Chemical Exposure-Induced Developmental Neurotoxicity in Head-Regenerating Schmidtea mediterranea

Abstract

The growing number of commercially used chemicals that are under-evaluated for developmental neurotoxicity (DNT) combined with the difficulty in describing the etiology of exposure-related neurodevelopmental toxicity has created a reticent threat to human health. Current means of screening chemicals for DNT are limited to expensive, time-consuming, and labor-intensive traditional laboratory animal models. In this study, we hypothesize that exposed head-regenerating planarian flatworms can effectively and efficiently categorize DNT in known developmental neurotoxins (ethanol and bisphenol A [BPA]). Planarian flatworms are an established alternative animal model for neurodevelopmental studies and have remarkable regenerative abilities allowing neurodevelopment to be induced via head resection. Here, we observed changes in photophobic behavior and central nervous system (CNS) morphology to evaluate the impact of exposure to low concentrations of ethanol, BPA, and BPA industry alternatives bisphenol F, and bisguaiacol on neurodevelopment. Our studies show that exposure to 1% v/v ethanol during regeneration induces a recoverable 48-h delay in the development of proper CNS integrity, which aligns with behavioral assessments of cognitive ability. Exposure to BPA and its alternatives induced deviations to neurodevelopment in a range of severities, distinguished by suppressions, delays, or a combination of the 2. These results suggest that quick and inexpensive behavioral assessments are a viable surrogate for tedious and costly immunostaining studies, equipping more utility and resolution to the planarian model for neurodevelopmental toxicity in the future of mass chemical screening. These studies demonstrate that behavioral phenotypes observed following chemical exposure are classifiable and also temporally correlated to the anatomical development of the CNS in planaria. This will facilitate and accelerate toxicological screening assays with this alternative animal model.

Less than 20% of chemicals used in the United States have been evaluated systematically for safety and risk to human health (Hartung, 2009). With over 80 000 chemicals in use commercially, this leaves an excess of nearly 65 000 chemicals that have not been assessed thoroughly (Hartung, 2009). Exposure to known toxins has been shown to affect neurodevelopmental processes in periods of development from conceptus through infancy (Grandjean and Landrigan, 2006, 2014; Paparella et al., 2020), including the widely publicized discovery of fetal alcohol syndrome (Clarren and Smith, 1978; Jones and Smith, 1973; Streissguth, 1997). Despite this knowledge, most industrial chemicals do not have to be tested for neurodevelopmental impacts before incorporation into consumer goods (Hartung, 2009), and it is possible that deviations of unknown etiology in the normal development of nervous system structures and functions could be attributed to exposure to unknown toxins from among these incompletely assessed chemicals, thereby creating a reticent threat to neurodevelopmental integrity. Therefore, there is a need to further study the impact of chemical exposure on development.

Among the poorly evaluated chemicals is a class of about 40 bisphenols, including the most widely known, bisphenol A (BPA). BPA is a commonly used plasticizer and is present in a spanning array of consumer goods, including thermal receipt paper and epoxy resins (Vandenberg et al., 2007). The leading routes of exposure to BPA are dermal absorption and the consumption of food and beverage products contaminated by their packaging (Pang et al., 2019) where the hydrolysis of polycarbonate chain ester bonds leads to leaching of the BPA subunit directly into the consumer products (Vandenberg et al., 2007). Furthermore, through processing and daily application of these products, large quantities of BPA are continually fed into the environment, enabling the global capacity of BPA to exceed 6 billion pounds per year (Cooper et al., 2011; Vandenberg et al., 2007; Vom Saal and Hughes, 2005; Xiao et al., 2020). BPA has been measured in human urine, amniotic fluid, placental tissue, and various other human samples in the range of 2–11 parts per billion (Gray et al., 2004; Ikezuki et al., 2002; Schönfelder et al., 2002; Todaka and Mori, 2002; Yamada et al., 2002). Knowledge of the effects of BPA in low doses is poor, but it is known to be an endocrine disruptor acting through estrogen receptor competition, and both pre- and postnatal studies have shown some correlation between BPA and developmental delays (Gray et al., 2004, Vandenberg et al., 2007; Della Seta et al., 2005; Kubo et al., 2004; Rochester, 2013; Gyimah et al., 2021; Liang et al., 2020; Mustieles and Fernández, 2020; Yilmaz et al., 2020).

