The Case for BPA as an Obesogen: Contributors to the Controversy.

Since the inception of the term endocrine disruptor, the idea that the environment is an important determinant of phenotype has motivated researchers to explore the effect of low dose exposure to BPA during organogenesis. The syndrome observed was complex, affecting various endpoints such as reproduction and reproductive tissues, behavior, mammary gland development and carcinogenesis, glucose homeostasis, and obesity. This constellation of impacted endpoints suggests the possibility of complex interactions among the multiple effects of early BPA exposure. One key finding of our rodent studies was alterations of energy and amino-acid metabolism that were detected soon after birth and continued to be present at all time points examined through 6 months of age. The classical manifestations of obesity and associated elements of metabolic disease took a longer time to become apparent. Here we examine the validity of the often-mentioned lack of reproducibility of obesogenic effects of BPA, starting from the known environmental causes of variation, which are diverse and range from the theoretical like the individuation process and the non-monotonicity of the dose-response curve, to the very pragmatic like housing, feed, and time and route of exposure. We then explore environmental conditions that may hinder reproducibility and discuss the effect of confounding factors such as BPA-induced hyperactivity. In spite of all the potential sources of variation, we find that some obesogenic or metabolic effects of BPA are reproducibly observed when study conditions are analogous. We recommend that study authors describe details of their study conditions including the environment, husbandry, and feed. Finally, we show that when experimental conditions are strictly maintained, reproducibility, and stability of the obese phenotype is consistently observed.

Perinatal BPA exposure and reproductive axis function in CD-1 mice.

Perinatal Bisphenol-A (BPA) exposure reduces fertility and fecundity in mice. This study examined effects of early BPA exposure on activation of gonadotropin releasing hormone (GnRH) neurons in conjunction with a steroid-induced luteinizing hormone (LH) surge, characterized patterns of estrous cyclicity and fertility over time, and assessed the ovarian follicular reserve to further explore factors responsible for the reduced fertility we previously described in this model. The percent activated GnRH neurons was reduced in BPA-exposed females at 3-6 months, and periods of persistent proestrus were increased. These data suggest that perinatal exposure to BPA reduces GnRH neuronal activation required for the generation of the LH surge and estrous cyclicity. Assessments of anti-Müllerian hormone (AMH) levels failed to suggest a decline in the follicular reserve at the BPA exposure levels examined.

Perinatal BPA exposure alters body weight and composition in a dose specific and sex specific manner: The addition of peripubertal exposure exacerbates adverse effects in female mice.

Body weight (BW) and body composition were examined in CD-1 mice exposed perinatally or perinatally and peripubertally to 0, 0.25, 2.5, 25, or 250µg BPA/kg BW/day. Our goal was to identify the BPA dose (s) and the exposure window(s) that increased BW and adiposity, and to assess potential sex differences in this response. Both perinatal exposure alone and perinatal plus peripubertal exposure to environmentally relevant levels of BPA resulted in lasting effects on body weight and body composition. The effects were dose specific and sex specific and were influenced by the precise window of BPA exposure. The addition of peripubertal BPA exposure following the initial perinatal exposure exacerbated adverse effects in the females but appeared to reduce differences in body weight and body composition between control and BPA exposed males. Some effects of BPA on body weight and body composition showed a non-linear dose response.

Low-dose BPA exposure alters the mesenchymal and epithelial transcriptomes of the mouse fetal mammary gland.

