Bidirectional placental transfer of Bisphenol A and its main metabolite,Bisphenol A-Glucuronide,in the isolated perfused human placenta

The widespread human exposure to Bisphenol A (BPA), an endocrine disruptor interfering with developmental processes, raises the question of the risk for human health of BPA fetal exposure. In humans, highly variable BPA concentrations have been reported in the feto-placental compartment. However the human fetal exposure to BPA still remains unclear. The aim of the study was to characterize placental exchanges of BPA and its main metabolite, Bisphenol A-Glucuronide (BPA-G) using the non-recirculating dual human placental perfusion. This high placental bidirectional permeability to the lipid soluble BPA strongly suggests a transport by passive diffusion in both materno-to-fetal and feto-to-maternal direction, leading to a calculated ratio between fetal and maternal free BPA concentrations of about 1. In contrast, BPA-G has limited placental permeability, particularly in the materno-to-fetal direction. Thus the fetal exposure to BPA conjugates could be explained mainly by its limited capacity to extrude BPA-G.     

Effects of bisphenol A on breast cancer and its risk factors

The incidence of breast cancer in Korea has been increasing for the last two decades (1983–2005), and now, breast cancer is ranked the leading cause of cancer in Korean women. Along with other endocrine disrupting chemicals (EDCs), bisphenol A (BPA) has been suspected as a potential risk factor for breast cancer. We studied potential associations between BPA exposure and breast cancer risks in Korean women by performing biomonitoring of BPA among breast cancer patients and controls (N = 167). Blood samples were collected between 1994 and 1997 and kept over 10 years in a freezer under well controlled conditions. The blood BPA levels determined by HPLC/FD, ranged between LOD (0.012 μg/L) and 13.87 μg/L (mean ± SD, 1.69 ± 2.57 μg/L; median, 0.043 μg/L). In age-matched subjects (N = 152), there were some associations between BPA levels and risks of breast cancer, such as age at first birth and null parity. However, there were no significant differences in blood BPA levels between the cases and the controls (P = 0.42). Considering interactions between BPA exposure and risks of breast cancer, we suggest further enlarged biomonitoring studies of BPA to provide effective prevention against breast cancer.     

Fetal bisphenol A and ethinylestradiol exposure alters male rat urogenital tract morphology at birth: Confirmation of prior low-dose findings in CLARITY-BPA

Bisphenol A (BPA) is a contaminant in virtually all Americans. To examine BPA’s adverse effects, the FDA-NCTR, NIEHS, and 14 groups of academic scientists formed a consortium: CLARITY-BPA. The purpose of our study was to investigate the effects of a wide range of doses of BPA on fetal development of the NCTR CD-SD male rat urogenital sinus (UGS). Pregnant rats were administered BPA or positive control ethinylestradiol (EE2) daily, via oral gavage, from gestational day 6 through parturition. Tissues were collected on postnatal day 1 and the UGS was analyzed using computer-assisted 3-D reconstruction. Importantly, only low doses of BPA, as well as EE2, significantly changed birth weight and UGS morphology, including an increased size of the colliculus and decreased size of the urethra, consistent with prior reported BPA and EE2 effects. Our findings provide further evidence that BPA mediates nonmonotonic developmental effects on the fetal urogenital sinus.     

Gestational and lactational xenoestrogen exposure disrupts morphology and inflammatory aspects in mammary gland of gerbil mothers during involution

In the mammary gland (MG), the developmental window for gestational/lactational differentiation and growth is highly vulnerable to hormonal disruption. Here we describe that the MG involution process in female gerbil mothers is delayed by bisphenol A (BPA) exposure during gestation and lactation. The process is directly influenced by changes in expression of extracellular matrix proteases MMP-2, MMP-9, and FAP, and the incidence of collagen and elastin is reduced after 7 and 14 days of weaning. A pro-inflammatory environment in the late involution process was confirmed by higher expression of TNF-α, COX-2 and phospho-STAT3 n the MG stroma, allied to increases in the incidence of macrophages and mast cells. These aspects impacted the proliferative pattern of epithelial cells, which decreased on the 14th post-weaning day. These data confirm that the milk production window of susceptibility is vulnerable to the impact of BPA, which promotes a suggestive pro-tumoral microenvironment during mammary involution.     