BPA imparts favorable performance properties in a wide variety of end-use applications, and thus scientists and engineers have explored similar bisphenols such as bisphenol F (BPF) and bisguaiacol (BG; Figure  1) as potential replacements. Although the performance properties imparted by these alternatives are comparable (Jana et al., 2005; Nicastro et al., 2018), most efforts in assessing toxicity have been limited to in vitro cytotoxicity and estrogenic effects (Pelch et al., 2019; Švajger et al., 2016), which may not represent in vivo toxicity, and cannot assess impacts on dynamic developmental processes. In particular, the assessment of developmental neurotoxicity (DNT) is predominantly retrospective and time-intensive in humans and higher-level model organisms. There is, therefore, a pressing need to rapidly assess neurodevelopmental effects of known and novel chemicals prior to widespread use, which can complement the in vitro cytotoxicity studies and assist in better understanding the full impact of these chemicals, and others, on living organisms.

Figure 1.

Chemical structures of bisphenol A (BPA), bisphenol F (BPF), 17β-estradiol, and bisguaiacol (BG). BPF and BG have been proposed as BPA alternatives. These molecules retain desirable mechanical properties in end-use applications, but the effects of their small structural changes on toxicity have not been evaluated comprehensively.

In this work, Schmidtea mediterranea (Smed) planarian flatworms provide a novel screening tool for neurodevelopmental toxicity. Planarian flatworms have a brain-like structure known as the cephalic ganglia, and a unique regenerative ability that stems from an enriched population of pluripotent stem cells, termed neoblasts (Cebrià, 2007, 2008; Cebrià et al., 2002; Newmark and Sánchez Alvarado, 2002). Proliferation of these neoblasts can reconstruct not only this brain-like structure, but entire worms from pieces as small as 1/279th the original size, all within a matter of days (Morgan, 1898). The complexity of the cephalic ganglia allows for comparisons to the human nervous system with visual, thermosensory, chemosensory, and mechanosensory neurons all present in planaria (Buttarelli et al., 2008; Cebrià, 2007; Cebrià et al., 2002; Hagstrom et al., 2015, 2019; Inoue et al., 2015). The molecular and cellular framework of the planarian central nervous system (CNS) has also been well studied (Agata et al., 1998, 2003; Sánchez Alvarado and Newmark, 1999; Cebrià, 2007, 2008; Cebrià et al., 2002; Newmark and Sánchez Alvarado, 2002; Inoue et al., 2004). Animals regain full wild-type morphology following decapitation (Morgan, 1898) and the formation of the cephalic ganglia during this regenerative process has been described (Cebrià et al., 2002).

Smed have been used previously to examine behavioral and anatomical abnormalities in chemically exposed animals. Studies have shown behavioral effects of planaria exposed to exogenous chemicals and drugs (Hagstrom et al., 2015, 2019; Lowe et al., 2015; Raffa and Valdez, 2001; Raffa et al., 2001; Rawls et al., 2008). When subjected to ethanol solutions, head-regenerating planaria displayed a delayed reacquisition of innate behavior, aligning with evidence in humans that brain and cognitive development is strongly affected by ethanol exposure (Best and Morita, 1982; Cochet-Escartin et al., 2016; El Shawa et al., 2013; Hanna et al., 2015; Lowe et al., 2015; Tallarida et al., 2014; Zhou et al., 2011). The planarian behavioral model has shown the dangerous impacts of chemical exposure, yet despite the vast knowledge regarding the behavior and anatomy of planaria, these studies remain largely disconnected. Correlation between these 2 assessment techniques in response to external stimuli would strengthen the information obtained from the planarian model for DNT.

The reacquisition of planarian photophobic behavior following head resection has been linked to reestablishment of anatomical structures such as the cephalic ganglia, photoreceptors, and the neural link between these structures (Inoue et al., 2004). Several groups have utilized photophobic response as a surrogate indicator of developmental anomalies in planaria in response to an experimental perturbation, including toxicity studies involving exposure to exogenous chemicals (Hagstrom et al., 2015, 2019; Inoue et al., 2015; Li, 2015; Lowe et al., 2015; Rawls et al., 2008; Zhang et al., 2019a,b). Although the link between behavior and development has been shown in many contexts (eg, siRNA studies, etc.; Cebrià, 2007, 2008; Cebrià et al., 2002; Inoue et al., 2004), this link has not been completely established in a toxicological context. It has been demonstrated that ethanol exposure in head-regenerating planaria affected behavior and led to analogously altered nervous system development at several time points during regeneration (Hagstrom et al., 2015). Here, we present the first evidence that behavioral phenotypes observed following chemical exposure are temporally correlated to anatomical development of the CNS throughout regeneration, which suggests both that behavioral readouts are sufficient for toxicological assessment and that these chemicals affect nervous system regeneration directly.

