. Intriguingly, endothelial O(2)•- (superoxide anion) production was up-regulated by DES in vitro and in vivo, which was, however, attenuated by the ER (oestrogen receptor) antagonist ICI 182780, the XO (xanthine oxidase) inhibitor oxypurinol or the NOX (NADPH oxidase) inhibitor NSC23766. These agents also restored NO• production. DES alone in a cell-free system did not produce any ESR-sound O(2)•- signal. Of note, eNOS (endothelial NO synthase) mRNA and protein remained unchanged in response to DES. These results suggest that receptor-dependent activation of XO or NOX, and subsequent production of O(2)•-, mediate DES-induced NO• deficiency. This could represent a previously unrecognized mechanism that is responsible for cardiovascular complications of DES administration. Importantly, DES-induced suppression of LNCaP cell invasion and apoptosis were not affected by XO or NOX inhibitor. Therefore combinatorial therapy of DES and XO/NOX inhibitor may prove to be an innovative and useful therapeutic option in eliminating cardiovascular complications of DES, while preserving its anti-cancer effects, benefiting patients with advanced cancer who do not respond well to any other treatments but DES.

Generation of reactive oxygen species in sperms of rats as an earlier marker for evaluating the toxicity of endocrine-disrupting chemicals

Bisphenol A (BPA) and diethylstilbestrol (DES) have been reported to cause sperm toxicity. To identify an earlier marker of toxicity of environmental substances or food additives, this study determined whether the levels of reactive oxygen species (ROS) in sperms could serve as indices for the prediction of sperm toxicity and quality. Male Wistar rats were given drinking water containing various doses of BPA or DES for 8 weeks. Some rats were treated with 0.45% N-acetyl cysteine (NAC) for 2 days prior to the administration of DES or BPA. Administration of BPA or DES to rats for 1 week dose-dependently increased the production of ROS, even at doses and time points which had no effect on sperm motility. 4-Hydroxy-2-nonenal modified proteins increased in sperms 8 weeks after BPA or DES treatment. NAC reversed oxidative stress and prevented the loss of sperm function in the DES or BPA-treated group. During observation, changes in the sperm motility, sperm count and morphology were not correlated to the increase in ROS levels. These results suggest that ROS levels may be used as an early indicator of sperm count and quality decreases which result from chronic toxicity.

Histological changes in the genital tract of the female calf after administration of diethylstilbestrol and hexestrol.