A new approach to synergize academic and guideline-compliant research: The CLARITY-BPA research program

Recently, medical research has seen a strong push toward translational research, or “bench to bedside” collaborations, that strive to enhance the utility of laboratory science for improving medical treatment. The success of that paradigm supports the potential application of the process to other fields, such as risk assessment. Close collaboration among academic, government, and industry scientists may enhance the translation of scientific findings to regulatory decision making. The National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), and U.S. Food and Drug Administration (FDA) developed a consortium-based research program to link more effectively academic and guideline-compliant research. An initial proof-of-concept collaboration, the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), uses bisphenol A (BPA) as a test chemical. The CLARITY-BPA program combines a core perinatal guideline-compliant 2-year chronic toxicity study with mechanistic studies/endpoints conducted by academic investigators. Twelve extramural grantees were selected by NIEHS through an RFA-based initiative to participate in the overall study design and conduct disease-relevant investigations using tissues and animals from the core study. While the study is expected to contribute to our understanding of potential effects of BPA, it also has ramifications beyond this specific focus. Through CLARITY-BPA, NIEHS has established an unprecedented level of collaboration among extramural grantees and regulatory researchers. By drawing upon the strengths of academic and regulatory expertise and research approaches, CLARITY-BPA represents a potential new model for filling knowledge gaps, enhancing quality control, informing chemical risk assessment, and identifying new methods or endpoints for regulatory hazard assessments.     

Triclosan exacerbates the presence of C-bisphenol A in tissues of female and male mice

Current human generations are commonly exposed to both triclosan (TCS), an antimicrobial agent, and bisphenol A (BPA), the monomer of polycarbonate plastics and epoxies. Both are readily absorbed into circulation and found distributed among diverse tissues. Potential interactions between TCS and BPA are largely unstudied. We investigated whether TCS exposure affects the distribution of ingested ¹⁴C-BPA in select tissues. CF-1 mice were each subcutaneously injected with TCS then orally administered 50 μg/kg ¹⁴C-BPA. Females received 0, 0.2, 0.6, 1, 2, or 18 mg TCS (equivalent respectively to 0, 6.3, 16.9, 30.1, 60.5, and 558.9 mg/kg). Males received 0, 0.2, 2, or 18 mg TCS (equivalent respectively to 0, 5.3, 53.4, and 415.0 mg/kg). Levels of radioactivity were measured through liquid scintillation counting in blood serum and brain, reproductive, and other tissues. Significantly elevated levels of radioactivity were observed following combined TCS and ¹⁴C-BPA administration, with minimally effective TCS doses being tissue-dependent (Females: lungs, 0.6 mg; uterus, 1 mg; heart, muscle, ovaries, and serum, 18 mg. Males: serum, 0.2 mg; epididymides, 2 mg). Subsequently, we found that 2 or 6 mg TCS increased radioactivity in the ovaries and serum of females orally given only 5 μg/kg ¹⁴C-BPA. These data indicate that TCS can interact with BPA in vivo, magnifying its presence in certain tissues and serum. The data are consistent with evidence that TCS utilizes enzymes that are critical for metabolism and excretion of BPA. Further research should investigate the mechanisms through which these two chemicals interact at environmentally-relevant doses.     

CLARITY‐BPA academic laboratory studies identify consistent low‐dose Bisphenol A effects on multiple organ systems

Bisphenol A (BPA) is a high‐production chemical used in a variety of applications worldwide. While BPA has been documented as an endocrine‐disrupting chemical (EDC) having adverse health‐related outcomes in multiple studies, risk assessment for BPA has lagged due to reliance on guideline toxicology studies over academic ones with end‐points considered more sensitive and appropriate. To address current controversies on BPA safety, the United States National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP) and the Food and Drug Administration (FDA) established the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY‐BPA) using the NCTR Sprague‐Dawley rats. The goal of CLARITY‐BPA is to perform a traditional regulatory toxicology study (Core study) in conjunction with multiple behavioural, molecular and cellular studies by academic laboratories focused on previously identified BPA‐sensitive organ systems (Academic studies). Combined analysis of the data from both study types will be undertaken by the NTP with the aim of resolving uncertainties on BPA toxicity. To date, the Core study has been completed and a draft report released. Most of the academic studies have also been finalized and published in peer‐reviewed journals. In light of this important milestone, the PPTOX‐VI meeting held in the Faroe Islands, 27‐30 May 2018 devoted a plenary session to CLARITY‐BPA with presentations by multiple investigators with the purpose of highlighting key outcome. This MiniReview synthesizes the results of three academic studies presented at this plenary session, evaluates recently published findings by other CLARITY‐BPA academic studies to provide an early combined overview of this emerging data and places this in the context of the Core study findings. This co‐ordinated effort revealed a plethora of significant BPA effects across multiple organ systems and BPA doses with non‐monotonic responses across the dose range utilized. Remarkably consistent across most studies, including the Core study, are low‐dose effects (2.5, 25 and 250 μg BPA/kg body‐weight). Collectively, the findings highlighted herein corroborate a significant body of evidence that documents adverse effects of BPA at doses relevant to human exposures and emphasizes the need for updated risk assessment analysis.     