MATERIALS and METHODS

 

 

Planaria care and maintenance

Asexual Smed CIW4 worms were obtained from the Sánchez Alvarado laboratory (Stowers Institute, Kansas City, Missouri). Smed were used for all studies, except for the behavioral assessment of ethanol toxicity, where brown planaria (Dugesia dorotocephala, Carolina Biological Supply Company, Burlington, North Carolina) were selected to demonstrate similar results across different species of planaria. All planaria were kept in environments described previously (Cebrià et al., 2002; Lowe et al., 2015; Newmark and Sánchez Alvarado, 2002), that includes 200–500 planaria in 250 ml planaria water (ddH₂O with 1.6 mM NaCl, 1 mM CaCl₂, 1 mM MgSO₄, 0.1 mM MgCl₂, 0.1 mM KCl, and 1.2 mM NaHCO₃) in a loosely covered 500 ml capacity BPA-free plastic container. Planaria were fed calf liver puree weekly and, when not being fed, cleaned, or observed, were stored in the dark in a Thermo Scientific incubator at 22°C. Worms 5–7 mm in length were selected for all studies and starved for 7 days prior to testing based on standard practice (Guedelhoefer and Sánchez Alvarado, 2012; Sandmann et al., 2011). For behavioral experiments, planaria were housed individually in the dark at 22°C in separate loosely covered 35 mm petri dishes containing the respective test solutions throughout the experimental period (approximately 10 days), except during daily testing when individual planaria were moved to the testing chamber (also containing the respective test solution). For immunostaining experiments, planaria were kept under the same conditions as behavioral experiments (n = 30 per exposure group). Until randomly selected for fixation at a prescribed time interval (see below).

Acquisition of bisphenols

Acetonitrile [high-performance liquid chromatography (HPLC) grade), methanol (HPLC grade), and glacial acetic acid were all purchased from VWR. Chloroform-d (CDCl₃, 99.8 atom % d) was purchased from Acros Organics. BPA (97%, pellets) was purchased from Sigma Aldrich (St Louis, Missouri). The p, p-BPF, 98%) was obtained from Sigma Aldrich. BPA and BPF were further purified by recrystallization in a 1:1 solution of glacial acetic acid and purified water (Mauck et al., 2018). Analytical standards for BPA (certified reference material, ≥ 99%) and BPF (≥ 98%’p, p’-isomer) were acquired from Millipore Sigma and used exclusively for calibration curves. BG was synthesized and purified to a mixture of the p, p’-, m, p’-, and o, p’- isomers, as previously reported (Hernandez et al., 2016). All other chemicals were used as received. Purities of BPA, BPF, and BG were confirmed by different purity assays.

Assessment of bisphenol purity

Purities of the bisphenols were assessed using ultraviolet HPLC, differential scanning calorimetry, and Proton Nuclear Magnetic Resonance. Full methods and results are available in the Supplementary materials.

Testing solution preparation

1% v/v ethanol solutions were diluted from Absolute Ethanol (Sigma-Aldrich) in planaria water, as described previously (Lowe et al., 2015).

BPA, BPF, BG, and estradiol (MP Biomedicals, Irvine, California) testing solutions were made by dissolving crystals in planaria water to a concentration of 2 ppm and stirred on a 90°C hot plate until complete assimilation of all crystals. Prepared solutions were cooled to room temperature and stored at 22°C. The 2 ppm concentration was selected as the highest screening concentration that was uniformly sublethal to intact, nonregenerating planaria across all compounds included.

Design of experiments

To induce regeneration, heads were resected with a sterilized scalpel posterior to the photoreceptors and auricles on day 0 of experimentation. Intact and head-regenerating tail pieces were exposed continuously to testing solutions or control planarian water for the next 9 days (n = 10 per treatment group). As illustrated in Figure  2, the samples designated for behavioral testing were continuously exposed to their testing solutions, and behavioral testing was conducted on the same worms every 24 h, for days 4–9 postamputation (4–9 days postamputation). Operators of the behavioral testing were blinded to the identity of the test solution groups to eliminate bias. Worms were checked for survival each day prior to behavioral testing by agitating the petri dish and watching for signs of movement including scrunching, curling, and stretching out (Cochet-Escartin et al., 2015). Fatalities were documented and discarded from further observations. Separate worms designated for immunostaining were sacrificed and fixed on each day (4–9). Unlike the planaria utilized for behavioral testing, these worms were not tested sequentially.

