Ana M. Soto

Perinatal Exposure to Environmentally Relevant Levels of Bisphenol A Decreases Fertility and Fecundity in CD-1 Mice

Background Perinatal exposure to low-doses of bisphenol A (BPA) results in alterations in the ovary, uterus, and mammary glands and in a sexually dimorphic region of the brain known to be important for estrous cyclicity. Objectives We aimed to determine whether perinatal exposure to environmentally relevant doses of BPA alters reproductive capacity. Methods Female CD-1 mice that were exposed to BPA at 0, 25 ng, 250 ng, or 25 μg/kg body weight (BW)/day or diethylstilbestrol (DES) at 10 ng/kg BW/day (positive control) from gestational day 8 through day 16 of lactation were continuously housed with proven breeder males for 32 weeks starting at 2 months of age. At each delivery, pups born to these mating pairs were removed. The cumulative number of pups, number of deliveries, and litter size were recorded. The purity of the BPA used in this and our previous studies was assessed using HPLC, mass spectrometry, and nuclear magnetic resonance. Results The forced breeding experiment revealed a decrease in the cumulative number of pups, observed as a nonmonotonic dose–response effect, and a decline in fertility and fecundity over time in female mice exposed perinatally to BPA. The BPA was 97% pure, with no evidence of contamination by other phenolic compounds. Conclusions Perinatal exposure to BPA leads to a dose-dependent decline in the reproductive capacity of female mice. The effects on the cumulative number of pups are comparable to those previously reported in mice developmentally exposed to DES, a compound well known to impair reproduction in women. This association suggests the possibility that early BPA exposure may also affect reproductive capacity in women.

Stromal Regulation of Neoplastic Development : Age-Dependent Normalization of Neoplastic Mammary Cells by Mammary Stroma

There is mounting evidence that the stroma plays a crucial role in mammary gland carcinogenesis. Here, we report that mammary gland stroma from mature and multiparous rats prevents neoplastic development and encourages normal ductal growth of grafted epithelial cancer cells. Fifty thousand epithelial cancer cells were injected into the cleared fat pads of virgin hosts at 24, 52, 80, and 150 days of age and of hosts that had undergone two cycles of pregnancy, lactation, and involution. Six months after inoculation, tumor incidence was 75%, 100%, 50%, and 18.2% in 24-, 52-, 80-, and 150-day-old virgin rats, respectively, and 0% in the twice-parous animals. Most remarkably, these neoplastic cells appeared to form normal ducts in all hosts-Ha-ras-1 mutation served as a marker to identify the tumor origin of the outgrowths. The tumor development pattern suggests a parallel to the phenomenon of age- and reproductive state-dependent susceptibility and resistance to chemical carcinogens. As susceptibility to carcinogenesis decreases, the ability of the stroma to reprogram neoplastic epithelial cells increases. Thus, the neoplastic phenotype is context-dependent, and it therefore offers the intriguing possibility that the process of carcinogenesis is amenable to normalization or cure once the mechanisms of stroma-mediated normalization are elucidated and manipulated.

Regulatory decisions on endocrine disrupting chemicals should be based on the principles of endocrinology

For years, scientists from various disciplines have studied the effects of endocrine disrupting chemicals (EDCs) on the health and wellbeing of humans and wildlife. Some studies have specifically focused on the effects of low doses, i.e. those in the range that are thought to be safe for humans and/or animals. Others have focused on the existence of non-monotonic dose-response curves. These concepts challenge the way that chemical risk assessment is performed for EDCs. Continued discussions have clarified exactly what controversies and challenges remain. We address several of these issues, including why the study and regulation of EDCs should incorporate endocrine principles; what level of consensus there is for low dose effects; challenges to our understanding of non-monotonicity; and whether EDCs have been demonstrated to produce adverse effects. This discussion should result in a better understanding of these issues, and allow for additional dialog on their impact on risk assessment.

