Effects of pre- and post-natal exposure to bisphenol A on the stress system

ABSTRACT

Evidence is accumulating that developmental exposure to Bisphenol A (BPA), an abundant endocrine disrupting chemical (EDC), affects neurodevelopment and later brain function, including cognitive performance, social and affective behaviors. However, an impact of BPA exposure on the stress system regulators has only recently come into focus. This mini-review aims to summarize and discuss the available findings from animal studies regarding BPA effects on the stress system and the hypothalamic-pituitary- adrenal (HPA) axis in particular. It appears that perinatal exposure of rodents to low doses of BPA, around the human tolerable daily intake, can modify the levels, expression and epigenetic patterning of key stress mediators at later-life stages in a sex-specific way. These observations provide evidence that developmental exposure to one of the most highly produced chemicals today may contribute to the regulation of the stress axis. Given the importance of the HPA axis in coordination of the organisms' global homeostasis and its implications in stress-related disorders, further studies are needed to delineate the underling mechanisms of these BPA actions.

KEYWORDS:

• anxiety
• Bisphenol A
• DNA methylation
• estrogen receptors
• epigenetics
• Fkbp5
• glucocorticoids
• gonadal hormones
• HPA axis
• stress

Introduction

Increasing evidence shows that perinatal exposure to endocrine disrupting chemicals (EDC), such as Bisphenol A (BPA), is particularly harmful for brain function as it induces long-lasting alterations in sex hormone receptors function, neurotransmitter signaling and related behaviors. However, little is known about the effects of EDCs on the stress system, and the few initial studies refer to lead effects in children¹ or to effects of nonylphenol, phthalates² and recently of BPA³ in aquatic organisms. With respect to BPA, initial indirect indications for its impact on the stress response system of mammals derived from studies reporting changes in the anxiety state of pubertal or adult rats and mice exposed perinatally to the agent (these studies are summarized below). Recent observations in children also associate urinary BPA concentrations of either the children or their pregnant mothers with increased anxiety and other behavioral aberrations in childhood (for a systematic review see Ejaredar et al.⁴). However, direct indications for the effects of BPA exposure on the hypothalamic-pituitary-adrenal (HPA) axis of rats appeared only recently.^5-10 These observations denote that perinatal exposure to this abundant EDC impacts on several components of the HPA axis that could modify stress responsiveness. Contemporary data suggest that the exposure window is critical for the detected BPA effects on the stress system.^11,12 Due to the ample exposure of organisms to BPA and the importance of the HPA axis in the maintenance of organisms' homeostasis, their possible interactions are worth further attention.

The scope of this mini-review is to present and discuss the available experimental data on this theme. The focus was set to the impact of BPA exposure on the HPA axis regulators. Accordingly, the literature search was bounded to recent studies that, in addition to behavioral observations, had examined components of the limbic-HPA axis involved in the stress response. The numerous studies reporting alterations in anxiety levels following BPA exposure are beyond the scope of the present mini-review and are not fully presented herein. Reviewing the relevant literature provided 8 studies (Table 1). All studies were conducted in rats, however their protocols differ in terms of the daily dose (2 or 40 μg BPA/Kg bw), the administration route (oral vs. injected), exposure windows (perinatal/long-term vs. pubertal/short-term) and time of outcome evaluation (puberty vs. adulthood). Some of the above parameters may significantly influence the detected effects and will be considered in the presentation of the results.

Table 1. Effects of BPA in the stress axis of rats.