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%). The K _m values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The V _max for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA. Received: 26 January 1998 / Accepted: 26 February 1998     

Detection of endocrine-modulating effects of the antithyroid acting drug 6-propyl-2-thiouracil in rats, based on the "Enhanced OECD Test Guideline 407"

The antithyroid acting drug propylthiouracil (PTU) was administered to male and female Wistar rats at 0, 0.1, 1, or 10mg/kg body weight for 4 weeks according to the draft protocol of the "Enhanced OECD Test Guideline 407" (enhanced TG 407) in order to investigate its suitability to detect endocrine-mediated effects. The study was conducted with two identical subsets of five animals per sex and dose each to provide data on sensitivity. The modified protocol includes the investigation of additional organ weights, pathology, and histopathology, of thyroid hormones, of spermatozoa, and of estrus cycle. At time of sacrifice, all females were in the diestrus stage as prescribed. Adverse effects were observed in the thyroid gland (hypertrophy/ hyperplasia) and the pituitary gland (hyperplasia of basophilic cells, hypoplasia of acidophilic cells) together with dose-related decreased serum triiodothyronine (T3) and thyroxine (T4) levels and increased thyroid stimulating hormone (TSH) levels. Other effects of PTU included decrease of organ weights, anaemia, impaired blood coagulation, and reduced activity of enzymes. Hence, some of the additional examined endpoints of the enhanced TG 407, e.g., examination of pituitary gland and thyroid hormones, were suitable to detect endocrine-modulating effects of propylthiouracil. Treatment of five animals provides sufficient sensitivity to detect the described adverse effects of propylthiouracil. The enhanced TG is currently under investigation in several laboratories, evaluation of all the results will allow determining its practicability as well as the most suitable additional endpoints.     

Fetal and neonatal exposure to three typical environmental chemicals with different mechanisms of action: Mixed exposure to phenol,phthalate, and dioxin cancels the effects of sole exposure on mouse midbrain dopaminergic nuclei

A major question is whether exposure to mixtures of low-dose endocrine disruptors (EDs) having different action mechanisms affects neurodevelopment differently than exposure to EDs individually. We therefore investigated the effects of fetal and neonatal exposure to three typical EDs – bisphenol A (BPA), di-(2-ethylhexyl)-phthalate (DEHP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) – on the midbrain dopaminergic system associated with functions – including motor activity, emotion, and cognition – affected by neuropsychiatric diseases such as attention-deficit/hyperactivity disorder. ICR mouse dams and their pups were orally treated with BPA (5 mg/(kg day)), DEHP (1 mg/(kg day)), or TCDD (8 ng/kg) individually, or with mixtures thereof, to compare the effects between sole and mixed administration. We analyzed tyrosine hydroxylase (TH)- and Fos-immunoreactive (ir) neurons as markers of dopamine and neuronal activation, respectively. The numbers of TH- and/or Fos-ir neurons and the intensity of TH-immunoreactivity within midbrain dopaminergic nuclei (A9, A10, and A8) of each sole administration group significantly differed from controls at 2, 4, and 6 weeks of age. In contrast, no significant differences were detected in the mixture groups, suggesting counteractions among those chemicals. These results indicate that ED mixtures as pollution have unique and elusive effects. Thyroid hormones and/or aryl hydrocarbon receptor-related mechanisms may be responsible for this counteraction.     

