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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Neurotoxicology. 2021 Sep 28;87:156–166. doi: 10.1016/j.neuro.2021.09.006

Developmental toxicity of bisphenol S in Caenorhabditis elegans and NODEF mice

Callie M McDonough a, Daniel J Guo b, Tai L Guo a,*
PMCID: PMC8595854  NIHMSID: NIHMS1745598  PMID: 34597708

Abstract

The growing concern surrounding bisphenol A (BPA) has led to increased industrial production and application of its analog bisphenol S (BPS). The goals of this study were: (1) To examine the generational effects in the nematode C. elegans for up to three generations following developmental exposure to BPS (0.1, 1.0, 5.0 and 10.0 μM), and (2) To examine the neurotoxicity and metabolic toxicity in NODEF mouse offspring exposed to BPS (3 μg/kg BW) in utero throughout gestation once/day via oral pipette. First, worms were exposed to BPS developmentally for a single period of 48 hours and then propagated for 2 additional generations. Exposure to 0.1 and 1.0 μM BPS decreased lifespan and the number of progeny with an ability to recover in subsequent generations. In contrast, worms exposed to 5.0 or 10.0 μM BPS exhibited a continuous effect in the second generation, e.g., decreased lifespan and reduced number of progeny. Only worms exposed to 10.0 μM BPS continued to have a significant long-term effect (e.g., decreased lifespan) through the third generation. In addition, worms developmentally exposed to BPS at 5.0 μM and 10.0 μM also showed decreases in body bends. In contrast, worms exposed to 0.1 μM BPS exhibited a significant increase in head thrashes. When the multigenerational effects were examined by exposing worms to BPS for 48 hours developmentally at each generation for three generations, an accumulative effect was observed in worms treated with 0.1 or 1.0 μM BPS for two generations, but not for three generations, suggesting a threshold existed. Worms exposed to either 5.0 or 10.0 μM BPS demonstrated accumulative effects through two and three generations. When the developmental effects of BPS were studied in NODEF mice, offspring exposed gestationally exhibited behavioral deficits at 12, but not at 3, weeks of age. Specifically, female offspring had decreases in working and short-term memories while male offspring showed increases in hyperactivity and anxiety-like behaviors. In summary, this study demonstrates the sex-related effects of BPS in NODEF mouse offspring exposed in utero, along with the generational effects observed in C. elegans.

Keywords: Bisphenol S, behavior, glucose, generational effects, C. elegans, NODEF mouse, developmental toxicity

1. Introduction

The growth of fetuses and infants depends on the production and secretion of several tightly regulated hormones during the periods of gestation and early development, making them susceptible to the exposure of endocrine disrupting chemicals (Gore et al. 2015). Bisphenol A (BPA) is an industrial chemical often found in polycarbonate plastics for making containers to store food and beverages and epoxy resins for coating the inside of metal products, such as food cans, bottle tops and water supply lines. BPA may act through several different receptor-mediated mechanisms of action to disrupt the endocrine system. It is a xenoestrogen that binds to and activates estrogen receptors (ERs), both ER alpha and beta, to potentially cause system-wide toxicity (Xu et al. 2016), including, but are not limited to, neurological deficits, exacerbation of type 1 diabetes (T1D), developmental and reproductive toxicity (Castro et al. 2015; Jandegian et al. 2015; Mersha et al. 2015; Manshack et al. 2016; Qiu et al. 2016; Mersha et al. 2018; Schirmer et al. 2021). Growing public concerns over BPA have led to a shift towards “BPA-free” products, which contain bisphenol analogues, with bisphenol S (BPS) being the most common. BPA and BPS are structurally related, and recent studies have shown that BPS also binds to ERs, resulting in similar toxicities as BPA (Naderi et al. 2014; Castro et al. 2015; Mersha et al. 2015; Qiu et al. 2016; LaPlante et al. 2017; Zhao et al. 2017; Gingrich et al. 2018; Gu et al. 2019; Xu, Huang, Guo 2019; Naderi et al. 2020). However, detailed studies on the neurological and reproductive toxicities following developmental exposure of BPS are still lacking.

