Bisphenol-A analogs induce lower urinary tract dysfunction in male mice.

Bisphenol-A (BPA), an estrogenic endocrine disrupting chemical, significantly impacts numerous diseases and abnormalities in mammals. Estrogens are known to play an important role in the biology of the prostate; however, little is known about the role of bisphenols in the etiology of prostate pathologies, including benign prostate hyperplasia (BPH) and associated lower urinary tract dysfunction (LUTD). Bisphenol-F (BPF) and bisphenol-S (BPS) are analogs often used as substitutes for BPA; they are both reported to have in vitro and in vivo estrogenic effects similar to or more potent than BPA. The objective of this study was to assess the role of these bisphenols in the development of LUTD in adult male mice. In adult mice exposed to BPA, BPS or BPF, we examined urinary tract histopathology and physiological events associated with urinary dysfunction. Mice treated with bisphenols displayed increased bladder (p < 0.005) and prostate (p < 0.0001) mass, and there was an increased number of prostatic ducts in the prostatic urethra (p < 0.05) and decreased size of the urethra lumen (p < 0.05) compared to negative controls. After two months of bisphenol exposure, mice displayed notable differences in cystometric tracings compared to controls, consistent with LUTD. Treatment of male mice with all bisphenols also induced voiding dysfunction manifested by detrusor instability and histologic changes in the prostatic urethra of male rodents, consistent with LUTD. Our results implicate BPA and its replacements in the development and progression LUTD in mice and provide insights into the development and progression of BPH/LUTS in men.

TRIENNIAL REPRODUCTION SYMPOSIUM: Environmental programming of reproduction during fetal life: Effects of intrauterine position and the endocrine disrupting chemical bisphenol A.

During critical periods in fetal life, there is an increased vulnerability to perturbations in endocrine function due to environmental factors. Small shifts in concentrations of hormones that regulate the differentiation of organs, such as estradiol and testosterone, can have permanent effects on morphology, enzymatic activity, and hormone receptors in tissues as well as neurobehavioral effects. These changes can lead to effects throughout life, including impacting the risk for various diseases (referred to as the Developmental Origins of Adult Health and Disease hypothesis). The intrauterine position phenomenon concerns the consequence for fetuses of randomly implanting next to embryos of the same or opposite sex. An intrauterine position next to males vs. females results in small differences in serum testosterone and estradiol during fetal life that are associated with marked effects on life history (such as lifetime fecundity) in both males and females born in litters (mice, rats, gerbils, rabbits, and swine) as well as human twins. Research with mice subsequently demonstrated that a very small experimental change in fetal serum estradiol levels altered organogenesis and caused permanent changes in organ function. Taken together, these findings led to the hypothesis that environmental chemicals that mimic or antagonize hormone action (e.g., endocrine disrupting chemicals) could also be causing harm at very low exposures (the "low dose" hypothesis) within the range of exposure of humans, domesticated animals, and wildlife. There is now extensive evidence from experimental laboratory animals, sheep, and humans that fetal exposure to very low (presumably safe) doses of the endocrine disrupting chemical bisphenol A (BPA), which exhibits estrogenic activity, can cause permanent changes that can increase the risk of a wide array of diseases. The reasons that federal regulatory agencies are ignoring the massive literature showing adverse effects of BPA and other endocrine disrupting chemicals are discussed.

Endocrine-disrupting chemicals and public health protection: a statement of principles from The Endocrine Society.

An endocrine-disrupting chemical (EDC) is an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action. The potential for deleterious effects of EDC must be considered relative to the regulation of hormone synthesis, secretion, and actions and the variability in regulation of these events across the life cycle. The developmental age at which EDC exposures occur is a critical consideration in understanding their effects. Because endocrine systems exhibit tissue-, cell-, and receptor-specific actions during the life cycle, EDC can produce complex, mosaic effects. This complexity causes difficulty when a static approach to toxicity through endocrine mechanisms driven by rigid guidelines is used to identify EDC and manage risk to human and wildlife populations. We propose that principles taken from fundamental endocrinology be employed to identify EDC and manage their risk to exposed populations. We emphasize the importance of developmental stage and, in particular, the realization that exposure to a presumptive "safe" dose of chemical may impact a life stage when there is normally no endogenous hormone exposure, thereby underscoring the potential for very low-dose EDC exposures to have potent and irreversible effects. Finally, with regard to the current program designed to detect putative EDC, namely, the Endocrine Disruptor Screening Program, we offer recommendations for strengthening this program through the incorporation of basic endocrine principles to promote further understanding of complex EDC effects, especially due to developmental exposures.