Exposure of rodent fetuses to low doses of the endocrine disruptor bisphenol A (BPA) causes subtle morphological changes in the prenatal mammary gland and results in pre-cancerous and cancerous lesions during adulthood. To examine whether the BPA-induced morphological alterations of the fetal mouse mammary glands are a) associated with changes in mRNA expression reflecting estrogenic actions and/or b) dependent on the estrogen receptor α (ERα), we compared the transcriptomal effects of BPA and the steroidal estrogen ethinylestradiol (EE2) on fetal mammary tissues of wild type and ERα knock-out mice. Mammary glands from fetuses of dams exposed to vehicle, 250 ng BPA/kg BW/d or 10 ng EE2/kg BW/d from embryonic day (E) 8 were harvested at E19. Transcriptomal analyses on the ductal epithelium and periductal stroma revealed altered expression of genes involved in the focal adhesion and adipogenesis pathways in the BPA-exposed stroma while genes regulating the apoptosis pathway changed their expression in the BPA-exposed epithelium. These changes in gene expression correlated with previously reported histological changes in matrix organization, adipogenesis, and lumen formation resulting in enhanced maturation of the fat-pad and delayed lumen formation in the epithelium of BPA-exposed fetal mammary glands. Overall similarities in the transcriptomal effects of BPA and EE2 were more pronounced in the epithelium, than in the stroma. In addition, the effects of BPA and EE2 on the expression of various genes involved in mammary stromal-epithelial interactions were suppressed in the absence of ERα. These observations support a model whereby BPA and EE2 act directly on the stroma, which expresses ERα, ERß and GPR30 in fetal mammary glands, and that the stroma, in turn, affects gene expression in the epithelium, where ERα and ERß are below the level of detection at this stage of development.

Effects of low doses of bisphenol A on the metabolome of perinatally exposed CD-1 mice.

BACKGROUND: Bisphenol A (BPA) is a well-known endocrine disruptor used to manufacture polycarbonate plastics and epoxy resins. Exposure of pregnant rodents to low doses of BPA results in pleiotropic effects in their offspring. OBJECTIVE: We used metabolomics--a method for determining metabolic changes in response to nutritional, pharmacological, or toxic stimuli--to examine metabolic shifts induced in vivo by perinatal exposure to low doses of BPA in CD-1 mice. METHODS: Male offspring born to pregnant CD-1 mice that were exposed to vehicle or to 0.025, 0.25, or 25 µg BPA/kg body weight/day, from gestation day 8 through day 16 of lactation, were examined on postnatal day (PND) 2 or PND21. Aqueous extracts of newborns (PND2, whole animal) and of livers, brains, and serum samples from PND21 pups were submitted to (1)H nuclear magnetic resonance spectroscopy. Data were analyzed using partial least squares discriminant analysis. RESULTS: Examination of endogenous metabolic fingerprints revealed remarkable discrimination in whole extracts of the four PND2 newborn treatment groups, strongly suggesting changes in the global metabolism. Furthermore, statistical analyses of liver, serum, and brain samples collected on PND21 successfully discriminated among treatment groups. Variations in glucose, pyruvate, some amino acids, and neurotransmitters (γ-aminobutyric acid and glutamate) were identified. CONCLUSIONS: Low doses of BPA disrupt global metabolism, including energy metabolism and brain function, in perinatally exposed CD-1 mouse pups. Metabolomics can be used to highlight the effects of low doses of endocrine disruptors by linking perinatal exposure to changes in global metabolism.

The male mammary gland: a target for the xenoestrogen bisphenol A.

Males of some strains of mice retain their mammary epithelium even in the absence of nipples. Here, we have characterized the mammary gland in male CD-1 mice both in whole mounts and histological sections. We also examined the effects of bisphenol A (BPA), an estrogen mimic that alters development of the female mouse mammary gland. BPA was administered at a range of environmentally relevant doses (0.25-250µg/kg/day) to pregnant and lactating mice and then the mammary glands of male offspring were examined at several periods in adulthood. We observed age- and dose-specific effects on mammary gland morphology, indicating that perinatal BPA exposures alter the male mammary gland in adulthood. These results may provide insight into gynecomastia, the most common male breast disease in humans, where proliferation of the mammary epithelium leads to breast enlargement.

Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects.

Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. This compound is a building block of polycarbonate plastics often used for food and beverage storage, and BPA is also a component of epoxy resins that are used to line food and beverage containers. Studies have shown that BPA can leach from these and other products in contact with food and drink, and as a result, routine ingestion of BPA is presumed. This compound is also found in an enormous number of other products that we come into contact with daily, and therefore it is not surprising that it has been detected in the majority of individuals examined. BPA is a known endocrine disruptor. Although initially considered to be a weak environmental estrogen, more recent studies have demonstrated that BPA may be similar in potency to estradiol in stimulating some cellular responses. Moreover, emerging evidence suggests that BPA may influence multiple endocrine-related pathways. Studies in rodents have identified adverse effects of BPA at levels at or below the current acceptable daily intake level for this compound. The various reported adverse effects of BPA are reviewed, and potential mechanisms of BPA action are discussed. Much more investigation is needed to understand the potential adverse health effects of BPA exposure in humans and to understand the multiple pathways through which it may act. Although many questions remain to be answered, it is becoming increasingly apparent that exposure to BPA is ubiquitous and that the effects of this endocrine disruptor are complex and wide-ranging.

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.

Interpreting endocrine disruption from an integrative biology perspective.

The ability of reductionism to advance our understanding of complex biological phenomena is limited. The ecological developmental biology (eco-devo) movement rejects the notion that development is merely the unfolding of a genetic program. Fetal exposure to environmental endocrine disruptors may contribute to the increased incidence of male genital tract malformations, decreased sperm quality, several neoplasms, and altered body weight. Here we discuss problems hindering the study of endocrine disruption (reductionist stance, technically driven research biases, and study of single end points, chemicals and exposure periods). We propose the study of both upward and downward causation and a Systems Biology approach to develop quantitative mathematical models for use in computer simulations that would generate testable predictions. This integrative approach will allow the simultaneous consideration of organismal (systemic) effects and effects on various organ systems. It will promote the identification of similar and unique effects of different endocrine disruptors, and their inter-relationships.

Bisphenol A: Perinatal exposure and body weight.

Bisphenol A (BPA) is a component of polycarbonate and other plastics including resins that line food and beverage containers. BPA is known to leach from products in contact with food and drink, and is therefore thought to be routinely ingested. In a recent cross sectional study, BPA was detected in urine samples from 92.6% of the US population examined. The potential for BPA to influence body weight is suggested by in vitro studies demonstrating effects of BPA on adipocyte differentiation, lipid accumulation, glucose transport and adiponectin secretion. Data from in vivo studies have revealed dose-dependent and sex dependent effects on body weight in rodents exposed perinatally to BPA. The mechanisms through which perinatal BPA exposure acts to exert persistent effects on body weight and adiposity remain to be determined. Possible targets of BPA action are discussed.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A.

BACKGROUND: In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES: We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION: Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., "good science"). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS: Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Bisphenol-A and the great divide: a review of controversies in the field of endocrine disruption.

In 1991, a group of 21 scientists gathered at the Wingspread Conference Center to discuss evidence of developmental alterations observed in wildlife populations after chemical exposures. There, the term "endocrine disruptor" was agreed upon to describe a class of chemicals including those that act as agonists and antagonists of the estrogen receptors (ERs), androgen receptor, thyroid hormone receptor, and others. This definition has since evolved, and the field has grown to encompass hundreds of chemicals. Despite significant advances in the study of endocrine disruptors, several controversies have sprung up and continue, including the debate over the existence of nonmonotonic dose response curves, the mechanisms of low-dose effects, and the importance of considering critical periods of exposure in experimental design. One chemical found ubiquitously in our environment, bisphenol-A (BPA), has received a tremendous amount of attention from research scientists, government panels, and the popular press. In this review, we have covered the above-mentioned controversies plus six additional issues that have divided scientists in the field of BPA research, namely: 1) mechanisms of BPA action; 2) levels of human exposure; 3) routes of human exposure; 4) pharmacokinetic models of BPA metabolism; 5) effects of BPA on exposed animals; and 6) links between BPA and cancer. Understanding these topics is essential for educating the public and medical professionals about potential risks associated with developmental exposure to BPA and other endocrine disruptors, the design of rigorously researched programs using both epidemiological and animal studies, and ultimately the development of a sound public health policy.