Endocrine-mediated effects of two benzene related compounds, 1-chloro-4-(chloromethyl)benzene and 1,3-diethyl benzene,based on subacute oral toxicity studies using rats

The purpose of this study was to investigate the endocrine-mediated effects of the benzene-related compounds with reference to Organization for Economic Co-operation and Development (OECD) Test Guideline No. 407. Rats were orally gavaged with 0, 10, 50, and 250 mg/kg/day of 1-chloro-4-(chloromethyl)benzene, and 0, 25, 150, and 1000 mg/kg/day of 1,3-diethyl benzene for at least 28 days, beginning at 8 weeks of age. Thyroid dysfunction was observed in rats given the 1,3-diethyl benzene. Serum T4 values increased in all groups of male rats and in the 1000 mg/kg group of female rats, and TSH values also increased in the 1000 mg/kg groups of both sexes after 28 days’ administration. Decreased T3 values were observed in the 1000 mg/kg group of female rats after 28 days’ administration, and hormone values increased in the 1000 mg/kg groups of both sexes after the 14-day recovery period. In addition, thyroid weight increased in the 1000 mg/kg groups and thyroid follicular cell hyperplasia was detected in one male rat from the 1000 mg/kg group after 28 days’ administration. Endocrine-mediated effects, including thyroid dysfunction were not observed in any groups of rats treated with 1-chloro-4-(chloromethyl)benzene. Our results indicated that endocrine-mediated effects such as thyroid dysfunction were associated with some benzene-related compounds.     

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.     

Comparison of endocrine-mediated effects of two bisphenol A related compounds, 2,2-bis(4-cyanatophyenyl)propane and 4,4'-cyclohexylidenebisphenol, based on subacute oral toxicity studies using rats

The purpose of this study was to compare endocrine-mediated effects of bisphenol A related compounds, 2,2-bis(4-cyanatophyenyl)propane and 4,4′-cyclohexylidenebisphenol with reference to OECD Test Guideline No. 407. Rats were orally gavaged with 0, 4, 20, and 100 mg/kg/day of 2,2-bis(4-cyanatophyenyl)propane, and 0, 30, 100, and 300 mg/kg/day of 4,4′-cyclohexylidenebisphenol for at least 28 days beginning at 8 weeks of age. Endocrine-mediated effects were not observed in rats given 2,2-bis(4-cyanatophyenyl)propane. Male accessory sex organ weights decreased in the 4,4′-cyclohexylidenebisphenol 300 mg/kg group and serum T4 values increased in all male groups treated with this compound. Our results suggest that endocrine-mediated changes caused by the present bisphenol related compound can be divided into estrogenic or thyroid hormonal effects, and estrogenic effects observed in the repeated-dose study were related to their estrogenic potency confirmed by uterotrophic assay.     

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL).α-ZOL exhibited the strongest estrogenic potency (EC₅₀ 0.022 ± 0.001 nM), slightly less potent than 17-β estradiol (EC₅₀ 0.015 ± 0.002 nM). ZEN was ∼70 times less potent than α-ZOL and twice as potent as β-ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, α-ZOL or β-ZOL.ZEN, α-ZOL or β-ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 μM. At 100 μM, cell viability decreased and levels of hormones were significantly reduced except for progesterone. β-ZOL increased estradiol concentrations more than α-ZOL or ZEN, with a maximum effect at 10 μM, with β-ZOL (562 ± 59 pg/ml) > α-ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.     

A broad-spectrum organophosphate pesticide O,O-dimethyl O-4-nitrophenyl phosphorothioate (methyl parathion) adversely affects the structure and function of male accessory reproductive organs in the rat

