Beverly S. Rubin

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.

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.

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.

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.

Long-Term Effects of Fetal Exposure to Low Doses of the Xenoestrogen Bisphenol-A in the Female Mouse Genital Tract

Abstract Developmental exposure to estrogenic chemicals induces morphological, functional, and behavioral anomalies associated with reproduction. Humans are routinely exposed to bisphenol-A (BPA), an estrogenic compound that leaches from dental materials and plastic food and beverage containers. The aim of the present study was to determine the effects of in utero exposure to low, environmentally relevant doses of BPA on the development of female reproductive tissues in CD-1 mice. In previous publications, we have shown that this treatment alters the morphology of the mammary gland and affects estrous cyclicity. Here we report that in utero exposure to 25 and 250 ng BPA/ kg of body weight per day via osmotic pumps implanted into pregnant dams at Gestational Day 9 induces alterations in the genital tract of female offspring that are revealed during adulthood. They include decreased wet weight of the vagina, decreased volume of the endometrial lamina propria, increased incorporation of bromodeoxyuridine into the DNA of endometrial gland epithelial cells, and increased expression of estrogen receptor-α (ERα) and progesterone receptor in the luminal epithelium of the endometrium and subepithelial stroma. Because ERα is known to be expressed in these estrogen-target organs at the time of BPA exposure, it is plausible that BPA may directly affect the expression of ER-controlled genes involved in the morphogenesis of these organs. In addition, BPA-induced alterations that specifically affect hypothalamic-pituitary-gonadal axis function may further contribute to the anomalies observed at 3 mo of age, long after the cessation of BPA exposure.

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.

Induction of estrous behavior in ovariectomized rats by sequential replacement of estrogen and progesterone to the ventromedial hypothalamus.

The present experiment was designed to determine whether sequential replacement of estrogen and progesterone to the ventromedial hypothalamus (VMH) would be sufficient to induce estrous behavior in ovariectomized rats. Bilateral cannulae containing 17 beta-estradiol (E2) diluted with cholesterol (1:250) were lowered into the VMH, preoptic area or midbrain and left in place for 4 days. On day 5, the E2 inserts were removed and P-filled cannulae were lowered into half of the subjects. The remaining females received systemic progesterone (500 micrograms). This steroid regimen was repeated 2 weeks later with the mode of progesterone administration reversed. All subjects were tested for estrous behavior twice after progesterone treatment. In a second experiment, 3H-P:P-filled cannulae were lowered into the VMH of estrogen-primed females in order to estimate the extent of hormone spread from full-strength P-filled cannulae. Results indicated that estrogen and progesterone stimulation of the VMH is sufficient to activate estrous behavior in spayed female rats, however, precise localization of the hormone implants within the VMH is essential. 9 of the 11 females with both cannulae located within or at the border of the ventromedial nucleus (VMN) exhibited estrous behavior whereas only half of the females with only one implant resting in the VMN exhibited estrous responsiveness. Subjects with neither cannula located within or at the border of the VMN did not exhibit the behavior. The facilitative effects of P appeared to result from hormonal stimulation of the VMH and not from leakage of the steroid into other brain regions or into the systemic circulation. Following placement of tritiated progesterone implants into the VMH, high levels of radioactivity were recovered only from the mediobasal hypothalamus. The low levels of radioactivity measured in other brain regions, pituitary, uterus and blood indicate that relatively little if any hormone reached these tissues.

EXAMINATION OF GUINEA PIG LUTEINIZING HORMONE-RELEASING HORMONE GENE REVEALS A UNIQUE DECAPEPTIDE AND EXISTENCE OF TWO TRANSCRIPTS IN THE BRAIN

We sequenced the complementary DNA (cDNA) encoding guinea pig LHRH from an expression library derived from the preoptic area-anterior hypothalamus. Data from in situ hybridization and RNase protection assays verified that the cloned cDNA was complementary to guinea pig LHRH messenger RNA. The architecture of the deduced precursor resembles that of LHRH precursors identified in other species. In contrast, the predicted sequence of the decapeptide differs from mammalian LHRH by two amino acid substitutions in positions 2 and 7. This is a novel finding, because the amino acid sequence that comprises LHRH decapeptide is identical in all mammals studied to date. Moreover, the predicted substitution in amino acid position 2 is unique among vertebrates. A second observation of potential significance is the existence of two subspecies of LHRH messenger RNA differing only in the length of their 3′ untranslated regions. These two transcripts were verified by sequence analysis of positive clones from the cDNA librar...

Corticotropin releasing hormone mRNA is elevated on the afternoon of proestrus in the parvocellular paraventricular nuclei of the female rat

We investigated changes during the estrous cycle in cellular levels of corticotropin-releasing hormone (CRH) mRNA in parvocellular neurons of the hypothalamic paraventricular nucleus, using in situ hybridization. Intact female rats with 4 day cycles were sacrificed at 11 different times during the cycle at 09.00 h and 16.00 h on each day, with additional collection times at 14.00 h, 18.00 h, and 20.00 h on the day of proestrus. Twelve microns coronal sections of fresh-frozen brains were made through the paraventricular nuclei (PVN) and placed on gelatin-coated slides. A 48 base oligodeoxynucleotide probe complementary to the coding region for rat CRH was used to measure CRH mRNA. There was a sharp increase (P less than 0.01) in CRH mRNA in the ventral PVN between 14.00 and 16.00 h on the day of proestrus, at the approximate time of the ovulatory surge. Following this rise, there was an even larger decline (P less than 0.01) between P 16.00 h and P 20.00 h. Levels of CRH mRNA did not change greatly on other days of the cycle, nor were there significant changes in the dorsal PVN. Given the known effects of CRH on GnRH secretion, these changes occur at a time when they could serve to modulate the midcycle luteinizing hormone (LH) surge.

Low dose effects of bisphenol A

In 2007, a group of experts critically analyzed hundreds of publications on bisphenol A (BPA), including the evidence for low dose effects. Here, we have updated these evaluations to determine the strength of the evidence for low dose effects of BPA. Based on the cut-offs for “low doses” established previously (i.e., the lowest observed adverse effect level [LOAEL], or 50 mg/kg/day for mammalian studies), we identified more than 450 low dose studies. Using an integrative approach, we examined five endpoints in depth that had evidence from two or more study types (in vitro, in vivo laboratory animal, and human). Based on all available studies, we are confident that consistent, reproducible, low dose effects have been demonstrated for BPA. We conclude that the doses that reliably produce effects in animals are 1–4 magnitudes of order lower than the current LOAEL of 50 mg/kg/day and many should be considered adverse.