Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.

Skin diseases of the breast and nipple: Benign and malignant tumors.

The evaluation and management of dermatologic diseases of the breast and nipple requires an understanding of the unique anatomy of the breast and nipple and an awareness of the significant emotional, cultural, and sexual considerations that may come into play when treating this anatomic area. The first article in this continuing medical education series reviews breast anatomy, congenital breast anomalies, and benign and malignant breast tumors. An emphasis is placed on inflammatory breast cancer and breast cancer with noninflammatory skin involvement and on cutaneous metastases to the breast and from breast cancer. Familiarity of the dermatologist with the cutaneous manifestations of breast cancer will facilitate the diagnosis of breast malignancy and assist with staging, prognostication, and evaluation for recurrence. This article also discusses genodermatoses that predispose to breast pathology and provides imaging recommendations for evaluating a palpable breast mass.

Gli activity is critical at multiple stages of embryonic mammary and nipple development.

Gli3 is a transcriptional regulator of Hedgehog (Hh) signaling that functions as a repressor (Gli3(R)) or activator (Gli3(A)) depending upon cellular context. Previously, we have shown that Gli3(R) is required for the formation of mammary placodes #3 and #5. Here, we report that this early loss of Gli3 results in abnormal patterning of two critical regulators: Bmp4 and Tbx3, within the presumptive mammary rudiment (MR) #3 zone. We also show that Gli3 loss leads to failure to maintain mammary mesenchyme specification and loss of epithelial Wnt signaling, which impairs the later development of remaining MRs: MR#2 showed profound evagination and ectopic hairs formed within the presumptive areola; MR#4 showed mild invagination defects and males showed inappropriate retention of mammary buds in Gli3(xt/xt) mice. Importantly, mice genetically manipulated to misactivate Hh signaling displayed the same phenotypic spectrum demonstrating that the repressor function of Gli3(R) is essential during multiple stages of mammary development. In contrast, positive Hh signaling occurs during nipple development in a mesenchymal cuff around the lactiferous duct and in muscle cells of the nipple sphincter. Collectively, these data show that repression of Hh signaling by Gli3(R) is critical for early placodal patterning and later mammary mesenchyme specification whereas positive Hh signaling occurs during nipple development.

Case report: ectopic nipple on the sole of the foot, an unexplained anomaly.

Supernumerary nipples are common congenital anomalies, most often occurring along the embryonic milk lines. We present a patient with an ectopic nipple on the foot. We are unable to explain the aetiology of this anomaly; however, several theories have been proposed. They are also associated with disorders of the renal and cardiovascular systems as well as pathology that affect normal breast tissue.

Endocrine-disrupting activities in vivo of the fungicides tebuconazole and epoxiconazole.

The triazole fungicides tebuconazole and epoxiconazole were investigated for reproductive toxic effects after exposure during gestation and lactation. Rats were dosed with epoxiconazole (15 or 50 mg/kg bw/day) or tebuconazole (50 or 100 mg/kg bw/day) during pregnancy from gestational day (GD) 7 and continued during lactation until postnatal day (PND) 16. Some dams were randomly chosen for cesarean section at GD 21 to evaluate effects on sexual differentiation in the fetuses. Other dams delivered normally, and the pups were examined (e.g., anogenital distance [AGD] and hormone levels) at birth, at PND 13 or PND 16, and semen quality was assessed in adults. Both tebuconazole and epoxiconazole affected reproductive development in the offspring after exposure in utero. Both compounds virilized the female offspring as shown by an increased AGD PND 0. Furthermore, tebuconazole had a feminizing effect on male offspring as shown by increased nipple retention. This effect was likely caused by the reduced testosterone levels seen in male fetuses. Tebuconazole increased the testicular concentrations of progesterone and 17alpha-hydroxyprogesterone in male fetuses, indicating a direct impact on the steroid synthesis pathway in the Leydig cells. The high dose of epoxiconazole had marked fetotoxic effects, while the lower dose caused increased birth weights. The increased birth weights may be explained by a marked increase in testosterone levels in dams during gestation. Common features for azole fungicides are that they increase gestational length, virilize female pups, and affect steroid hormone levels in fetuses and/or dams. These effects strongly indicate that one major underlying mechanism for the endocrine-disrupting effects of azole fungicides is disturbance of key enzymes like CYP17 involved in the synthesis of steroid hormones.

Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats.

In mammals, abnormal increases in fetal androgens disrupt normal development of the female phenotype. Due to the recent concern regarding environmental androgen-active chemicals, there is a need to identify sources of fetal androgen variation and sensitive developmental markers for androgenic activity in female rats. Anogenital distances (AGD), nipple retention, reproductive tract, and external genitalia are morphological parameters organized by prenatal androgens and are predictive of altered masculinized/defeminized phenotype in adult female mice and rats. The objectives of this study were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the intrauterine position (IUP) effect, (2) characterize the permanent effects of prenatal androgen exposure on female rats, and (3) determine the ability of AGD and areolas to predict these permanent androgenic alterations in female rats. Untreated male fetal rats had higher tissue testosterone (T) concentrations than females in the amniotic fluid, reproductive tract, gonad, and fetal body. The intrauterine position (IUP) of male and female fetuses did not affect T concentrations or AGD in male or female rats at gestational day (GD) 22. Female offspring exposed to 0, 1.5, and 2.5 mg/kg/day testosterone propionate (TP) on GDs 14-18 displayed increased AGD at postnatal day (PND) 2 and decreased nipples at PND 13 and as adults. TP-induced changes in neonatal AGD and infant areola number were reliable indicators of permanently altered adult phenotype in female rats. Further, females in the two high-dose groups displayed increased incidences of external genital malformations and the presence of prostatic tissue, not normally found in female rats.

Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz.

The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17alpha-hydroxylase/17,20-lyase (P450c17) and a reduction in 17beta-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level.

Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development.

Prior reports have demonstrated that both parathyroid hormone-related protein (PTHrP) and the type I PTH/PTHrP receptor are necessary for the proper development of the embryonic mammary gland in mice. Using a combination of loss-of-function and gain-of-function models, we now report that PTHrP regulates a series of cell fate decisions that are central to the survival and morphogenesis of the mammary epithelium and the formation of the nipple. PTHrP is made in the epithelial cells of the mammary bud and, during embryonic mammary development, it interacts with the surrounding mesenchymal cells to induce the formation of the dense mammary mesenchyme. In response, these mammary-specific mesenchymal cells support the maintenance of mammary epithelial cell fate, trigger epithelial morphogenesis and induce the overlying epidermis to form the nipple. In the absence of PTHrP signaling, the mammary epithelial cells revert to an epidermal fate, no mammary ducts are formed and the nipple does not form. In the presence of diffuse epidermal PTHrP signaling, the ventral dermis is transformed into mammary mesenchyme and the entire ventral epidermis becomes nipple skin. These alterations in cell fate require that PTHrP be expressed during development and they require the presence of the PTH/PTHrP receptor. Finally, PTHrP signaling regulates the epidermal and mesenchymal expression of LEF1 and (&bgr;)-catenin, suggesting that these changes in cell fate involve an interaction between the PTHrP and Wnt signaling pathways.

Supernumerary breast tissue: historical perspectives and clinical features.

The presence of extra nipples and breasts, polythelia and polymastia respectively, is not uncommon. Such supernumerary breast tissue usually is found within the milk line extending from the axilla to pubic region. It was once thought that this condition was a symbol of increased fertility and femininity. Anne Boleyn was said to have a third breast. Ancient goddesses of fertility had row upon row of breasts on their chests. Polythelia is seen congenitally. Ectopic breast tissue and polymastia may not appear until enhanced by sex hormones during puberty or early pregnancy. The same pathology that can affect normally positioned breasts, including carcinoma, can occur in supernumerary breast tissue. Renal and other organ system anomalies are associated with supernumerary breast tissue. Further research is needed to establish the clinical significance of supernumerary breast tissue in light of its reported associated conditions. Appropriate treatment is yet to be refined.

Supernumerary nipples.

Supernumerary nipples are common anomalies, and their significance is usually limited to cosmetic concerns. However, a high index of suspicion should be maintained during physical examinations, because any disease process that involves anatomically normal breasts may affect aberrantly located breasts or nipples as well. These anomalies may be associated with several systemic disorders, particularly urinary tract abnormalities.

Unilateral athelia with a subcutaneous dermoid cyst.

A case of unilateral athelia with a subcutaneous dermoid cyst in the area in question is reported. The histologic features of the cyst wall suggest that it is derived from a base in the nipple-areola complex. Dozens of cases of congenital absence of the breast have been reported, some of them associated with congenital anhidrotic ectodermal dysplasia, others with additional musculoskeletal abnormalities, and still others normal in other respects. But to our knowledge, the combination of athelia and corresponding subcutaneous dermoid cyst has not been reported.

Nipple differentiation in fetal male rats treated with an inhibitor of the enzyme 5 alpha-reductase: definition of a selective role for dihydrotestosterone.

In the rat, androgens are responsible for sexually dimorphic nipple differentiation. Nipples are expressed in the female, while in the male, the nipple anlage regress prenatally. Mammary gland development is present in both sexes. Treatment of pregnant Sprague-Dawley rats from days 12-21 of gestation with the aza-steroid 17 beta-N,N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, a competitive inhibitor of the enzyme 5 alpha-reductase, resulted in nipple development in male offspring. Additionally, there was feminization of the external genitalia, with urethral displacement to the base of the phallus. The role of androgens in suppression of nipple anlage in the male rat fetus is known. This study, however, suggests for the first time a selective role for 5 alpha-dihydrotestosterone in regression of the nipple anlage in utero. Thus, 5 alpha-dihydrotestosterone may be critical not only for masculinization of the external genitalia, but also for inhibition of nipple development in the male rat fetus.

Lumen formation in the developing mouse mammary gland.

The mammary gland is a system of hollow interconnecting tubes which develops from an invasive branching cord of epithelial cells. This ultrastructural study of the developing mammary gland focuses on how the lumen forms and establishes the polarized epithelial lining of the gland. The earliest signs of lumen formation are many small cavities and crevices lined with microvilli which appear at scattered sites throughout the branching cords and neck of the gland. It is suggested that these initial small lumina form quite simply by separation of cells whose opposing faces are non-adhesive. The continuous central lumen of the gland develops by fusion and enlargement of the many small lumina. The cells adjacent to the developing lumen will form the polarized epithelial lining of the gland. Excess, more basal, epithelial cells degenerate. The lumen begins to appear when the branching pattern is almost complete. Thus, during morphogenesis, invasion by the mammary gland epithelium involves penetration of the mesenchyme by a solid cord of cells. We suggest that this cellular organization may be a fundamental characteristic of invasive epithelia and that a crucial step in the development of malignant epithelial tumours is a change in cell organization from a polarized cell sheet to a solid cord of cells which can invade.