Figure 2.

Workflow diagram of experimental procedure. A, Test animals were amputated postauricle and prepharynx using a sterilized scalpel on day 0 of each experiment. B, Worms selected for behavioral experiments were kept in their designated test solutions for the entire testing period and cognitive function was observed using the planarian light avoidance assay every 24 h, starting 4 days postamputation. C, For anatomical experiments, head-regenerating worms were selected from a population in each designated testing solution for each day of the testing period, starting on day 4 postamputation. Worms were then killed, fixed, stained, and imaged to assess central nervous system structure.

Immunostaining

Immunostaining was conducted with minor adaptations from previously published methods from Cebrià (2008) and Pearson et al. (2009). Briefly, at the conclusion of the exposure period, worms (n = 5 per experimental group) were killed in 2% HCl (Sigma-Aldrich Inc.) on ice. Carnoy’s solution (Spectrum Chemical MFG Corp., New Brunswick, New Jersey) was used to fix the worms, and after subsequent bleaching, samples were frozen in 100% Methanol (Sigma-Aldrich Inc.). Smed worms were rehydrated from storage in 100% methanol solution and blocked in PBSTB (PBS, 0.3% Triton X-100, and 2.5 mg/ml bovine serum albumin) solution before administration of the primary antibody anti-SYNORF (3C11, Developmental Studies Hybridoma Bank, 1:50), and secondary antibody (goat antimouse conjugated with Dylight 488 VWR, 1:200). Samples were DNA counterstained with Hoescht 33342 (1:50) and worms were mounted in Vectashield (VWR, Radnor, Pennsylvania). Imaging was done using a Nikon A1 confocal microscope. Images presented are representative of all images in the treatment group.

Behavioral testing

Planaria have an instinctual behavior to avoid light. The cognitive ability of the test planaria can be quantified by comparing the planaria’s negative phototactic behavior to its locomotive ability. Mobility testing and light avoidance behavior testing were conducted simultaneously by combining techniques described in Lowe et al. (2015) and Raffa et al. (2001). Modifications include using a mini-Maglite LED flashlight (1150 average lux in the light area and 400 lux in the dark area) and sectioning the 3- × 2-inch testing chamber into target and nontarget halves instead of quadrants.

Behavioral testing quantification was done via the assignment of a cognitive function score (CFS; Lowe et al., 2015) for each 90-s test. CFS= % time in target areaNo. of lines crossed

The CFS quantifies behavioral reacquisition. Fully developed worms would have a high CFS, representing the cognitive ability to travel directly to the target area and remain there until the conclusion of the test. For visualization and comparison, CFS’s of experimental groups were normalized to the percent maximum of control CFS’s for each testing day or percent maximum of treatment group CFS’s for each testing day.

Low CFS’s could be attributed to an inability to move, rather than inadequate cognitive function to facilitate movement. Lack of movement in planarians was investigated to further discern the nature of the chemicals’ effects. Studies have shown that exposure to elevated concentrations of ethanol causes both cilia-mediated and cilia-independent locomotion defects (Cochet-Escartin et al., 2016; Stevenson and Beane, 2010). Therefore, immunostaining of ventral cilia using Smed 1H11 antibody (DSHB University of Iowa, Iowa City, Iowa) was performed on worms in each experiment group 6 and 7 days posthead resection to rule out possible cilia burnoff skewing behavioral results. Normal presence of the ventral cilia (Supplementary Figure 2) indicates that exposed worms are not deciliated in these conditions.

Statistical analysis

A mixed-model ANOVA was used to assess differences of behavioral data measurements across treatment groups. Both treatment group and days postamputation were included as factors, and the interaction of these factors suggests time-dependent effects of chemical exposure. Tukey’s Honestly Significant Difference (Tukey’s HSD) post hoc testing was used for pairwise comparison of treatment groups. Two-tailed Student’s t tests were used for time point specific comparisons between treatment groups (eg, control vs BPA on 4 days postamputation). A 95% confidence level was used for all statistical comparisons. Error bars on all figures represent the standard error of the mean for visualization purposes.