Summary The changes in the genital tract of the female calf after the administration of two estrogens, diethylstilbestrol and hexestrol, at the age of 8 weeks were investigated. At slaughter at the age of 16 weeks alterations were found in the tuba, the cervix, the vagina, the glands of Bartholin and the mammary glands. The changes are similar to those found during estrus. All animals which had received DES showed changes in all organs at slaughter with the exception of the tuba, where only 40 out of 50 animals displayed alterations. In animals which had received HEX only the glands of Bartholin showed alterations in all animals while changes in the other organs could be found in only some of the animals. In the genital tract of 225 female calves slaughtered at various slaughterhouses in the Netherlands, no alterations could be observed in the cervix and the glands of Bartholin, indicating that these animals had not been treated with estrogens. The histological examination of the glands of Bartholin is proposed as a method to determine the administration of estrogens to female fattening calves. Zusammenfassung Histologische Veränderungen am Genitaltrakt des weiblichen Kalbes nach der Applikation von Diäthylstilböstrol und Hexöstrol Es wurden die Veränderungen am Genitaltrakt weiblicher Kälber nach der Verabreichung von zwei Oestrogenen (Diäthylstilböstrol, Hexöstrol) im Alter von 8 Wochen untersucht. Bei der Schlachtung im Alter von 16 Wochen wurden Veränderungen am Eileiter, an der Zervix, an der Vagina, an den Bartholinischen Drüsen und an der Milchdrüse gefunden. Die Veränderungen sind denen während der Brunst ähnlich. Alle Tiere, welche Diäthylstilböstrol erhielten, zeigten mit Ausnahme des Eileiters an allen Organen Veränderungen. Am Eileiter konnten nur in 40 von den 50 Tieren Alterationen festgestellt werden. Bei den Tieren, welche Hexöstrol erhielten, wiesen nur die Bartholinischen Drüsen bei allen Tieren Veränderungen auf. Nur einige Tiere zeigten auch an den andern Organen Veränderungen. Am Genitaltrakt von 225 weiblichen Kälbern, welche an verschiedenen Orten der Niederlande geschlachtet wurden, konnten Alterationen weder an der Zervix noch an den Bartholinischen Drüsen festgestellt werden, was dafür spricht, daß die Tiere nicht mit Oestrogenen behandelt wurden. Die histologische Untersuchung der Bartholinischen Drüsen kann als Test für eine Oestrogenisierung weiblicher Kälber zum Zwecke der Mastförderung angewendet werden. Résumé Modifications histologiques du tractus génital chez le veau femelle, après administration de diéthylstilboestrol et d'hexoestrol On examine les modifications du tractus génital de veaux femelles après administration de deux oestrogènes (diéthylstilboestrol, hexoestrol) à l'âge de 8 semaines. Après abattage à l'âge de 16 semaines, on trouve des modifications des oviductes, du cervix, du vagin, des glandes vulvo-vaginales et des glandes mammaires. Les modifications ressemblent à celles que l'on observe pendant le rut. Tous les animaux ayant reçu du diéthylstilboestrol présentent des modifications dans tous les organes, à l'exception des oviductes. On ne trouve des modifications des oviductes que chez 40 sur 50 animaux. Chez les veaux ayant reçu de l'hexoestrol, seules les glandes vulvo-vaginales présentent des modifications constantes. Quelques animaux seulement ont des modifications dans les autres organes. En examinant le tractus génital de 225 veaux femelles abattus dans différents endroits des Pays-Bas, on n'a trouvé des modifications ni dans le cervix, ni dans les glandes vulvo-vaginales, ce qui signifie que les animaux n'ont pas été traités aux oestrogènes. L'examen histologique des glandes vulvo-vaginales peut donc servir de test pour mettre en évidence l'oestrogénisation des veaux femelles à des fins d'engraissement. Resumen Alteraciones histológicas en el tracto genital de terneras tras la aplicación de dietilestilbestrol y hexestrol Se estudiaron las alteraciones producidas en el tracto genital de terneras tras la administración de dos estrógenos (dietilestilbestrol, hexestrol) a la edad de 8 semanas. Tras el sacrificio a las 16 semanas de edad, se hallaron transformaciones en la trompa de Falopio, cuello del útero, vagina, glándulas de Bartholin y en la glándula mamaria. Las alteraciones son semejantes a las apreciadas durante el celo. Todos los animales que recibieron dietilestilbestrol presentaban alteraciones en todos los órganos, excepción hecha por la trompa de Falopio. En esta última, solo en 40 de 50 animales se pudieron reconocer modificaciones. En los animales que recibieron hexestrol, solo las glándulas de Bartholin presentaron alteraciones en todos los animales. Pocas terneras nada más también mostraban alteraciones en otros órganos. En el tracto genital de 225 terneras, que fueron sacrificadas en los más diversos puntos de los Países Bajos, no se pudieron apreciar modificaciones ni en el cuello del útero ni en las glándulas de Bartholin, lo cual aboga a favor de que estos animales no fueron sometidos a estrógenos. El examen histológico de las glándulas de Bartholin se puede utilizar como prueba elucidativa de la estrogenizacion de terneras con fines de fomentar el engorde.

Histological changes in the uterus of the hens after embryonic exposure to bisphenol A and diethylstilbestrol

Many employed chemicals in industries have estrogenic hormone effects on organisms, and these are called as environmental estrogens. Environmental estrogens have adverse effects on development and function of reproductive organs of the birds. Bisphenol A (BPA) is one of the best known environmental estrogens widely found in plastic products. In this study, we injected BPA and the synthetic estrogen diethylstilbestrol (DES) in ovo and then examined and compared the effects of those on the uteri (shell gland) of the adult hens by histological methods. Five groups have been designed in the current study. Only vehicle substance was given in ovo to the control group and BPA (67 or 134 μg/g egg) and DES (0.02 or 0.2 μg/g egg) were administered in the experimental groups. Tissue specimens were taken from uteri of hens at 21 weeks of age, prior to the laying period. Estrogen receptor alpha (ERα) was immunohistochemically stained. It was observed that the hatching proportion in BPA (67 μg and 134 μg/g) was lesser than the other groups (P<0.01). Uterine tubular glandular density and thickness of tunica mucosa were found to have reduced (P<0.01) in BPA (134 μg/g) and DES (0.2 μg/g) groups, in comparison with those of the control and the other experimental groups. Uterine gland epithelium revealed positive immunoreaction for ERα. These findings suggested that administration of BPA and DES at high doses affected embryonic development in a negative way, and this adverse effect was seen less in adult period.