Exposure protocol and offspring's age at evaluation	Effect on stress axis-related components per sex (f: female; m: male)	Effect on stress-related behavior	Ref.
40μg BPA/Kg/dG1-PND22Orally via food Puberty(PND46)	Increased plasma CORT (f)	Reduced exploration (f, m), mostly in females	Poimenova et al 2010
40μg BPA/Kg/dG1-PND22Orally via food Puberty(PND46)	Increased adrenal weight (f).Hyperplastic adrenal zona fasciculata (f, m).Underdeveloped zona reticularis (m).Increased basal CORT (f). Impaired increase following stress (lower in f; enhanced in m).Reduced basal GR in hippocampus and hypothalamus (f), no change post-stress.Increased pituitary CRHR1 and POMC mRNA (m).	Increased anxiety (f)	Panagiotidou et al 2014
2μg BPA/Kg/dG10-PND7Subcutaneous Injections Adulthood(PND80)	Increased basal CORT, ACTH and CRH (m).Decreased basal hippocampal GR mRNA (m).Increased basal GR mRNA in PVN and hippocampus (f).Increased hippocampal MR, nNOS, p-CREB (f), but decreased in (m).Decreased plasma testosterone and hypothalamic ERβ, increased ERα (m).	Reduced anxiety (f)Increased anxiety (m)Depression-like behavior (m)	Chen et al 2014
40μg BPA/Kg/dPuberty (PND42-49)Subcutaneous Injections(PND 49-77)	No impact on CORT levels following stress (m, f)		Bowman et al 2014
40μg BPA/Kg/dG1-PND22Orally via food Puberty(PND46)	Increased basal CORT and ACTH at circadian peak and nadir (f).Reduced basal GR mRNA in hippocampus (f).Impaired Dexamethasone suppression (f).	Increased anxiety (f)	Zhou et al 2015
40μg BPA/Kg/dG1-PND22Orally via food Puberty(PND46)	Increased DNA methylation in intron 5 of FKBP5 and reduced protein levels in the hippocampus at basal conditions (m).		Kitraki et al 2015
2μg BPA/Kg/dG10-PND7Orally via food Adulthood(PND80)	Increased basal CORT, ACTH and CRH (m).Decreased basal hippocampal GR mRNA (m).Increased hippocampal MR, nNOS, p-CREB (m). Decreased plasma testosterone (m).		Chen et al 2015
40μg BPA/Kg/dPuberty(PND42-49)Subcutaneous InjectionsAdulthood (PND91)	No impact on CORT levels following stress (m, f)	No BPA effects in anxiety (m, f)Reduced exploration (f, m)	Bowman et al 2015

The HPA axis

The organism's response to any situation that threatens homeostasis is driven by the stress system that comprises the sympathomedullary and the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is the neuroendocrine effector of the system that mediates both enhancement and termination of the stress response. Following stress, the corticotrophin releasing hormone (CRH) and arginine-vasopressin (AVP), released from paraventricular hypothalamic neurons, trigger the secretion of the adrenocorticotropic hormone (ACTH) from the anterior pituitary, which in turn stimulates the release of glucocorticoids from the adrenal cortex. Extra-hypothalamic brain areas, such as the hippocampus, amygdala and prefrontal cortex, are also mutually interacting with the HPA axis, both at basal and under stress conditions. Glucocorticoids, the axis' final effectors, play key roles in both the potentiation of the response and its termination and return to homeostasis. By binding to their receptors (GR), they exert negative feedback in axis' activation at the level of the pituitary, the hypothalamus and the hippocampus (Fig. 1). Unsuccessful adaptation to stress is linked to severe pathophysiology including depression, obesity and chronic inflammation. Notably, HPA axis components have been identified as targets for epigenetic modifications induced by early life adversities that can persist throughout life.¹³

Figure 1. The hypothalamic-pituitary-adrenal axis (HPA) and BPA's effects under basal conditions. Hypothalamic and higher brain centers such as the hippocampus control HPA axis function. Corticotrophin releasing hormone (CRH) from the paraventricular hypothalamic area stimulates the release of adrenocorticotrophin hormone (ACTH) from the anterior pituitary, which in turn triggers the release of glucocorticoids (corticosterone in rodents) from the adrenal cortex. Following stress, the highly secreted glucocorticoids, via their receptors (GR), are responsible for the termination of the axis activation through a negative feedback loop exerted on the central axis components. GR activity is tightly regulated by the co-chaperon FKBP51 that inhibits GR hormone binding affinity and nuclear translocation via an ultra-short feedback loop within the HPA axis. The figure illustrates the reported effects of BPA on the HPA axis under basal conditions. Perinatal BPA exposure appears to affect most HPA axis regulators, in a sex-specific manner, including FKBP51 expression.