RESULTS

 

Ethanol-Induced Delay in Behavioral Reacquisition of Photophobic Behavior Mirrors Regeneration of CNS Morphology

We have shown that ethanol exposure during head regeneration induces a delay in the reacquisition of photophobic ability in Smed (Lowe et al., 2015). Recently, Ireland et al. (2020) reported exposure-induced behavioral differences among planarian species. In order to assess the consistency of the ethanol-induced delay in regeneration, here we tested brown planaria (D. dorotocephala), which were selected for their commercial availability and biological diversity. Ethanol has already been shown to affect various species of planaria (Best and Morita, 1982; Byrne, 2018; Hanna et al., 2015; Ireland et al., 2020; Tallarida et al., 2014). Here, we found similar behavioral responses to ethanol exposure during head regeneration as those observed previously, specifically the delay in reacquisition of natural photophobic behavior (Figure  3A). The average CFS of head-regenerating planaria exposed to 1% v/v ethanol are significantly lower than the control group on 3 and 4 days postamputation (post hoc t test, p < .05). This initial deficit in cognitive ability is later recovered in the ethanol-exposed planaria that obtained CFS similar to the control group on 5 through 9 days postamputation. It is important to note that observed delays are relative in magnitude and timing to species-matched controls. Changes to CFS calculation methods (eg, differences between Lowe et al., 2015 and herein) and/or average mobility (eg, differences across species—Ireland et al., 2020) may affect absolute values.

Figure 3.

Ethanol-induced delays in anatomical regeneration mimic behavioral reacquisition delays (A). Cognitive function scores (CFSs) of ethanol-exposed worms show a delay in photophobic behavior in brown planaria, consistent with previously reported reacquisition delays in Schmidtea mediterranea (Smed). B, Immunohistochemistry (IHC) of regenerative period (4–9 days) of Smed exposed to ethanol or control. C, Forty-eight-hour time offset of CFS data in (A) shows that ethanol exposure imparts temporal effects on reacquisition of behavior. D, Forty-eight-hour time offset of (B) shows that ethanol-induced delays in anatomical regeneration mimic behavioral reacquisition delays. E, IHC of neurons in nonhead regenerating, adult naïve Smed. F, IHC of neurons in nonhead regenerating, adult ethanol-exposed (>9 days) Smed. # indicates statistical significance (p < .05) between treatment groups. Error bars = SEM. For (B) and (D–F), anti-SYNORF primary and Dylight (488) conjugated secondary (green) with Hoescht 33342 counterstain (blue). Scale bar is 100 µm.

Immunostaining neurons in ethanol-exposed Smed show similar delays in cephalic ganglia regeneration (Figure  3B). The stages of planarian neurodevelopment defined in Cebrià et al. (2002) were utilized to contrast these neurodevelopmental differences. In exposed and naïve flatworms 4 days postamputation, the new brain primordium can be seen in the anterior of the regenerating blastema (Figure  3B). In naïve flatworms 4 days postamputation, the brain primordium has developed into a small bilobed-brain connected in the most anterior region; some lateral branches projecting into the new head periphery can be observed; however, this is absent in the ethanol-exposed group. Original CNS structure, with new spongy brain and connections through several transverse commissures, can be observed in control worms 6 days postamputation; this is not observed in ethanol-exposed worms until 8 days postamputation. This delayed development of normal brain morphology aligns with the delay in reacquisition of photophobic behavior in planarian flatworms.

Behavioral (Figure  3A) and anatomical (Figure  3B) differences between naïve and ethanol-exposed animals suggest that ethanol exposure induces a recoverable 48-h delay in neuroregeneration. There is no statistical difference when comparing the 48-h accelerated ethanol group to the control group (ie, data from day 6 of the ethanol-exposed group is compared with data from day 4 of the control group, etc.; p > .05; Figure  3C). Time offset of nervous system staining also rectifies differences in morphology (Figure  3D).

Mature worms are not anatomically or behaviorally affected by ethanol exposure (Figs.  3E and 3F and Supplementary Figure 1). Nonhead-regenerating, adult planarians were left continuously exposed for a full 9 days for direct comparison of effects on neurological morphology and CFS to head-resected samples. Robust cephalic ganglia are present in both control and ethanol-exposed nonregenerating worms. Eyespots indicate a visual system in the tested specimens that are interconnected with the bilobed brain region. Therefore, the mechanism of chemical exposure is detrimental to the sensitive regenerative period of planaria and not a result of prolonged exposure.