Uterine responsiveness to estradiol and DNA methylation are altered by fetal exposure to diethylstilbestrol and methoxychlor in CD-1 mice: effects of low versus high doses.

We examined the effects on female CD-1 mice of fetal exposure to low doses of the drug diethylstilbestrol (DES) (0.1 microg/kg/day) and the insecticide methoxychlor (MXC) (10 microg/kg/day) as well as 1000-fold higher doses: 100 microg/kg/day DES and 10,000 microg/kg/day MXC. Pregnant females were administered these chemicals on gestation days 12-18. At 7-8 months of age, female offspring were ovariectomized and implanted for 7 days with a Silastic capsule containing estradiol. Relative to controls, females exposed to the 0.1 microg DES dose showed significantly heavier uteri, while females exposed to the 100 microg DES dose showed significantly lighter uteri. Females exposed prenatally to the 10 microg/kg dose of MXC had significantly heavier uteri relative to females exposed to the 10,000 microg/kg dose of MXC, but neither group differed significantly from controls. Liver weight for females exposed to both doses of DES was significantly greater than controls. Using a microarray approach to analyze DNA methylation, an increase in ribosomal DNA (rDNA) methylation was observed. Sequence data and Southern analysis indicate an increase in 18S rDNA and 45S pre-rDNA methylation in uterine samples exposed prenatally to low and high doses of DES. We thus found opposite effects of fetal exposure to a low and a high dose of DES on the uterine response to estradiol (inverted-U dose-response relationship). In contrast, there was a monotonic dose-response relationship found for prenatal DES exposure on both liver weight and ribosomal DNA hypermethylation.

Ethological methods to study the effects of maternal exposure to estrogenic endocrine disrupters: a study with methoxychlor.

There has been increasing interest, both at the scientific and regulatory level, in the use of ethological methods for evaluating neural effects of endocrine disrupters. We present a series of ethological studies on the effects of maternal exposure to low, environmentally relevant doses (0.02, 0.2, and 2 microg/g mother bw/day) of the estrogenic pesticide methoxychlor (MXC) on behavior. From gestation day 11 to 17, female mice spontaneously drank oil with or without MXC; their maternal behavior was examined from postpartum days 2 to 15. MXC treatment during pregnancy produced slight changes in the expression of maternal behavior: females fed the lower MXC dose spent less time nursing the pups as compared to control dams. Their maternally exposed offspring were subjected to a series of behavioral tests at different ages. Maternal exposure to MXC affected behavioral responses to novelty in both sexes at periadolescence. The onset of male intrasex aggression was delayed in males prenatally exposed to low doses of MXC, since exposed males showed low levels of aggressive interactions during early adolescence but not after they reached adulthood. When adults, MXC-exposed females, but not males showed increased exploration in an unfamiliar open-field. While a sex difference was observed in the control group, with males being significantly more active in the open field than females, prenatal treatment with some MXC doses tended to decrease the sexual dimorphism in activity levels in the novel environment. Ethology, as the evolutionary study of behavior, may provide a framework for integrating a functional perspective (i.e., evolutionary significance) to studies on proximate mechanisms that can account for behavioral alterations induced by developmental exposure to endocrine disrupters.

Effects of prenatal exposure to low doses of diethylstilbestrol, o,p'DDT, and methoxychlor on postnatal growth and neurobehavioral development in male and female mice.

