Evidence is mounting in rodent model systems that developmental BPA exposure at human-relevant levels results in behavioral abnormalities and learning deficits (Jones et al., 2011
; Nakamura et al., 2011
; Palanza et al., 2008
). We demonstrated here that low-dose BPA exposure during CNS development caused larval hyperactivity and adult learning delays, establishing the zebrafish model as an additional resource for exploring the mode of action underlying BPA's neurobehavioral effects.
While other groups have utilized the embryonic zebrafish model to assess the developmental toxicity of both BPA and E2 (Duan et al., 2008
; Fei et al., 2010
; Gibert et al., 2011
; Kishida et al., 2001
; McCormick et al., 2010
), these groups’ assays differed from ours in exposure volume, duration, vehicle used, or endpoints assessed, leading to discrepancies among the study outcomes () and highlighting a need to standardize testing methods. Quantifying stock concentrations is one way to compare results across research groups. Here, we derived a concentration-response curve using a BPA stock concentration that was clearly defined by HPLC analysis in order to most accurately link larval neurobehavioral endpoints to exposure concentration.
Table 2 Comparison of bisphenol A (BPA) and 17β-estradiol (E2) toxicity studies conducted in the larval zebrafish model. Note: the caudal fin defect observed by Duan et al. (2008) was the only abnormality from among the other studies that was not observed (more ...)
A second key component of our study was to measure BPA tissue dose associated with exposure concentrations using a novel application of HPLC heretofore not used in larval zebrafish studies. One major advantage of using this method is that it circumvented the need to use radio-labeled BPA standards. The main limitation of this assay was an inability to detect BPA in samples of 50 pooled embryos exposed to <1μM BPA, necessitating extrapolation of tissue dose from exposure concentrations below 1μM. Nevertheless, the uptake measurements reported here provide an important approximation in the embryonic zebrafish model associating waterborne exposure concentrations with tissue dose. Interestingly, the measured dose was less than the expected tissue concentration (e.g., 1 μM exposure concentration ≈ 228 μg/L ≈ 228 μg/kg (given the embryo is composed of >70% water (Hagedorn et al., 1997
)); however, 1 μM exposure resulted in ~10 μg/kg tissue dose, ~20x less than 228 μg/kg). This suggests that the entire amount of BPA in the well was not absorbed by the embryo over the course of exposure and/or metabolism effectively reduced the measured tissue level.
Although unaccounted for factors such as absorption and metabolism make extrapolation between species a rough estimate, at best, it is important to note that the tissue doses associated with exposure concentrations ≤1 μM were in the range of human-relevant BPA levels (e.g., 1 μM waterborne exposure yielded roughly 10 μg/kg tissue dose, which is near the average level detected in human placenta (11.2 ng/g) (Schonfelder et al., 2002
) and close to the maximum daily BPA intake of a bottle fed infant (11 μg/kg) (NTP, 2008
)). Taken together, the larval behavior tests (roughly 8-58 hpf exposure) and the 8-58 hpf tissue dose approximation demonstrate that BPA doses ≤1μg/kg measured shortly after peak CNS development (i.e., 16 – 36 hpf) were sufficient to produce significant hyperactivity in larval zebrafish. Thus, the concentrations to which fetuses and infants are potentially exposed (Braun et al., 2009
; NTP, 2008
; Schonfelder et al., 2002
; Vandenberg et al., 2010
) are associated with hyperactivity in this model.
BPA's developmental toxicity is commonly assumed to be mediated through ER agonism (e.g., Ben-Jonathan and Steinmetz, 1998
). Thus, endogenous estrogen (17β-estradiol, E2) is an appropriate control for studies aimed at testing BPA's estrogen mimicry. Only one published study was found describing windows of exposure that elicit morphological defects following developmental E2 exposure in zebrafish (Kishida et al., 2001
). Despite reported differences in E2 potency, the types of observed effects were consistent between the Kishida et al. (2001)
and current studies (). The most noticeable consistency was a distinct curved body axis most apparent in our study in embryos exposed to 20μM E2. Kishida and colleagues noted that this “curved tail down phenotype” has been associated with defects in CNS development, supporting the idea that the CNS is the target organ system underlying behavioral abnormalities observed in E2-exposed embryos (Brand et al., 1996
). Additionally, a recent study showed that E2 has a significant role in maintaining neuromuscular function and thus also impacts the peripheral nervous system (Houser et al., 2011
). Although E2 is an appropriate control for all studies investigating the possibility that BPA causes neurobehavioral effects through estrogen mimicry, it is prudent to also include ligands of alternative receptors for which BPA exhibits high binding affinity.