Bisphenols Attenuate Reacquisition of Photophobic Behavior and CNS Morphology

In order to assess the effects of bisphenol exposure on planarian neuroregeneration, head-regenerating Smed were exposed to 2 ppm BPA, BPF, or BG throughout the regeneration period (Figure  4). It has been previously reported that 17β-estradiol (estradiol) and BPA have comparable in vitro estrogenic activity (Singleton et al., 2004), so estradiol (2 ppm) was included as a positive control. Chemical exposure induced statistically significant time-dependent changes in the reacquisition of behavior. All factors (treatment, time) and interactions (treatment: time) were significant in a mixed model ANOVA (p < 1 × 10⁻⁹, p < 1 × 10⁻¹⁶, p < .02, respectively) and post hoc Tukey HSD analyses identified significant differences between treatment groups.

Figure 4.

Bisphenols attenuate reacquisition of photophobic behavior and central nervous system morphology. A, Cognitive function scores (CFSs) of planaria exposed to bisphenols show a range of impacts on photophobic behavior [Control—gray, Estradiol—blue, bisphenol A (BPA)—yellow, bisphenol F (BPF)—green, and bisguaiacol (BG)—red]. All data are scaled to the maximum average control CFS (day 6) and data points are staggered slightly for visualization purposes. For each chemical n = 10. Error bars represent the standard error of the mean. * indicates statistical significance between BG and control (p < .05). ^ indicates statistical significance between BPA and control (p < .05). + indicates statistical significance between BPF and control (p < .05). B, Immunohistochemistry of the planarian neuropil and synaptic clefts show stark differences in the anatomy of worms exposed to bisphenols during head regeneration A condensed regenerative span of 4–7 days shows the crucial period of cephalic ganglia regrowth, when the lateral branching of neural synapses should occur. Cephalic ganglia region is located in the anterior region of the worms and is connected ventrally to the animals’ nerve cords. Anti-SYNORF primary and Dylight (488) conjugated secondary (green) with Hoescht 33342 counterstain (blue). Scale bar is 100 µm.

Immunohistochemistry analysis of the planarian neuropil and synaptic clefts show stark differences in the anatomy of worms exposed to bisphenols during head regeneration (Figure  4B). The naïve CNS shows a dense connection of commissures at the most anterior region after 6 days of regeneration. Large clusters of neurons are seen in the bilobed brain region indicating the maturation of this group.

All bisphenols induced attenuations to neuroregeneration (Figure  4), both in behavioral reacquisition (Figure  4A) and CNS morphology (Figure  4B). Effects can be broadly classified into 2 nonmutually exclusive categories: suppression (magnitude) and delay (temporal).

Small Structural Changes Amongst Bisphenols Lead to Distinguishable Impacts on Neurodevelopment

As illustrated in Figure  1, the selected bisphenols differ due to small variations in conformation or included functional groups but maintain similar chemical structures. However, exposure during head regeneration led to differential behavioral and morphological deviations.

BPF is one of the first industry BPA-alternative plasticizers and is currently in widespread commercial use (Liao and Kannan, 2013). Exposure to BPF induced a delay in reacquisition of both behavioral and CNS morphology without suppression (Figure  4). Conversely, BPA and BG induced both suppressive and delayed reacquisition of photophobic behavior (Figure  4A). Interestingly, the behavioral manifestations of BG exposure are not mirrored in CNS morphology (Figure  4B). BG is a bio-based bisphenolic analog and potential sustainable alternative in the production of epoxy thermosets (Hernandez et al. 2016), among other polymeric materials.

BPA Exposure Induces Delayed and Suppressed Neuroregeneration

Exposure to 2 ppm BPA produces drastic delays and suppression of planarian neuroregeneration (Figs.  4 and 5). However, the time-shifted and magnitude-scaled behavioral responses are indistinguishable from developing naïve worms (Figure  5A). This suggests that the underlying processes of neurodevelopment are conserved across treatment groups. Moreover, the cephalic ganglia of BPA-exposed planaria failed to regenerate completely within the observed developmental period (Figure  5B). Estradiol-, BPF-, and BG-exposed planaria have commissures connecting the 2 hemispheres of the cephalic ganglia 6 days postamputation. BPA, however, shows a severe lack of connection between nerve cords and longitudinal spreading of the brain primordia into the head periphery. This provides a potential explanation for the behavioral manifestation and suggests that the photophobic behavioral reacquisition metric is capable of describing complex exposure-induced effects on neuroregeneration.