The nematode C. elegans is a common model used to study reproductive toxicity and lifespan. To date, there are a limited number of studies examining the reproductive effects of BPS in C. elegans (Mersha et al. 2015; Zhou 2018; Xiao et al. 2019), including only one multigenerational study. In addition, the only study examining more than one generation employed a continuous exposure regimen (Xiao et al. 2019), and thus the generational effects of BPS in subsequent generations after a single exposure are unknown. One goal of this study was to investigate the reproductive and behavioral effects of BPS in C. elegans by exposing age-synchronized eggs to BPS for 48 hours, a developmental stage prior to the time when worms reach maturity and are able to lay eggs (e.g., the L4 stage), for one or multiple generations. The period of 48 hours was chosen to ensure that worms in the lifespan assay would have enough time to absorb the reproductive inhibitor 5-Fluoro-2′-deoxyuridine (FUDR) and that all offspring of the worms used in the fertility assay were counted. Although bioamine neurotransmitters such as dopamine and serotonin have been implicated in worm locomotive activities (Mesce and Pierce-Shumomura 2010), there are limitations in using C. elegans to examine the neurological changes. Previous studies have also shown that developmental BPA exposure could exacerbate T1D in female offspring of non-obese diabetic (NOD) mice (Bodin et al. 2014). To assist in the evaluation of metabolic toxicity and neurotoxicity, the NOD excluded flora (NODEF) mice were additionally utilized to examine glucose homeostasis and behavioral changes in mouse offspring exposed in utero throughout gestation. NODEF mice, which have an excluded flora that is protective against T1D, are promoted by Taconic Biosciences to replace NOD mice, a strain previously used as a model for T1D, with the hope of increasing the rate of T1D incidence.

Previous studies have shown that exposure to BPA at an external concentration of 1 mM resulted in an uptake of approximately 2 μg BPA/g worm extract (~2 ppm) in C. elegans (Allard and Colaiacovo 2011). Based on these results and taking into consideration the BPS doses used by Xu et al. (2019) in their mouse studies, environmentally relevant BPS concentrations (0, 0.1, 1.0, 5.0 or 10.0 μM) in C. elegans and a BPS dose at 3 μg/kg body weight (BW) in NODEF mice were selected for exposure in our studies. The 3 μg/kg BW dose used in our gestational mouse study is approximately equivalent to an external exposure of 1.5 μM in C. elegans according to the reported measurements (Allard and Colaiacovo 2011). It was hypothesized that C. elegans exposed to BPS for one generation would exhibit a decreased lifespan and lowered fertility in the subsequent generations, while worms exposed for multiple generations would have accumulative decreases in these endpoints. In addition, BPS-exposed mice would have a sex-related effect on the incidence of T1D, glucose sensitivity, and behavioral changes.

2. Methods

2.1. C. elegans Model

2.1.1. Preparation of nematode growth medium (NGM) petri plates and BPS exposure

BPS or 4,4′-sulfonyldiphenol (purity: 98%, stable at room temperature) was obtained from Sigma (St. Louis, MO). BPS was dissolved in ethanol and further diluted with K buffer (3.04 g/L NaCl, 2.39 g/L KCl) to make a 100 mM stock solution containing 10% ethanol as previously described by Mersha et al. (2018). NGM agar (2.3 g/L NaCl, 15 g/L agar, 20 g/L peptone, 1 mM KH2PO4 [pH=7.0]) was prepared and autoclaved according to the WormBook (Stiernagle 2006). Once the agar cooled to 55°C, cholesterol (1 mM), CaCl2 (1 mM), and MgSO4 (1mM) were added and mixed into the agar along with BPS at various concentrations to prepare plates with BPS at final concentrations of 0 (vehicle), 0.1, 1.0, 5.0, and 10.0 μM. Plates were then seeded with E. coli OP50 and incubated at 37 °C overnight. There were no apparent differences between the control and BPS-containing plates in terms of E. coli growth.