We examined effects of a wide range of doses of three man-made estrogenic chemicals during fetal life on neurobehavioral changes during early postnatal life in mice. Pregnant mice were fed a 4-log range of o,p'DDT, methoxychlor (MXC), and the drug diethystilbestrol (DES) from gestation days 11 to 17. Offspring were examined for changes in postnatal growth and the development of neuromuscular reflexes. Fetal exposure to the estrogenic chemicals altered the number of live pups per litter, the sex ratio of the litters, the anogenital distance of male and female offspring at birth (a bioassay for fetal androgen action), and the body weight of offspring at birth and during the first 5 days of postnatal life. In most cases, however, the dose-response relationships were complex (non-monotonic), with effects at the highest dose examined being opposite to effects seen at lower doses. The two markers of neurobehavioral development, righting and cliff avoidance reflexes, were not sensitive indicators of prenatal estrogen exposure. Only maternal exposure to the lowest MXC dose produced an increase in reactivity in righting and cliff avoidance tests in offspring.

Altered prostate growth and daily sperm production in male mice exposed prenatally to subclinical doses of 17alpha-ethinyl oestradiol.

Approximately 2 million women in the USA and Europe continue taking oral contraceptives each year during undetected pregnancy due primarily to non-compliance and also to individual variation in sensitivity to hormones in the contraceptives. Prenatal exposure to oral contraceptives containing 17alpha-ethinyl oestradiol (EE) has generally not been associated with an increased incidence of externally observable malformations at birth. The purpose of this study was to assess effects on reproductive organs in adult male mice that had been exposed during gestation day 0 through 17 (equivalent to gestation week 16 in humans) to clinically relevant (approximately 0.5 microg/kg/day) and lower doses of EE. Doses used in this study ranged from 0.002 to 2 microg/kg/day. By 5 months of age, prostate weight was significantly (P < 0.05) higher than controls in most treatment groups of EE (0.02-2 microg/kg). Prostatic androgen receptor populations were significantly elevated only in the 0.02 microg/kg group, suggesting different mechanisms for the increase in prostate weight at different doses. Daily sperm production (DSP) and DSP per gramme of testis were reduced in all treatment groups during adolescence, but not later in adulthood. These findings are consistent with prior studies showing that prenatal exposure of mice to very low doses of a number of oestrogenic chemicals can alter the adult male reproductive system without causing gross external malformations.

Prenatal exposure to low doses of the estrogenic chemicals diethylstilbestrol and o,p'-DDT alters aggressive behavior of male and female house mice.

Exposure to estrogenic chemicals during critical periods in fetal life can alter the development of reproductive organs, the neuroendocrine system, and subsequent behavior. We examined the effects of prenatal exposure to the estrogenic chemicals, o,p'-DDT (the estrogenic contaminant in commercial DDT) and the drug diethystilbestrol (DES), as a positive control, on different forms of aggressive behavior in both male and female house mice. We also examined effects of these chemicals on male reproductive organs. From gestation days 11-17 female mice were fed an average concentration (dissolved in oil) 0.018 and 0.18 ng/g body weight of DES. Doses of o,p'-DDT were 18 and 180 ng/g body weight, based on the prediction that the in vivo potency of o,p'-DDT would be approximately 1000-times lower than DES. We found that prenatal exposure to DES increased the frequency of both males and females that responded aggressively to a same-sex conspecific. Preputial glands in males exposed to the 0.018 ng/g dose of DES were significantly enlarged relative to controls. Males exposed to the 18 ng/g dose of DDT had smaller testes than controls. The possible implications of perturbing the development of social behaviors, such as aggression, on individuals reproductive success and social structure of the population are discussed.

Prenatal exposure to endocrine disrupting chemicals: effects on behavioral development.