Perinatal exposure to the xenoestrogen bisphenol-A induces mammary intraductal hyperplasias in adult CD-1 mice.

Humans are routinely exposed to bisphenol-A (BPA), an estrogenic compound that leaches from consumer products. Given the sensitivity of the developing organism to hormones, exposure of fetuses and infants is a concern. Here, CD-1 mice were exposed to environmentally relevant doses of BPA during gestation and the lactational period (gestational day 8 through postnatal day 16). At 3, 9 and 12-15 months of age, mammary glands from exposed offspring were examined for structural changes. BPA-exposed females demonstrated altered mammary phenotypes including the appearance of alveolar buds. Additionally, intraductal hyperplasias were observed exclusively in BPA-exposed females. These lesions had the appearance of "beaded" ducts, with epithelial cells present inside the ductal lumen and increased proliferation indexes compared to normal ducts. Similar structures have also been observed following exposure to other estrogens. These results are further evidence that perinatal BPA exposure can alter the morphology of the rodent mammary gland in adulthood.

In vivo effects of bisphenol A in laboratory rodent studies.

Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/(kg day), in laboratory exposures with mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol and ethinylestradiol. For most effects, the potency of BPA is approximately 10-1000-fold less than that of diethylstilbestrol or ethinylestradiol. Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long lasting, organizational effects in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely, but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that developmental effects occur in the female reproductive system.

Perinatal bisphenol A exposure increases estrogen sensitivity of the mammary gland in diverse mouse strains.

BACKGROUND: Studies of low-dose effects of xenoestrogens have yielded conflicting results that may be attributed to differences in estrogen sensitivity between the rodent strains examined. Perinatal exposure of CD-1 mice to low doses of the xenoestrogen bisphenol A (BPA) alters peripubertal mammary gland development. Future studies to assess the role of estrogen receptors as mediators of BPA action require estrogen receptor knock-out mice that were generated on a C57Bl6 background. The sensitivity of the C57Bl6 strain to estradiol and BPA is unknown. OBJECTIVES: In the present study we examined whether the mammary glands of CD-1 and C57Bl6 mice exhibited similar responses to 17beta-estradiol (E(2)) and whether perinatal exposure to BPA equally enhanced sensitivity of the mammary glands to E(2) at puberty. METHODS: Immature mice were ovariectomized and treated for 10 days with one of eight doses of E(2). Morphological mammary gland parameters were examined to identify doses producing half-maximal effects. Mice were exposed perinatally to 0 or 250 ng BPA/kg body weight (bw)/day from gestational day 8 until postnatal day (PND) 2. On PND25, female offspring were ovariectomized and given an estrogen challenge of 0, 0.5, or 1 microg E(2)/kg bw/day for 10 days. Morphometric parameters of the mammary gland were compared between strains. RESULTS: Both strains exhibited similar responses to E(2). Perinatal BPA exposure altered responses to E(2) at puberty for several parameters in both strains, although the effect in CD-1 was slightly more pronounced. CONCLUSION: Both mouse strains provide adequate models for the study of perinatal exposure to xenoestrogens.

Anterior pituitary gene expression with reproductive aging in the female rat.