One of the HPA axis' regulators that is sensitive to environmental factors and epigenetic changes is FKBP51. This co-chaperone (the protein product of the Fkbp5 gene), is a negative regulator of GR translocation to the nucleus.^14,15 Interestingly, Fkbp5 is itself a GR target and glucocorticoids induce its expression.¹⁶ Chronic exposure to glucocorticoids persistently changes Fkbp5 expression by altering DNA methylation of the gene in the mouse hippocampus and hypothalamus.^17,18 Similar epigenetic changes have been detected in patients with post-traumatic stress disorder and bipolar disorder,^19,20 highlighting the importance of this GR coregulator in stress related disorders.²¹

BPA

BPA is one of the most highly produced chemicals today,²² used in the production of polycarbonate plastics and epoxy- resins. Common BPA-containing products include water bottles, food and beverage cans, personal care products, thermal receipt paper, and medical equipment.²³ Consequently, humans and animals are exposed to BPA on a daily basis and biomonitoring studies estimate that over 90% of the population has measurable urinary BPA levels.²³ Early toxicokinetic studies suggested that BPA is rapidly metabolized (6 h in plasma after oral administration).^24,25 However, it seems that exposure route and type (acute vs. chronic), age, sex, and genetic background can affect BPA bioaccumulation and/or excretion from the organism. Epidemiological studies show higher exposure and urine BPA accumulation in children compared to adults.²³ Moreover, BPA readily passes the placenta and accumulates in the fetus due to its immature drug-metabolizing system.²⁶ It particularly accumulates in the fetal brain,^27,28 where its average concentration is higher in males than in females.²⁷

The tolerable daily intake, based on a classical dose-response relationship, is set to 50 μg/kg in USA and 4 μg/kg in Europe. However, BPA at biologically relevant levels has repeatedly shown non-monotonic effects in different cell and animal models and even in humans.²⁹ The reason for these non-monotonic, low-dose effects may reside in BPA's numerous mechanisms of action at the cellular level. The most studied mechanism is via the 2 estrogen receptors, ERα and ERβ. BPA acts as partial agonist for these receptors, albeit with 10,000-100,000 fold lower affinity than estradiol. Its potency differs for the 2 ERs and is cell type-dependent.³⁰ However, it is now clear that BPA affects other targets in the cell, thereby interfering with additional signaling pathways. For example, it can induce fast non-genomic responses via ERα or the transmembrane G-protein coupled receptor GPR30.^31-36 Further, both in vitro³⁷ and in vivo³⁸ studies have shown that BPA binds and activates estrogen related receptor gamma (ERRγ), an orphan receptor that interferes with ER signaling.³⁹ Additionally, BPA is a weak androgen receptor (AR) antagonist,³⁰ it interferes with thyroid hormone signaling³⁰ and activates GR.⁴⁰

BPA effects on the stress system of rodents

Initial observations

Indirect evidence for a potential BPA effect on the stress response derived from several studies showing BPA-induced changes in anxiety-related behavior of rats and mice. The effects vary depending on the dose, time of exposure and evaluation, and the animal's sex and species. A handful of these studies, mostly conducted in mice, report increased anxiety levels upon perinatal BPA exposure^41-44 or pubertal exposure⁴⁵ while others find no changes^46-50 or even anxiolytic effects.^51-53 Importantly, in the study applying several exposure doses, non-monotonic effects for this BPA action were detected.⁵³

Although anxiety-like behavior is often expressed during stress exposure and the anxiety status can modify stress coping, it cannot be directly, or solely, associated with the function of the neuroendocrine stress axis, since anxiety is often the outcome of changes in other circuits and neurotransmitters. Indeed BPA-induced affective and neurotransmitter changes have been reported in previous animal studies. For example, in perinatally exposed mice the altered anxiety state has been related to changes in dopaminergic⁴³ and glutamatergic^42,52 systems.

Therefore, we and others initiated studies directly addressing potential BPA effects on the key regulators of the stress response.