Numerous chemicals released into the environment by man are able to disrupt the functioning of the endocrine system by binding to hormonal receptors. Exposure to estrogenic endocrine disruptors during critical periods in fetal life can alter the development of reproductive organs, the neuroendocrine system and subsequent behavior. We present a series of studies on the effects of exposure during fetal life to low, environmentally relevant doses of two pesticides, o,p'DDT and methoxychlor, and of low doses of the synthetic estrogen, diethylstilbestrol on subsequent neuro-behavioral development in house mice. The main findings can be summarized as follows: (1) Mice prenatally exposed to methoxychlor showed changes in reflex development. Exposure to a very low dose of methoxychlor appeared to produce an increased reactivity during early postnatal life. (2) Methoxychlor exposed periadolescent mice showed a decreased reaction time exploring both a novel environment and a novel object. (3) The onset of male intrasex aggression appeared to be delayed in males prenatally exposed to low doses of methoxychlor, since exposed males showed low levels of aggressive interactions during early adolescence but not after they reached adulthood. (4) The rate of depositing urine marks in a novel environment was increased in males prenatally exposed to DES, and also to o,p'DDT and methoxychlor. (5) The proportion of both males and females attacking a same-sex conspecific was increased in mice prenatally exposed to low doses of DES and, marginally, to o,p'DDT. This effect appeared to be related to a decreased latency to attack. However, males prenatally exposed to o,p'DDT displayed a decreased intensity of aggression. The possible implications of perturbing the hormonal milieu during fetal development on the modulation of developmental turnpoints and future behavioral responses are discussed.

Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum.

Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [3H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium divided by the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.

Evaluating the effects of endocrine disruptors on endocrine function during development.

The major concerns with endocrine disruptors in the environment are based mostly on effects that have been observed on the developing embryo and fetus. The focus of the present manuscript is on disruption of three hormonal systems: estrogens, androgens, and thyroid hormones. These three hormonal systems have been well characterized with regard to their roles in normal development, and their actions during development are known to be perturbed by endocrine-disrupting chemicals. During development, organs are especially sensitive to low concentrations of the sex steroids and thyroid hormones. Changes induced by exposure to these hormones during development are often irreversible, in contrast with the reversible changes induced by transient hormone exposure in the adult. Although it is known that there are differences in embryonic/fetal/neonatal versus adult endocrine responses, minimal experimental information is available to aid in characterizing the risk of endocrine disruptors with regard to a number of issues. Issues discussed here include the hypothesis of greater sensitivity of embryos/fetuses to endocrine disruptors, irreversible consequences of exposure before maturation of homeostatic systems and during periods of genetic imprinting, and quantitative information related to the shape of the dose-response curve for specific developmental phenomena.

Prostate gland growth during development is stimulated in both male and female rat fetuses by intrauterine proximity to female fetuses.

In rodents, steroid hormones are transported between adjacent fetuses, and male or female fetuses that develop in utero between female fetuses (2F males or 2F females) have higher serum levels of estradiol and lower serum levels of testosterone relative to siblings of the same sex that develop between two male fetuses (2M males or 2M females). The present study was prompted by the prior unexpected finding that as adults, 2F male mice have an enlarged prostate, and increased numbers of prostatic androgen receptors relative to 2M males. We examined prostate development in both male and female rat fetuses from different intrauterine positions using computer-assisted, 3-dimensional reconstruction of the urogenital complex. In males, this included the prostate, seminal vesicles and utricle (a remnant of the Müllerian ducts), while in females it included development of prostatic glandular buds. The mean cross-sectional area of developing prostatic epithelial buds, utricle and seminal vesicles was significantly increased in 2F male relative to 2M male fetuses. In female fetuses, prostatic bud development was significantly more likely to occur in 2F (67%) than in 2M (29%) animals. These findings suggest that the transport of a small supplement of estrogen from adjacent female fetuses enhances androgen-dependent accessory organ development. We also found that mRNAs encoding receptors for both estrogen and androgen were located in the mesenchyme of the developing male prostate. The localization of estrogen and androgen receptor mRNA in this region further suggests that the mesenchymal induction of prostatic epithelial growth involves both hormones. The cranial dorsolateral prostatic buds exhibited the greatest enlargement in 2F males. This region of the developing prostate in rats is comparable (that is the embryonic homologue) to the region exhibiting benign prostatic hyperplasia (BPH) during aging in men. We propose that the potential for pathological regrowth of the prostate during aging is imprinted by estradiol during fetal development.