Figure 5.

Exposure to 2 ppm bisphenol A (BPA) leads to complex, drastic delays and suppression. A, Time-offset and magnitude-scaled behavioral responses of naïve (black) and BPA-exposed (yellow) planaria suggest a common trajectory where BPA induces delay and suppression. Error bars represent the SEM. B, Immunohistochemistry images of BPA 7–9 days postamputation shifted to align with Control 4–6 days postamputation, as in (A), elucidate complexity beyond delay that likely manifests as suppression in the behavioral assay. Anti-SYNORF primary and Dylight (488) conjugated secondary (green) with Hoescht 33342 counterstain (blue). Scale bar is 100 µm.

Bisphenolic Effects on Neuroregeneration Are Nonestrogenic

Due to some structural homology (see Figure  1), unconjugated BPA has been shown to act as a weak xenoestrogen and disrupt endocrine activity (Akash et al., 2020). Bioactive BPA binds both nuclear estrogen receptors (ESR1 and ESR2) with 0.1%–to 0.01% the affinity of its endogenous agonist, estradiol, and induces ESR1- and ESR2-mediated gene expression (Wetherill et al., 2007). BPA can also activate gene expression and production of ESR1 itself (an effect that seems to be conserved across species) and activate rapid signaling via nonnuclear receptors with equivalent potency as estradiol (Bhandari et al., 2015). BPA alternatives and other bisphenols have also been shown to induce estrogenic effects (Cano-Nicolau et al., 2016). Although planaria possess robust estrogenic systems, estradiol exposure did not affect planarian neuroregeneration in either behavorial or morphological assessments (Figure  4). This suggests that the bisphenol-induced effects are nonestrogenic.

DISCUSSION

Planaria have been used to investigate exposure-induced effects on regeneration and development, primarily via surrogate measures of behavioral alterations (Hagstrom et al., 2015, 2019; Lowe et al., 2015; Zhang et al., 2019a,b). Here, we present the first evidence that behavioral phenotypes observed following chemical exposure are temporally correlated to anatomical development of the planarian CNS. This substantiates serial behavioral assessment as a viable, sensitive approach for discovering and classifying exposure-induced disruptions to the complex processes of neuroregeneration in planaria.

Exposure to low concentrations of ethanol during head regeneration has been shown to delay reacquisition of photophobic behavior in Smed (Lowe et al., 2015). Here, we show that this delay is also observed in brown planaria and that it is of the same approximate magnitude and relative timing as the delayed development of the cephalic ganglia in ethanol-exposed Smed. Since photophobic behavior of planaria is related to the integrity of the axonal connections of visual neurons to CNS structures in naïve planaria (Inoue et al., 2004), and ethanol exposure-induced delays are temporally correlated in behavior and CNS morphology (Figure  3), this indicates that exposure-induced changes to photophobic behavior are sufficient to identify DNT effects in ethanol.

Another compound with suspected DNT effects is BPA. In adults, approximately 80% of BPA is metabolized via the uridine 5′-diphospho-glucuronosyltransferase (UGT) system to BPA-glucuronide, and a small portion of BPA is converted to BPA-sulfate by cytosolic sulfotransferase; neither conjugated form binds to estrogen receptors (Taylor et al., 2011; Teeguarden et al., 2005; Völkel et al., 2002). As a result, bioactive BPA has a short residence time in the human body. However, expression and activity of UGT enzymes are reduced in human fetuses, and BPA and BPA-sulfate have been shown to be present in much higher concentrations than BPA-glucuronide in mid-gestation umbilical cord serum (Gerona et al., 2013; Hines, 2008). This has led to concern that accumulated bioactive BPA levels in developing fetuses could be considerably higher than those measured in adults, and this has precipitated studies to define and characterize the consequences of prenatal BPA exposure.