2.1.2. Age-synchronization and F0 exposure of C. elegans to BPS

The C. elegans strain N2 was obtained from Dr. Lili Tang (Dept. of Environmental Health Science at the University of Georgia), who originally procured it from the Caenorhabditis Genetics Center (Minneapolis, MN). Worms were grown and maintained at 20 °C. During assays, worms were in the laboratory that had a temperature range between 20–21 °C. Once the NGM plates had sufficient C. elegans growth, they were age-synchronized using the bleaching method (Stiernagle 2006). Briefly, a worm pellet was collected in a 15 mL conical tube and then exposed to a hypochlorite solution (1N) containing 5% household bleach. Tubes were vortexed until all worms appeared dead. The pellet was then washed with K buffer and centrifuged. The intact eggs remaining (F0) were transferred to seeded NGM plates containing 0, 0.1, 1.0, 5.0, or 10.0 μM BPS and allowed to hatch and grow for 48 hours. These F0 worms were used for behavioral, fertility, or lifespan assays following BPS exposure as described below (Figure 1A). As mentioned in the introduction, the period of 48 hours was chosen to allow sufficient time for worms to absorb FUDR in the lifespan assay and to ensure that all offspring used in the fertility assay were counted (described below).

Figure 1.

Figure 1.

Figure 1.

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Outlines for generational studies in C. elegans. (A) Behavioral, fertility and lifespan assays for F0 C. elegans following BPS exposure. (B) Recovery studies following a single exposure of BPS during the developmental stage. (C) Accumulative studies following developmental BPS exposure for multiple generations. To ensure there were no effects from bleaching, each generation of the study had a vehicle control (e.g., without BPS). Since there were no differences among them for the parameters measured, they were combined and presented as the control in Figures 2–5.

2.1.3. Behavioral assays in the F0 worms

After the eggs hatched and grew on the new NGM plates with or without BPS for 48 hours as described above, the F0 worms were transferred individually to a 96-well flat bottom plate containing 5 μL K buffer for the behavioral assays, with one worm per well (Figure 1A). Worms were allowed to acclimate to the new environment for one minute, and were then recorded for one minute using an iPhone and Gosky Universal Cell Phone Adapter Mount. The number of head thrashes in the given minute was manually counted and recorded. A head thrash is defined as a change in the direction of bending at the mid-body, observed as a change in direction of the upper pharynx along the Y-axis, assuming that the nematodes are moving along the X-axis (Wu et al. 2013). For the body bend assay, worms were recorded for 20 seconds using the same method described above following one minute of acclimation to the new environment, and the number of body bends was counted and recorded. Body bends are defined as a change in the direction of the posterior bulb part of the pharynx along the y axis when the worm is traveling along the x axis (Wu et. al 2013). In each experiment, there were at least 15 worms per treatment for both the head thrash and body bend measurements. Both assays were repeated 3 times to ensure accuracy.

2.1.4. Recovery study following a single exposure of BPS

For the recovery study, F0 worms exposed to BPS for 48 hours as described above were transferred to normal NGM plates without BPS and allowed to fully mature and lay eggs for another 48 hours. Worms were then age-synchronized to obtain the F1 progeny by transferring the eggs onto fresh NGM plates without BPS (Figure 1B). These eggs were allowed to hatch and grow for 48 hours. Thereafter, the F1 worms were used for lifespan and fertility assays. The remaining F1 worms (>100) were placed on fresh NGM plates and allowed to fully mature and lay eggs for 48 hours. The same steps were repeated for the F2 progeny (Figure 1B).