As BPA binds ERRγ with substantially greater affinity than it binds classical ERs (Okada et al., 2008
; Washington et al., 2001
), we included the synthetic ERRβ/γ agonist, GSK4716, as a second control for the early life stage toxicity and larval behavior assays (Zuercher et al., 2005
). This is the first study to describe the effects of GSK4716 exposure on developing vertebrates. The narrow margin of safety for GSK4716 (i.e., the mortality and abnormality concentration-response curves almost overlap) suggests that ERRβ/γ have significant roles in embryonic zebrafish development and exogenous activation of these receptors likely impacts multiple organ systems through pathways necessary for survival. The finding that ERRα, ERRβ, and ERRγ have significant roles in regulating energy metabolism (reviewed in Deblois and Giguere, 2011
) supports the idea that ERRβ/γ activation by GSK4716 leads to mortality by interfering with metabolism in multiple organ systems. While the behavioral effects observed at non-lethal doses may also be the result of altered metabolism, it is probable that mortality/abnormality and hyperactivity are elicited through diverging underlying molecular events. It is also worth noting that the narrow margin of safety observed for GSK4716 could be associated with off-target effects (i.e., activation of receptors other than ERRβ or ERRγ.). Since few studies have investigated alternative GSK4716 targets and, to our knowledge, no study has investigated the targets of GSK4716 exposure at concentrations higher than 10 μM, further study is needed to explain this compound's toxicity. Although little is known about the activity of GSK4716 at doses higher than 10 μM, effects of GSK4716 ≤10 μM in skeletal muscle have been shown to be ERRγ dependent through use of in vitro
siRNA control experiments (Wang et al., 2010
) demonstrating selectivity for its intended targets at the lower dose range.
This is the first study to examine the effects of low-dose BPA exposure on zebrafish behavioral endpoints. Preliminary testing determined that an exposure window between 10 and 58 hpf (48 hour exposure) was sufficient to produce a locomotor effect in 5 day old larvae (data not shown). This exposure window encompasses the primary wave of neurogenesis (16 – 36 hpf; (Kimmel et al., 1995
)) and was used for all larval behavior tests (Note: an 8 hpf start time was found to yield identical results on behavior tests as using larvae first exposed at 10hpf; the 8hpf exposure was used for some trials to increase the number of embryos available for testing). We demonstrated here that exposure to 0.01 or 0.1 μM BPA during neurogenesis resulted in hyperactivity in the dark by 5 dpf zebrafish larvae. Exposure to higher BPA concentrations did not affect larval activity. Thus, the locomotor activity concentration-response curve was nonmonotonic, consistent with the effects of BPA on in vivo
reproductive endpoints (Weltje et al., 2005
). The observation of an inverted U shape dose-response is consistent with that commonly observed for hormones such as E2 (reviewed in Kendig et al., 2010
). Although this type of curve has been explained as an adaptation to a toxic response (e.g., the lack of response at higher concentrations is the result of adaptive detoxification mechanisms), a recent review on nonmonotonic curves emphasizes that this is too simplistic of an explanation for what are certainly complex underlying events (Kendig et al., 2010
). In the case of BPA, it has been proposed that such a curve is the result of the disruption of endogenous estrogen (Weltje et al., 2005
). Without further testing beyond the scope of this study, all that can be concluded by the observation of this concentration-response is that the mode of action of BPA is complex at the low concentration range (i.e., ≤10 μM) and does not follow a sigmoidal dose-response curve, which depicts the saturation of a target receptor with increasing dose. Therefore, this data is consistent with the involvement of multiple receptor targets and the idea that the receptor activity levels associated with the hyperactivity phenotype are not representative of saturation of available target receptors, whether one or multiple types. It is also important to note that this response was observed because we were specifically looking for the effects of BPA on behavior at low and more relevant exposure concentrations. If we had only considered morphological abnormalities, or had not included the concentrations between 0.001 and 1 μM, then this nonmonotonic curve would not have been observed. We echo the warning by Kendig and colleagues (2010)
that the potential to overlook subtle, nonmonotonic responses is great and should be considered by researchers conducting low-dose BPA studies in zebrafish, particularly groups working from a definition of “low-dose” that may consider the lower concentrations tested herein irrelevant.
The evidence that BPA impacts neurodevelopment in the zebrafish model complements existing data from other model systems. While a range of behavioral effects have been reported in rodent models following prenatal BPA exposure (e.g., sex difference in mating behavior, anxiety, and learning and memory) (Negishi et al., 2004
; Palanza et al., 2002
)), it is nevertheless intriguing that several rodent studies have also shown increased locomotor activity following gestational BPA exposure. For example, both Ishido et al. (2004)
and Kiguchi et al. (2007) reported significant hyperactivity in rats several weeks following a single intracisternal dose of BPA delivered to 5 day old pups. Additionally, Palanza et al. (2008)
described greater activity by males in an open field test following prenatal low-dose (10 mg/kg) BPA exposure. It is important to note that zebrafish hyperactivity is a general swimming response that is not necessarily analogous to rodent or human hyperactivity. While it is possible this phenotype is representative of direct impact on CNS function, it could also be elicited through effects on neuromuscular functioning or impairment of sensory perception, any of which could be a manifestation of inappropriate ER or ERR activation.