Methyl parathion (MP) is an organophosphate pesticide used in agriculture, but also illegally used to spray homes and businesses to control insects. The present study was designed to investigate adverse effects of MP on accessory reproductive organs. Male Wistar rats aged 13–14 weeks were treated and sacrificed as follows. Experiment 1: 0.0 (water vehicle), 1.75, 3.5 or 7 mg/kg (i.p.) for 5 days and sacrificed on day 14; experiment 2: 0.0, 0.5 or 1 mg/kg (i.p.) for 12 days and sacrificed on day 130; experiment 3: 0.0, 0.5 or 1 mg/kg (i.p.) for 12 days and sacrificed on day 77; experiment 4: 0.0, 0.75 or 1.5 mg/kg (i.p.) for 25 days and sacrificed on day 17 and experiment 5: 0.0 or 3.5 mg/kg (p.o.) for 25 days and sacrificed on day 17, after the last exposure. The accessory reproductive organs were removed, weighed and processed for histopathological analysis. Structural qualitative changes such as epithelial cell morphology and luminal observations were carried out for each organ in all experiments. Epididymis of one side was homogenized and biochemical estimations of acid phosphatase (ACP), cholesterol, total protein, uric acid, and Vitamin C were conducted by calorimetric methods in experiments 4 and 5. In experiment 1 the organ weights did not change; in experiment 2, the epididymal weight increased (P < 0.001); in experiment 3, the weights of ductus deferens decreased at 1 mg/kg and that of seminal vesicle decreased at both dose-levels (P < 0.001). In experiments 4 and 5, weights of epididymis and prostate decreased, whereas in experiment 5, weights of ductus deferens and seminal vesicle increased (P < 0.05–0.001). The sperm density was normal in control, moderately decreased in experiment 1 at 3.5 and 7 mg/kg; in experiment 2 at 1 mg/kg, and in experiment 5 at 3.5 mg/kg, and severely decreased in experiment 3 at 1 mg/kg and in experiment 4 at both dose-levels. The epithelial necrosis and nuclear pyknosis were seen in experiments 1, 3, 4 and 5, whereas nuclear degeneration was seen in experiment 1 and 4 and germ cells in the lumina of epididymis were seen in experiment 4. The nuclear pyknosis in the ductus deferens was seen in all experiments, except at 1.75 mg/kg in experiment 1 and at 0.5 mg/kg in experiment 3. Brush border disruption in the ductus deferens was seen in experiments 1 and 4; sperms were seen in the lumen in experiment 1 at 7 mg/kg, and in experiments 4 and 5. The vacuoles in the epithelium were seen in experiments 1 and 4 and immature germ cells were seen in the lumen in experiment 4. The ACP and Vitamin C levels decreased in experiment 4 at both dose-levels, and in experiment 5 all biochemical parameters tested found decreased (P < 0.01–0.001). The present results indicate that MP affects the structure and function of accessory reproductive organs in the rat.     

Lack of maternal dietary exposure effects of bisphenol A and nonylphenol during the critical period for brain sexual differentiation on the reproductive/endocrine systems in later life

Two potential endocrine-disrupting chemicals, bisphenol A (BPA) and nonylphenol (NP), were assessed for their long-lasting effects on endocrine/reproductive systems following transplacental and lactational exposure to rat offspring during a time-window that included the critical period for brain sexual differentiation. Each chemical was mixed with diet at concentrations of 60, 600 and 3000 ppm and was provided to maternal Sprague-Dawley rats from gestational day (GD) 15 to postnatal day (PND) 10. Ethinylestradiol (EE) at 0.5 ppm was used as an estrogenic reference drug. During pregnancy and lactation, including the exposure period, a soy-free rodent diet was provided to eliminate possible modification of the study results by plant-derived phytoestrogens. Effects on endocrine/reproductive systems were evaluated by examining the anogenital distance, organ weights before puberty, onset of puberty, estrous cyclicity, and organ weights and histopathology of adult endocrine organs (at 11 weeks of age), as well as the volume of the sexually dimorphic nucleus of preoptic area. Both NP and BPA, at high doses, caused decreases in maternal body weights and retardation of offspring growth, but neither affected any of the endocrine/reproductive endpoints of offspring, whereas EE induced irreversible changes in estrous cyclicity and histopathology of ovaries and uterus of adult females. The results indicated that maternal dietary exposure to NP or BPA at concentrations up to 3000 ppm from GD 15 through PND 10 do not exert any apparent adverse effects on the endocrine/reproductive systems of offspring.     

Urinary bisphenol A and semen quality,the LIFE Study

Bisphenol A (BPA), a high-production volume industrial chemical found in several consumer products, has been negatively associated with sperm quality. This study aimed to estimate the association between BPA and 35 measures of semen quality among reproductive aged men recruited from 16 counties in Michigan and Texas, 2005–2009. Of 501 enrolled males, 418 (83.4%) provided a urine sample and at least one semen sample. Linear and logistic regression models assessed the association between urinary BPA levels and individual semen quality endpoints. Generalized estimating equations were used to account for repeated measures of semen quality and adjusted models accounted for 11 a priori covariates. Geometric mean total urinary BPA concentration among participants was 0.55 ng/mL (95% CI 0.49–0.63). A negative relation between BPA and DNA fragmentation was the sole significant finding in adjusted linear regression (β = −0.0544, p = 0.035) and suggestive of less sperm DNA damage.