Importance of dosage standardization for interpreting transcriptomal signature profiles: Evidence from studies of xenoestrogens

To obtain insights into similarities and differences in the biological actions of related drugs or toxic agents, their transcriptomal signature profiles (TSPs) have been examined in a large number of studies. However, many such reports did not provide proper justification for the dosage criteria of each agent. Using a well characterized cell culture model of estrogen-dependent proliferation of MCF7 human breast cancer cells, we demonstrate how different approaches to dosage standardization exert critical influences on TSPs, leading to different and even conflicting conclusions. Using quantitative cellular response (QCR)-based dosage criteria, TSPs were determined by Affymetrix microarray when cells were proliferating at comparable rates in the presence of various estrogens. We observed that TSPs of the xenoestrogens (e.g., genistein or bisphenol A) were clearly different from the TSP of 17β-estradiol; namely, the former strongly enhanced expression of genes involved in mitochondrial oxidative phosphorylation, whereas the latter showed minimal effects. In contrast, TSPs for genistein and 17β-estradiol were indistinguishable by using the marker gene expression-based dosage criteria, conditions in which there was comparable expression of the mRNA transcripts for the estrogen-inducible WISP2 gene. Our findings indicate that determination and interpretation of TSPs in pharmacogenomic and toxicogenomic studies that examine the transcriptomal actions of related agents by microarray require a clear rationale for the dosage standardization method to be used. We suggest that future studies involving TSP analyses use quantitative and objective dosage standardization methods, such as those with quantitative cellular response or marker gene expression-based dosage criteria.

Science and policy on endocrine disrupters must not be mixed: a reply to a “common sense” intervention by toxicology journal editors

The “common sense” intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

An updated review of environmental estrogen and androgen mimics and antagonists

For the last 40 y, substantial evidence has surfaced on the hormone-like effects of environmental chemicals such as pesticides and industrial chemicals in wildlife and humans. The endocrine and reproductive effects of these chemicals are believed to be due to their ability to: (1) mimic the effect of endogenous hormones, (2) antagonize the effect of endogenous hormones, (3) disrupt the synthesis and metabolism of endogenous hormones, and (4) disrupt the synthesis and metabolism of hormone receptors. The discovery of hormone-like activity of these chemicals occurred long after they were released into the environment. Aviation crop dusters handling DDT were found to have reduced sperm counts, and workers at a plant producing the insecticide kepone were reported to have lost their libido, became impotent and had low sperm counts. Subsequently, experiments conducted in lab animals demonstrated unambiguously the estrogenic activity of these pesticides. Man-made compounds used in the manufacture of plastics were accidentally found to be estrogenic because they fouled experiments conducted in laboratories studying natural estrogens. For example, polystyrene tubes released nonylphenol, and polycarbonate flasks released bisphenol-A. Alkylphenols are used in the synthesis of detergents (alkylphenol polyethoxylates) and as antioxidants. These detergents are not estrogenic; however, upon degradation during sewage treatment they may release estrogenic alkylphenols. The surfactant nonoxynol is used as intravaginal spermicide and condom lubricant. When administered to lab animals it is metabolized to free nonylphenol. Bisphenol-A was found to contaminate the contents of canned foods; these tin cans are lined with lacquers such as polycarbonate. Bisphenol-A is also used in dental sealants and composites. We found that this estrogen leaches from the treated teeth into saliva; up to 950 microg of bisphenol-A were retrieved from saliva collected during the first hour after polymerization. Other xenoestrogens recently identified among chemicals used in large volumes are the plastizicers benzylbutylphthalate, dibutylphthalate, the antioxidant butylhydroxyanisole, the rubber additive p-phenylphenol and the disinfectant o-phenylphenol. These compounds act cumulatively. In fact, feminized male fish were found near sewage outlets in several rivers in the U.K.; a mixture of chemicals including alkyl phenols resulting from degradation of detergents during sewage treatment seemed to be the causal agent. Estrogen mimics are just a class of endocrine disruptors. Recent studies identified antiandrogenic activity in environmental chemicals such as vinclozolin, a fungicide, and DDE, and insecticide. Moreover, a single chemical may produce neurotoxic, estrogenic and antiandrogenic effects. It has been hypothesized that endocrine disruptors may play a role in the decrease in the quantity and quality of human semen during the last 50 y, as well as in the increased incidence of testicular cancer and cryptorchidism in males and breast cancer incidence in both females and males in the industrialized word. To explore this hypothesis it is necessary to identify putative causal agents by the systematic screening of environmental chemicals and chemicals present in human foods to assess their ability to disrupt the endocrine system. In addition, it will be necessary to develop methods to measure cumulative exposure to (a) estrogen mimics, (b) antiandrogens, and (c) other disruptors.