Early embryonic losses in mice induced by diethylstilbestrol

Estrogens cause embryonic lethality and the disturbance of early placental development in mice. Diethylstilbestrol (DES) at 1, 10, or 100 microg/kg was orally administered to Institute of Cancer Research mice on gestational days (GD) 4 through 8, and the uterus and placenta were examined histopathologically on GD 9. Decidua of DES-treated mice showed insufficient development, and the uterine lumen at the implantation site did not effectively minimize. The trophoblast giant cell layer was not separated from the uterine lumen by the decidua capsularis, and hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted at the peripheral part of the decidua basalis. The results of the present study suggest that decidual hypoplasia and subsequent placental hemorrhage causes fetal death due to the administration of DES during the early stage of pregnancy.

Induction stage-dependent expression of vascular endothelial growth factor and aquaporin-1 in diethylstilbestrol-treated rat pituitary

The anterior pituitary gland undergoes tumourigenic changes in response to oestrogen treatment in several breeds of rats. We administered diethylstilbestrol (DES) to female Wistar rats and assessed whether the expression of vascular endothelial growth factor (VEGF) and aquaporin-1 (AQP-1) was altered at different time points following DES administration. In vivo magnetic resonance imaging (MRI) scans showed that the mass index corresponding to the mid-sagittal area of DES-treated pituitary was significantly higher than the vehicle-controlled pituitary (p<0.01) at three specific time points, accompanied by a significant reduction in body weight. Haematoxylin and eosin (HE) staining and immunohistochemical analysis demonstrated that during early stages of induction, DES increased cell proliferation and sprouting of endothelial cells, and VEGF expression transitioned from a vessel-surrounding pattern to a diffuse pattern. During later stages, angiogenesis was predominant, and VEGF expression decreased. In contrast to the early abundant expression of VEGF, endothelial expression of AQP-1 increased during later stages. Our data indicated a dynamic scenario of biological alterations in DES-treated pituitary tissue, in which VEGF and AQP-1 exert their functions at different stages of induction, and we provide novel insights into understanding oestrogen-related tumourigenesis in the anterior pituitary gland.

Induction stage-dependent expression of vascular endothelial growth factor and aquaporin-1 in diethylstilbestrol-treated rat pituitary

The anterior pituitary gland undergoes tumourigenic changes in response to oestrogen treatment in several breeds of rats. We administered diethylstilbestrol (DES) to female Wistar rats and assessed whether the expression of vascular endothelial growth factor (VEGF) and aquaporin-1 (AQP-1) was altered at different time points following DES administration. In vivo magnetic resonance imaging (MRI) scans showed that the mass index corresponding to the mid-sagittal area of DES-treated pituitary was significantly higher than the vehicle-controlled pituitary (p<0.01) at three specific time points, accompanied by a significant reduction in body weight. Haematoxylin and eosin (HE) staining and immunohistochemical analysis demonstrated that during early stages of induction, DES increased cell proliferation and sprouting of endothelial cells, and VEGF expression transitioned from a vessel-surrounding pattern to a diffuse pattern. During later stages, angiogenesis was predominant, and VEGF expression decreased. In contrast to the early abundant expression of VEGF, endothelial expression of AQP- 1 increased during later stages. Our data indicated a dynamic scenario of biological alterations in DES-treated pituitary tissue, in which VEGF and AQP-1 exert their functions at different stages of induction, and we provide novel insights into understanding oestrogen-related tumourigenesis in the anterior pituitary gland.

Effects of 4-n-Octylphenol on the Induction of Mammary Tumors Induced by 7,12-Dimethylbenz[a]anthracene in Rats

We previously reported that a neonatal administration of diethylstilbestrol or 17beta-estradiol affected the mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats. The aim of this present study was to investigate the effects of 4-n-octylphenol (OP), a weak estrogenic disruptor, on the induction of mammary carcinomas (MC) and benign proliferative lesions (PL) induced by DMBA in rats. All female rats were administered at 0, 0.1, 10, 100, and 1,000 microg OP once at birth, given 10 mg DMBA at 50 days after birth, and thereafter underwent necropsy at 351 days after birth. All male rats were given 10 mg DMBA at 28, 42, and 56 days after birth, and 0, 10, 100, and 1,000 ppm OP fed from day 70-153; they underwent necropsy at 153 days after birth. Neonatal single administration of OP in female rats showed no effects such as persistent estrus, anovulatory ovaries, or PL. A slight increase in numbers of rats with MC occurred at the highest dosage. Feeding a large dose of OP for a long period in male rats induced atrophy of testes and slightly increased numbers of affected MC but not increased numbers of males while it showed no effects on PL. These results suggested that administration of a large dose of OP for a long period may have had a minimal effect on mammary carcinogenesis in male rats.