Effects on the HPA axis effectors

Direct evidence for possible BPA effects on the HPA axis of rats upon perinatal exposure were first reported in 2010⁵ and further elaborated in the following years.^6-10 Based on the available literature (Table 1), the BPA-induced alterations in HPA axis of exposed animals differ depending on the exposure window, dose, sex and age at evaluation. All studies using perinatal BPA administration^6-10 report significant alterations in HPA axis' components at later life stages (i.e. puberty or adulthood) that appear dose-dependent and sex-specific. In contrast, short-term BPA exposure in adolescence revealed no significant alterations in any HPA axis component at either puberty or adulthood.^11,12 All studies used BPA doses (2 and 40 μg /kg /d) within the range of the current tolerable daily intake for humans (4-50 μg /kg /d).

Effects on adrenals and stress hormone levels

Perinatal exposure to 40 μg BPA /kg /d increased adrenal weight of female BPA-offspring at mid-puberty.⁶ Additionally, BPA-offspring of both sexes exhibited a hyperplastic zona fasciculata (the glucocorticoid synthesising zone), while male BPA-offspring had a diminished zona reticularis, where the non-gonadal androgens are synthesized. Although there are no further studies for BPA effects on adrenal glands, reports from other EDCs support an impact of xenoestrogens in adrenal physiology. Early life exposure to genistein⁵⁴ reduces adrenal steroidogenesis and expands zona fasciculata and reticularis of pubertal male offspring, while nonylphenol exposure leads to hyperadrenalism in both sexes.⁵⁵

Perinatal BPA exposure (40 μg BPA /kg /d) increased basal plasma corticosterone levels in female, but not male, offspring tested at puberty.⁶ By using the same protocol and BPA dose, Zhou et al.¹⁰ also detected increased corticosterone and ACTH concentrations in BPA-exposed pubertal females at circadian peak and nadir. These findings were further supported by behavioral data of the same studies showing increased anxiety of the adolescent female BPA-offspring in different anxiety tests.

Perinatal BPA exposure also led to sex-specific changes in the stress-induced corticosterone response of the offspring at puberty. Upon exposure to a 15-min swimming stress, female BPA-offspring exhibited lower stress-induced corticosterone levels than control females at 30 and 60 min post-stress, whereas male BPA-offspring showed higher stress-induced corticosterone levels at 30 and 120 min post-stress compared to BPA-treated females.⁶ In addition, Zhou et al.¹⁰ detected an inefficient response to the dexamethasone suppression test in pubertal female BPA-offspring, denoting an impairment of negative feedback inhibition in these animals. These data suggest that perinatal BPA exposure impacts corticosterone responses primarily in female offspring at puberty. These alterations imply HPA axis hyperactivity that in humans is related to the appearance of affective disorders with early onset in adolescence.⁵⁶

Interestingly, differential impact of perinatal BPA exposure on corticosterone levels has been reported when rats were exposed to 2 μg BPA/Kg /d, instead of 40μg, and evaluated in adulthood, instead of puberty.^7,8 BPA-exposed males, but not females, had increased corticosterone, ACTH and CRH levels at basal conditions. These changes were followed by increased anxiety and depression-like behavior in male BPA-offspring and reduced anxiety in female offspring. BPA-treated males had also higher corticosterone and ACTH levels immediately after stress. However, according to corticosterone stress kinetics,⁵⁷ hormonal determinations so close to stress onset (5 min) are not informative for the corticosterone stress response. Further studies are required, preferably by using the same group of animals in several sampling points following stress, to clarify the stress-induced hormonal responses.