Although reproductive aging in women is classically attributed to loss of ovarian follicles, recent data have suggested that the entire hypothalamic-pituitary-ovarian axis undergoes functional changes with time. The aim of this study was to characterize age-related changes in pituitary gene expression for factors with known importance for gonadotroph function, including 1) steroid hormone receptors (Esr and Pgr), 2) orphan nuclear receptors [Nr5a1 (steroidogenic factor-1) and Nr5a2 (liver receptor homologue-1)], and 3) pituitary-derived polypeptides (activin, inhibin, and follistatin), as well as 4) gonadotropin subunits and 5) GnRH receptors. We chose to utilize a middle-aged rat model for these studies. Young (Y; 3-mo-old) and middle-aged (MA; 9- to 12-mo-old) rats were ovariectomized, primed with estradiol, and injected with progesterone to induce an LH surge. The mRNA levels for the gonadotropin subunits and GnRH receptors were decreased in middle-aged females relative to young animals. Nr5a1 and follistatin mRNA levels were significantly greater in Y versus MA animals following ovariectomy. Furthermore, steroid-induced regulation of these genes was lost in the MA animals. Regulation of the Nr5a2, Inhba, and Inhbb transcripts was also limited to the young animals. In contrast, there were no significant differences in the mRNA levels of Esr or Pgr family members between age groups at any time point. Although this in vivo model normalizes ovarian steroid levels, it does not control for potential differences in GnRH stimulation with aging. Therefore, in a second set of experiments, we used an in vitro perifusion system to compare the effects of pulsatile GnRH in the two age groups. Nr5a1 mRNA expression was greater in Y than MA animals and was significantly decreased by GnRH pulses in both age groups. Follistatin mRNA levels increased significantly with GnRH treatment in Y animals but were not significantly changed in the MA females. Taken together, these data demonstrate gene-specific blunting of pituitary gene expression postovariectomy and during the steroid-induced surge in middle-aged rats. We propose that age-related changes in pituitary physiology may contribute to reproductive senescence.

Exposure to environmentally relevant doses of the xenoestrogen bisphenol-A alters development of the fetal mouse mammary gland.

Humans are routinely exposed to bisphenol-A (BPA), an estrogenic compound that leaches from dental materials, food and beverage containers, and other plastic consumer products. Effects of perinatal BPA exposure on the mouse mammary gland have been observed in puberty and adulthood, long after the period of exposure has ended. The aim of this study was to examine fetal mammary gland development at embryonic day (E)18 and assess changes in the tissue organization and histoarchitecture after exposure to an environmentally relevant dose of BPA. In unexposed fetuses, the relative position of the fetus with respect to its female and male siblings in the uterus influenced growth of the ductal tree, which was more developed in females placed between two males than in females placed between two females. Exposure of dams to 250 ng BPA per kilogram body weight per day from E8 to E18 significantly increased ductal area and ductal extension in exposed fetuses and obliterated positional differences. In the stroma, BPA exposure promoted maturation of the fat pad and altered the localization of collagen. Within the epithelium, BPA exposure led to a decrease in cell size and delayed lumen formation. Because mammary gland development is dependent on reciprocal interactions between these compartments, the advanced maturation of the fat pad and changes in the extracellular matrix may be responsible for the altered growth, cell size, and lumen formation observed in the epithelium. These results suggest that alterations in mammary gland phenotypes observed at puberty and adulthood in perinatally exposed mice have their origins in fetal development.

The mammary gland response to estradiol: monotonic at the cellular level, non-monotonic at the tissue-level of organization?

The role of hormones in mammary gland development has been studied in detail using surgical and genetic models. These studies have indicated roles for estrogen in ductal elongation and terminal end bud formation. However, no comprehensive study has quantified how different doses of estrogen affect morphological parameters of mammary gland development. Additionally, comparisons between the estrogen-responsiveness of the mammary gland and uterus, the model organ for estrogen action are incomplete. In this study, immature mice were ovariectomized and implanted with osmotic pumps releasing one of eight doses of 17beta-estradiol for 10 days. As expected from the classical uterotrophic assay, the uterus showed a monotonic dose-response curve for all measured endpoints. In contrast, the mammary gland showed a non-monotonic, inverted-U shaped response to estrogen with regard to morphometric parameters, and a monotonic response with regard to gene expression parameters. These results indicate that estrogen has opposing effects in mammary gland morphogenesis depending on estrogen dose, i.e. low to moderate doses induce terminal end bud formation and ductal elongation while higher doses inhibit these processes. This non-monotonic dose-response in the mammary gland may reflect complex interactions, where estrogen can act on multiple targets either as an agonist or antagonist.

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.