Analysis of plasma protein in newborn rats treated with diethylstilbestrol by using rapid micro two-dimensional electrophoresis

We examined the effect of diethylstilbestrol(DES) administration on newborn male rats by rapid micro two-dimensional polyacrylamide gel electrophoresis (RM2D-PAGE) under non-denaturing/denaturing conditions and a 10-17% second-dimensional gel. We began by visually analyzing the changes at each week over time (weeks 1-9) in plasma protein spots from the same individual DES-administered male rats. We found the tendency for mercaptoalbumin (reduced form) spots to increase in week 1 in DES-administered rats. We also analyzed the quantitative alterations in α2u-globulin (AUG), a biomarker of endocrine disruptors, and found a two-week delay in expression in DES-administered rats in comparison to those not given DES (control rats). We then investigated the reproducibility of these results by using samples taken from multiple male rats in weeks 5, 7 and 9 to perform electrophoretic analysis three times for each individual, and observed the changes in AUG. AUG production was not seen in week 5 in the control rats, but was present at weeks 7 and 9. In DES-administered rats, expression was not observed at week 5 or 7, but was present at week 9. In addition, we identified the spots of AUG by Western blotting after electrophoresis. We consequently confirmed that AUG production delayed by two weeks in DES-administered rats, in comparison with control rats, which suggests that the effect of either a decline in AUG synthesis function or its depletion results in stunting of the reproductive organs, which was confirmed through autopsy.

Effects of Postnatal Administration of Diethylstilbestrol on Puberty and Thyroid Function in Male Rats

To examine the effects of diethylstilbestrol (DES) on male pubertal development and thyroid function, juvenile male Sprague-Dawley rats were given DES daily by oral intubation at doses of 10, 20 and 40 microg/kg/day from postnatal day 33 for 20 days. Prepuce separation was significantly delayed at the dose of 20 microg/kg/day and above in the DES-treated rats. DES treatment induced a significant reduction in the weights of testes, epididymides, the ventral prostate, seminal vesicles plus coagulating glands and fluid, levator ani bulbocavernosus muscles, Cowper's glands and the glans penis. The weights of the liver and adrenals increased in the DES-treated animals. DES caused a dose-dependent reduction in germ cells; in particular the spermatids were mainly affected. The serum levels of testosterone and luteinizing hormone were significantly reduced in the DES-treated groups, but that of estradiol decreased. No differences were observed in the serum thyroxine levels of the control and DES-treated groups. In microscopic observation of the DES-treated animals, degeneration of germ cells and tubular atrophy in the testis were noted, but there were no microscopic changes in the thyroid. These results indicate that DES affected the pubertal development of juvenile male rats and that its mode of action may be related to alterations in hormone levels.

Effects of Neonatally Administered High-dose Diethylstilbestrol on the Induction of Mammary Tumors Induced by 7,12-Dimethylbenz[a]anthracene in Female Rats

The aim of this study was to investigate the effects of neonatal administration of a relatively high dose of diethylstilbestrol (DES) on the induction of mammary carcinomas (MCs) and benign proliferative lesions (PLs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female rats. Three different terms of daily administration of DES (10 microg) were used: 0-14, 0-5, and 6-14 days after birth. Control animals were administered solvent (oil) alone once daily from 0 to 14 days after birth. Rats were given DMBA (10 mg) at 50 days after birth. All rats administered DES showed persistent estrus and anovulatory ovaries. In rats administered DES from 0 to 14 and 0 to 5 days after birth there was protection from development of MCs and PLs, and serum levels of both estrogen and progesterone were significantly lower than in the control animals at 100 days after birth. In rats administered DES from 6 to 14 days after birth, the incidence of MCs was equal to that of the control animals (100%), and the number of PLs was significantly higher than in the control animals. The critical period for exposure to endocrine disruptors, such as DES, affecting the induction of MCs and PLs may be from 0 to 5 days after birth.