The aforementioned contrasting observations may rely on the existence of non-monotonic BPA actions previously reported for this agent.²⁹ Differential dose- and sex-specific behavioral effects of BPA have been detected among mice exposed in utero to 2, 20 and 200 μg of BPA/Kg/d.⁵⁸ Another explanation could reside in the differences of HPA axis physiology between puberty and adulthood, since puberty is a period of ongoing maturation of the axis.⁵⁹ Furthermore, short pubertal exposure of rats to 40 μg BPA/Kg /d had no effects on anxiety and corticosterone levels during stress at either puberty or adulthood^11,12 denoting that the perinatal period is probably the most susceptible for BPA effects on corticosterone levels, in agreement with observations in other systems.^27,28

Effects on stress hormone receptors

Upon perinatal BPA exposure (40 μg BPA/kg/d), glucocorticoid receptor levels were reduced in the hypothalami⁶ and hippocampi^5,10 homogenates from pubertal female rats. The decreases in GR levels may represent a tissue response to the increased corticosterone levels observed in these animals under basal conditions. The detected GR down-regulation, reportedly a stress-induced mechanism to protect the brain from excess of glucocorticoids, implies that these animals were in a stress-like state. Following stress, no further alterations were found in GR levels of female BPA-treated offspring, compared to basal conditions, whereas the receptor levels were down-regulated in the hypothalami of control females,⁶ suggesting possible disturbances in the negative feedback mediated by this receptor at the level of the hypothalamus. No effects of BPA were detected in the basal mRNA levels of pituitary proopiomelanocortin (POMC) and CRH receptor 1 (CRHR1) in adolescent rats of either sex, upon perinatal exposure to the dose of 40 μg /kg /d. However, following stress, CRHR1 and POMC levels were increased in male BPA-offspring, implying that perinatal BPA exposure heightens stress response at the level of the pituitary in a sex-specific manner.⁶

Similarly to the hormonal levels, different findings are reported for the adult rats upon perinatal exposure to 2μg (instead of 40μg) BPA /kg /d, that are however compatible with the hormonal and behavioral measures of the same animals. Decreased hippocampal GR and MR mRNA were detected in the adult male BPA-offspring, under basal conditions, whereas GR levels in the PVN and GR and MR in the hippocampus of female BPA-offspring were increased.⁷ Non-monotonic BPA actions²⁹ and differences between adolescent and adult brain physiology⁵⁹ could possibly account for these differences among studies, but further studies are required to delineate these findings.

HPA and gonadal hormone signaling

It is well accepted that HPA axis function can be modulated by gonadal hormones. Estrogens are enhancing, whereas androgens are inhibitory to the activation of the axis.⁶⁰ ERα and ERβ in the brain are implicated in affective behaviors⁶¹ whereby ERβ mediates anxiolytic actions.⁶² BPA can interfere with gonadal receptor signaling due to its agonistic properties on ERα and ERβ and antagonistic properties on AR.³⁰

Developmental BPA exposure reduces male steroidogenesis.⁶³ Low doses of perinatal BPA affect rodent estrogen receptor levels in a sexually dimorphic manner. Khurana and co-workers⁶⁴ observed that neonatal BPA exposure increases the expression of ERα in the female hypothalamus and the male pituitary. Another study reported sex- and BPA dose-dependent changes in ERα, ERβ and ERRγ mRNA levels in the hippocampus, hypothalamus, and prefrontal cortex of juvenile mice.⁵⁸ Chen et al.⁷ showed reduced circulating testosterone levels and decreased ERβ mRNA in adult male rats perinatally exposed to BPA, as well as increased ERα in the hypothalamus of both sexes. In the last study, the reduced testosterone levels along with the increased ERα and decreased ERβ levels, could confer to the enhanced HPA responsiveness and increased anxiety detected in these male BPA-offspring.

The existing evidence suggest that BPA effects on the stress system and related behaviors could be partly exerted through changes induced in the gonadal hormone signaling, however further studies are required to clarify the underlying mechanisms.