To begin to test whether BPA's neurobehavioral effects are mediated through inappropriate ER or ERRγ activation, we included agonists of these receptors in the larval zebrafish behavior tests. Consistent with our findings for BPA, we found that exposure to 0.1 μM E2 or GSK4716 during neurogenesis resulted in hyperactivity in the dark by 5 dpf zebrafish larvae. While this is the first study in any vertebrate model system to investigate whether ERRγ activation by developmental GSK4716 exposure elicits any behavioral effects, estrogen exposure has previously been associated with increased activity in adult rodents (Morgan and Pfaff, 2002
; Thomas et al., 1986
) and has been used to rescue “immobility” in a rat forced swimming test, substantiating its role in maintaining normal activity levels (Estrada-Camarena et al., 2003
). Although the above studies dealt with adult exposures, they provide background supporting the importance of E2's effects on nervous system function. An additional study specifically addressed E2's effects on neurobehavioral development; Dugard et al. (2001)
observed increased motor activity in offspring of rats exposed to 17α-ethinylestradiol during gestation. Although E2 has also been used to rescue the “listless” phenotype in the embryonic zebrafish model (Nelson et al., 2008
), hyperactivity following low concentration E2 exposure has not been previously described in this model. In contrast, one study noted no effects of E2 on larval movement at lower doses, but significant loss of movement at a higher E2 dose (Hamad et al., 2007
The hyperactivity following BPA exposure observed in our study is consistent with the increased activity observed in both rodents and larval zebrafish (this study) after E2 exposure. However, because GSK4716 exposure also elicited a hyperactive phenotype in our study, and GSK4716 is a selective ERRβ/γ agonist, this data is also consistent with an ERRγ agonism mode of action. More importantly, the similar phenotype following activation of different nuclear receptors demonstrates that zebrafish larval hyperactivity can be engendered through a variety of pathways. While ER and ERRγ activation is presumed to be central to the hyperactive phenotype elicited by E2 and GSK4716, respectively, many questions remain as to which organ systems are being affected and the identity of downstream gene targets. The hypothesis that larval hyperactivity is representative of a CNS effect is certainly bolstered by the adult data described here.
To investigate whether higher order brain functions such as learning are affected in adult fish by BPA exposure during critical periods of CNS development, we used a T-maze testing apparatus () commonly used in both rodent and zebrafish studies (e.g., Grossman et al., 2010
; Peitsaro et al., 2003
). We showed here that exposure to the same concentration of BPA that caused significant hyperactivity in larval zebrafish (0.1 and 1 μM) led to significant deficits in learning a reversal task in adults 8 months after the initial BPA exposure. Similar learning deficits in spatial memory and avoidance tests following prenatal low-dose BPA exposure have been reported for mice (Negishi et al., 2004
; Tian et al., 2010
; Xu et al., 2010
). Interestingly, no sex differences in behavior were detected in this study (further testing with a larger sample size will be required to confirm these findings). In contrast, there is a wealth of evidence for effects on sexually dimorphic behavior following prenatal BPA exposure (e.g., Jones et al., 2011
; Negishi et al., 2004
; Palanza et al., 2002
). While the molecular events underlying BPA's effects on learning and memory are actively being investigated, with some evidence that neurotransmitter receptor expression may be altered (Ishido et al., 2004
; Xu et al., 2010
), it is generally postulated that inappropriate activation of ERs is central to BPA's mode of action (Ben-Jonathan and Steinmetz, 1998
). The role of ERs in addition to other receptors of interest in eliciting BPA's effects on neurobehavioral development can be effectively investigated using the larval zebrafish model.
Here, we describe an early life-stage zebrafish assay that is ideal for investigating the mode of action by which BPA elicits long-term learning deficits, thus introducing a powerful tool to be used towards understanding BPA's neurobehavioral toxicity. Because transient BPA exposure during CNS development led to learning deficits in adults, we know that the events that permanently affected brain development and function were initiated during the early life-stage exposure. Therefore, although the persistence of neurobehavioral BPA toxicity is paramount to the relevance of this study, the power of this model lies in the identification of an early phenotype, larval hyperactivity, which resulted from exposure to the same concentrations that led to learning deficits in adults exposed during development. Although it is important to note that the larval and adult phenotypes are not necessarily representative of the same physiological events during the behavior tests, themselves (e.g., locomotion and learning are quite different behaviors), the larval phenotype can nevertheless be employed to further investigate the molecular events occurring during the developmental exposure that underlie BPA's neurobehavioral toxicity.
Taken together, the results of this study confirm the efficacy of using the larval zebrafish model to investigate the neurobehavioral effects of developmental BPA exposure. We have also demonstrated how the two compounds, E2 and GSK4716, can be used to investigate the roles of ERs and ERRγ, respectively, in eliciting the behavioral phenotypes described here.