Low-dose bioactivity of xenoestrogens in animals: fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice.

The hormonal activity of natural estrogens is influenced by the degree to which they bind to serum proteins. In the pregnant female and in the fetus, greater than 99% of estradiol may be bound by serum binding proteins. Therefore, even though total serum levels of estradiol appear very high in fetuses, we have found that in rodent fetuses, there is a very low free concentration of estradiol (0.2 pg/ml). Naturally occurring variation in fetal serum estradiol predicts differences in numerous postnatal traits, including prostate size. In addition, when this low level of free estradiol was experimentally increased from 0.2 to 0.3 pg/ml during the last third of fetal life, treated male mice showed an increase in adult prostate weight. Fetal exposure to low doses of xenobiotic estrogens by feeding to pregnant females, including the compounds methoxychlor (20 and 2000 micrograms/kg body weight), DES (0.02 to 2 micrograms/kg body weight) and bisphenol A (2 and 20 micrograms/kg body weight), also led to increased prostate weight in adulthood. In contrast, fetal doses of natural estradiol and DES above the physiological range of estrogenic activity, and within a toxicological dose range, led to the opposite outcome, a reduction in subsequent adult prostate weight. This indicates that it may be impossible to assess endocrine-disrupting activities in response to low doses within a physiological range of activity by using high, toxic doses of xenoestrogens in testing procedures. We have developed approaches in vitro to predict the potential estrogenic bioactivity of compounds in the physiologically relevant range in animals and humans. We address the following factors in predicting the final observed endocrine-disrupting effect in the animal: (1) the intrinsic estrogenic activity of a given molecule, (2) the effective free concentration determined by how the molecule is carried in serum, (3) partitioning between aqueous and lipid compartments in body and cell lipids, and (4) absorption and metabolism relative to the route of exposure. The studies and strategies we describe are important in developing criteria for a tiered testing system for the detection of estrogenic chemicals as well as endocrine-disrupting chemicals with different modes of action.

The effective free fraction of estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity.

The biological activity of natural estrogens is influenced by the degree to which they bind to serum proteins. To determine directly how serum affected the uptake of estradiol, we compared the whole cell uptake of [3H]estradiol in intact MCF-7 human breast cancer cells from serum-free medium with the uptake from 100% serum from adult men. In estrogen receptor saturation assays, 28.9 times more estradiol was required in serum to occupy the same number of estrogen receptors as was required in serum-free medium (SFM), suggesting that the effective free fraction of estradiol in adult male serum was 3.46% (1/28.9). Since most xenoestrogens are not available in tritium-labeled form, the cell uptake of unlabeled xenoestrogens could not be measured directly with saturation analysis. Therefore, we developed the relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of nonradioactive xenoestrogens to estrogen receptors within intact MCF-7 cells. Serum modified access (SMA) was calculated by dividing the relative binding affinity (RBA, relative to estradiol) measured in 100% serum, by the RBA measured in serum-free medium. An SMA > 1 indicated that the xenoestrogen had greater access to estrogen receptors than estradiol from serum. In contrast, an SMA < 1 indicated that the xenoestrogen had less access to estrogen receptors from serum than did estradiol. The synthetic estrogen diethylstilbestrol (DES) binds poorly to sex hormone binding globulin (SHBG), and DES showed enhanced access in serum, SMA = 6.2. Additional calculations through the Ki (inhibition constant) indicated that this corresponded to an effective free fraction of 26.9% for DES in serum. The phytoestrogens, coumestrol, genistein, and equol, showed substantial enhanced access in serum, over 10-fold relative to estradiol (SMA = 12.1, 10.3, and 11.3, respectively), and effective free fractions in serum of 47.8, 45.8, and 49.7%, respectively. Since most in vitro assays of xenoestrogens do not address how serum influences their bioactivity, the estrogenic activity of these phytoestrogens would be underestimated. Conversely, biochanin A showed decreased access from serum (SMA = 0.44) and had an effective free fraction of 2.4%; its estrogenic activity would be overestimated in serum-free assays.

A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior.

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 micrograms; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.