Epigenetic alterations

Brain maturation during puberty is under the influence of gonadal hormones, and epigenetic modifications are involved in this sex-specific process. Epigenetic mechanisms, apart from regulating the programmed brain development, are also responsive to environmental inputs that contribute to the final brain shaping.⁶⁵ Epigenetic mechanisms include DNA methylation, post-translational modification at histone tails, and regulation by non-coding RNAs. DNA methylation is considered the most stable epigenetic mark with a clear mechanism of heritability brought about by DNA methyltransferase 1 (DNMT1) that establishes the methylation pattern of the mother strand at the daughter strand during replication.⁶⁶ In various experimental systems, BPA was shown to induce gene-specific DNA methylation changes, among others in the brain.^58,67 To investigate possible epigenetic modifications in the HPA axis induced by perinatal BPA, we examined DNA methylation changes in the promoter of the GR gene and in intronic areas of its negative regulator Fkbp5. We found increased DNA methylation of Fkbp5 intron 5 and reduced protein levels in the hippocampus of exposed male rats. Similar effects were obtained in the male hippocampal cell line HT22 when exposed to BPA during differentiation. The mechanisms underlying epigenetic effects of BPA are still unknown. Several studies report that BPA changes expression of the DNMTs.^58,68,69 This alone, however, would lead to global rather than to gene-specific effects on DNA methylation. In our study, we demonstrated an involvement of ERβ in the epigenetic effect of BPA.⁹ In another study, we have found that ERβ is involved in regulating local DNA methylation by recruiting thymine DNA glycosylase (TDG), an enzyme involved in DNA demethylation, to specific genomic regions.⁷⁰ Thus a possible mechanism for how BPA exerts its effects on DNA methylation could involve the disruption of the interaction between ERβ and TDG, and thus the recruitment of this enzyme to specific genomic regions including intron 5 in Fkbp5 (Fig. 2).

Figure 2. Suggested model for the role of ERβ in the inhibitory effect of BPA on Fkbp5, based on observations in differentiating HT22 cells. In the absence of BPA treatment, ERβ prevents DNA-methylation of CpGs at the glucocorticoid response element (GRE) of the Fkbp5 gene. This might be mediated by ERβ's recruitment of thymine DNA-glycosylase (TDG) that is involved in active DNA demethylation. As a result, a transcriptional complex is formed that can cooperate with upstream promoter regions to mediate Fkbp5 expression by activated GRs.¹⁹ However, when the cells are exposed to BPA during differentiation, BPA might disrupt ERβ binding, resulting in no TDG recruitment, DNA hypermethylation and lower Fkbp5 expression. This, translated to the HPA axis, could result in enhanced GR activity.

Conclusions

The evidence summarized here implicates BPA in the regulation of an organism's stress system in addition to its known implication in the reproductive and metabolic tracts. The available literature indicates that perinatal BPA exposure of rats significantly impacts most HPA axis' regulators of the exposed offspring at 2 later life-stages (adolescence and adulthood), whereas adolescent exposure does not appear to impact corticosterone or anxiety levels. The perinatal BPA-induced alterations are associated with changes on related behaviors but appear dose- age- area- and sex-specific (Table 1, Fig. 1). The impact of BPA on the stress axis could be influenced by effects on the gonadal steroid hormone signaling and may additionally involve epigenetic changes induced by this endocrine disruptor. However, more studies are required to delineate the molecular events leading to these HPA axis alterations and to shed more light on possible non-monotonic effects. Most importantly, these studies conducted in rodents should be linked to epidemiological findings in order to better understand if developmental BPA exposure could be linked to stress-related disorders in humans.

Abbreviations

ACTH	=	adrenocorticotropic hormone
AR	=	Androgen receptor
AVP	=	arginine-vasopressin
BPA	=	Bisphenol A
Bw	=	body weight
CRH	=	corticotrophin releasing hormone
CRHR1	=	CRH receptor 1
DNMT	=	DNA methyltransferase
EDCs	=	endocrine disruptive chemicals
Erα	=	Estrogen receptor α
ERβ	=	Estrogen receptor β
ERs	=	Estrogen receptors
ERRγ	=	estrogen related receptor gamma
Fkbp5	=	FK506 binding protein 5
FKBP51	=	FK506 binding protein 51
GR	=	glucocorticoid receptor
GRE	=	Glucocorticoid responsive element
HPA	=	Hypothalamic Pituitary Adrenal
MR	=	mineralocorticoid receptor
POMC	=	proopiomelanocortin
TDG	=	thymine DNA glycosylase

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

JR receives funding from the Swedish Research Council FORMAS, JR and EK receive funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 634880.