Endocrine disruptors and reproductive health: The case of bisphenol-A

Epidemiological studies have reported that during the last 60 years the quantity and quality of human sperm has decreased and the incidence of male genital tract defects, testicular, prostate and breast cancer has increased. During the same time period, developmental, reproductive and endocrine effects have also been documented in wildlife species. The last six decades have witnessed a massive introduction of hormonally active synthetic chemicals into the environment leading some to postulate that the diverse outcomes documented in human and wildlife populations might be the result of extemporaneous exposure to xenoestrogens during development. The estrogen-mimic bisphenol-A (BPA) is used as a model agent for endocrine disruption. BPA is used in the manufacture of polycarbonate plastics and epoxy resins from which food and beverage containers and dental materials are made. Perinatal exposure to environmentally relevant BPA doses results in morphological and functional alterations of the male and female genital tract and mammary glands that may predispose the tissue to earlier onset of disease, reduced fertility and mammary and prostate cancer.

The stroma as a crucial target in rat mammary gland carcinogenesis

A complex network of interactions between the stroma, the extracellular matrix and the epithelium drives mammary gland development and function. Two main assumptions in chemical carcinogenesis of the mammary gland have been that carcinogens induce neoplasia by causing mutations in the DNA of the epithelial cells and that the alterations of tissue architecture observed in neoplasms are a consequence of this primary mutational event. Here, we use a rat mammary tissue recombination model and the chemical carcinogen N-nitrosomethylurea (NMU) to determine whether the primary target of the carcinogen is the epithelium, the stroma or both tissue compartments. Mammary epithelial cells were exposed in vitro either to the carcinogen or vehicle before being transplanted into the cleared fat pads of rats exposed to carcinogen or vehicle. We observed that neoplastic transformation of these mammary epithelial cells occurred only when the stroma was exposed in vivo to NMU, regardless of whether or not the epithelial cells were exposed to the carcinogen. Mammary epithelial cells exposed in vitro to the carcinogen formed phenotypically normal ducts when injected into a non-treated stroma. Mutation in the Ha-ras-1 gene did not correlate with initiation of neoplasia. Not only was it often found in both cleared mammary fat pads of vehicle-treated animals and intact mammary glands of untreated animals, but it was also absent in some tumors. Our results suggest that the stroma is a crucial target of the carcinogen and that mutation in the Ha-ras-1 gene is neither necessary nor sufficient for tumor initiation.

Environmental causes of cancer: endocrine disruptors as carcinogens

Environmental endocrine disrupting chemicals (EDCs), including pesticides and industrial chemicals, have been and are released into the environment producing deleterious effects on wildlife and humans. The effects observed in animal models after exposure during organogenesis correlate positively with an increased incidence of malformations of the male genital tract and of neoplasms and with the decreased sperm quality observed in European and US populations. Exposure to EDCs generates additional effects, such as alterations in male and female reproduction and changes in neuroendocrinology, behavior, metabolism and obesity, prostate cancer and thyroid and cardiovascular endocrinology. This Review highlights the carcinogenic properties of EDCs, with a special focus on bisphenol A. However, humans and wildlife are exposed to a mixture of EDCs that act contextually. To explain this mindboggling complexity will require the design of novel experimental approaches that integrate the effects of different doses of structurally different chemicals that act at different ages on different target tissues. The key to this complex problem lies in the adoption of mathematical modeling and computer simulations afforded by system biology approaches. Regardless, the data already amassed highlight the need for a public policy to reduce exposure to EDCs.

In Utero Exposure to Bisphenol A Alters the Development and Tissue Organization of the Mouse Mammary Gland

Abstract Exposure to estrogens throughout a womans life, including the period of intrauterine development, is a risk factor for the development of breast cancer. The increased incidence of breast cancer noted during the last 50 years may have been caused, in part, by exposure of women to estrogen-mimicking chemicals that are released into the environment. Here, we investigated the effects of fetal exposure to one such chemical, bisphenol A (BPA), on development of the mammary gland. CD-1 mice were exposed in utero to low, presumably environmentally relevant doses of BPA (25 and 250 μg/kg body weight), and their mammary glands were assessed at 10 days, 1 mo, and 6 mo of age. Mammary glands of BPA-exposed mice showed differences in the rate of ductal migration into the stroma at 1 mo of age and a significant increase in the percentage of ducts, terminal ducts, terminal end buds, and alveolar buds at 6 mo of age. The percentage of cells that incorporated BrdU was significantly decreased within the epithelium at 10 days of age and increased within the stroma at 6 mo of age. These changes in histoarchitecture, coupled with an increased presence of secretory product within alveoli, resemble those of early pregnancy, and they suggest a disruption of the hypothalamic-pituitary-ovarian axis and/or misexpression of developmental genes. The altered relationship in DNA synthesis between the epithelium and stroma and the increase in terminal ducts and terminal end buds are striking, because these changes are associated with carcinogenesis in both rodents and humans.