Several groups have investigated the impact of prenatal BPA exposure on neurodevelopmental outcomes in children (eg, Braun et al., 2009, 2011; Evans et al., 2014; Harley et al., 2013; Miodovnik et al., 2011; Perera et al., 2012; Yolton et al., 2011). Most of these studies found behavioral effects, but there seems to be a wide distribution of both the nature of effects across sexes and the degree of impairment. In her 2013 review, Rochester summarizes the outcomes of these studies with, “on the whole, the studies strongly suggest that BPA is associated with neurobehavioral problems in children” (Rochester, 2013), suggesting that BPA directly or indirectly modulates neurodevelopmental processes in humans. However, due to the ethical considerations involved in studying prenatal exposure in humans, human studies can only reveal correlations, not causation. Consequently, investigators have turned to using in vitro assays (eg, Kong et al., 2013; Trapphoff et al., 2013) and animal models to characterize the effects of prenatal BPA exposure, including studies in rodents (Angle et al., 2013; Mathisen et al., 2013), birds (Mathisen et al., 2013), and aquatic life (Bhandari et al., 2015; de Kermoysan et al., 2013; Hulak et al., 2013; Saili et al., 2012; Wang et al., 2013). From these studies, it is clear that prenatal BPA exposure has the capacity to alter developmental processes. Our data show that BPA has profound, nonestrogenic effects on planarian head regeneration and no observable effects in intact, nonregenerating worms exposed for a similar duration. This may lend insight into differential toxicity across lifecycles.

In general, bisphenols are a class of chemicals characterized by 2 hydroxyphenyl functionalities and are common plastic components and additives. Due to increased concern over the safety of bisphenols in consumer goods, many researchers and engineers have attempted to produce BPA analogs that preserve the advantageous material properties of the molecule while eliminating toxic activity. It is because these new alternatives are structurally similar to BPA that there is an increased need to evaluate any exposure-induced DNT related to them. It has been shown that small structural changes, including conformational isomers, between chemicals can lead to differences in toxic phenotypes (Blagg, 2006; Peng et al., 2018; Smith, 2009). Here, our data elicit distinct effects induced by exposure to structurally similar bisphenols on regeneration in planaria (Figure  4).

In contrast to some of the bisphenol-induced effects, DNT effects of ethanol exposure were recoverable delays; planaria exposed to 1% v/v ethanol during head regeneration returned to CFS levels statistically indistinguishable from control within 6 days postamputation. However, exposure to other exogenous chemicals induced additional behavioral phenotypes that elicited more severe DNT effects. BPA and BG led to both temporally delayed and consistently suppressed CFSs that were correlated with more substantial delays and defects in CNS regeneration (Figure  4). These effects were not observable in nonregenerating, intact animals exposed for a similar duration (Supplementary Figure 2) indicating that the chemicals affect regenerative processes directly. Importantly, some effects, especially temporal shifts, are not observable without serial testing throughout the regenerative period. For example, ethanol and BPF are statistically indistinguishable from control on day 9 but are affected by exposure during the regenerative period. Access to this sensitive behavioral readout throughout the regenerative period suggests that planaria are perfectly positioned to assess intermediate effects of chemical exposure and provide early indications for DNT potential.

We have shown that serial behavioral assessments accurately describe chemical exposure-induced changes to neurodevelopmental morphology in planaria. These results suggest that behavioral data, which can be collected rapidly and at low cost, may provide an acceptable surrogate to immunohistochemical studies, which are time-consuming, tedious, and expensive to produce. These low cost, high-yield behavioral studies provide an advantageous tool in advancing the future of chemical screening and can be easily adapted to high-throughput screening platforms. Implementing high-throughput screening techniques to planarian photophobic behavior analysis would allow for testing spanning the entire neurodevelopmental process following chemical exposure. The design of experiments can be augmented to describe dynamic changes to neurodevelopment and include more variables such as dose, duration, and timing of chemical exposure. This influx of exposure-induced toxicity data may open the door for better understanding of the etiology of chemical toxicity, as well as catalyze the informed design of safer “smart” industry alternatives to potentially toxic chemicals.

SUPPLEMENTARY DATA

Supplementary data are available at Toxicological Sciences online.

AUTHOR CONTRIBUTIONS

J.M., E.J.B., and M.M.S. conceived and planned the experiments. J.M., E.J.B., L.R., and H.B. carried out the experiments and interpreted results. J.M. planned and carried out the simulations. I.S. and J.F.S. led chemical purity assessments and efforts. J.F.S. and M.M.S. also contributed to the interpretation of the results. J.M. and E.J.B. led writing the article. All authors provided critical feedback and helped shape the research, analysis, and article.