2.1.5. Accumulative study following BPS exposure for multiple generations

For the accumulative study following BPS exposure for multiple generations (Figure 1C), F0 worms exposed to BPS for 48 hours as described above were transferred to normal NGM plates without BPS and allowed to fully mature and lay eggs for 48 hours. Plates were then age-synchronized to obtain the F1 progeny. The F1 eggs were transferred to fresh NGM plates with BPS and allowed to hatch and grow for 48 hours. Next the F1 worms were transferred via pipette and used for lifespan and fertility assays. The remaining F1 worms were placed on fresh NGM plates (without BPS) and allowed to fully mature and lay eggs for 48 hours. The same steps were repeated for the F2 progeny (Figure 1C).

2.1.6. Fertility assay

Following BPS exposure described above, the worms were used for the fertility assay (Figure 1). Specifically, worms were transferred to 96-well plates containing 5 μL K buffer and 0.5 μL concentrated OP50 suspended in LB broth (10 g/L Tryptone, 10 g/L NaCl, 5 g/L yeast extract, 1mM NaOH [pH=7]). The concentrated OP50 was prepared by incubating the OP50 in the LB broth overnight to allow for maximum growth. Each row of the 96-well plate had 2 worms on opposite sides. Twenty-four hours after transfer, each worm was examined for egg laying. Worms that had started laying eggs were transferred to the adjacent well. This was repeated daily until all worms had finished laying. Total offspring (number of offspring hatched + number of unhatched eggs), number of unhatched eggs, number of offspring hatched (L1) and lethality rates (number of unhatched eggs/total offspring × 100) were measured and recorded daily. Worms that failed to lay eggs, were excluded from study. For each experiment, approximately 16 worms were used per treatment. The experiment was repeated 3 times to ensure accuracy.

2.1.7. Lifespan assay

The reproductive inhibitor FUDR was obtained from MP Biomedicals, LLC (Irvine, CA) and dissolved in filtered DI water to create a 150 mM stock. Following a 48-hour BPS exposure described above (Figure 1), worms were transferred to wells in a 96-well plate in which each well contained 2 μL FUDR, 5 μL K buffer and 0.5 μL OP50-seeded LB broth. Twenty-four hours after transfer, worms were assessed for viability (dead or alive) with any eggs/offspring removed using a pipette tip, which was repeated daily until all worms had died. The seeded LB broth was replenished as needed. For each experiment, approximately 100–200 worms were used per treatment. The experiment was repeated 3 times to ensure accuracy.

2.2. NODEF Mouse Study

2.2.1. Animal husbandry and BPS exposure

NODEF mice were initially obtained from Taconic Biosciences (Germantown, NY). A breeding colony was established and housed in Coverdell animal facility at the University of Georgia (UGA) in polysulfone cages with irradiated laboratory animal bedding and Bed-r’Nest for enrichment (The Andersons Inc., Maumee, Ohio). Negligible amounts of BPA have been reported to leach from new or used polysulfone cages maintained at room temperature (Delclos et al. 2016; Johnson et al. 2016). They were kept at 22–25°C with a relative humidity of 50±20% and a 12-hour light/dark cycle. Filtered water was provided ad libitum through the animal facility’s automatic watering system. Breeder Chow (PicoLab Rodent Diet 5058) or a regular growth diet (PicoLab Rodent Diet 5053) was provided ad libitum. All animals were treated humanely and with regard to alleviating animal suffering. An approved animal protocol by the UGA Institutional Animal Care and Use Committee (IACUC) was followed for all procedures.

Eleven time-mated adult female NODEF mice were divided into the treatment (n=6) or vehicle (n=5) group. There were no significant differences between the two groups in the initial BWs and non-fasting blood glucose levels (BGLs). From breeding day one to parturition, pregnant mice were dosed daily with either vehicle (Control) or 3 μg/kg BW BPS via oral pipette. BPS was dissolved in 100% ethanol and added to corn oil at a final concentration of 0.05% ethanol. Offspring from both the control and BPS-exposed dams were weaned at postnatal day 21, and pups were sexed and distributed to separate cages by treatment at random. To eliminate litter effect, each cage had pups from different litters.

2.2.2. Body weight, blood glucose measurement, and diabetic incidence

BWs of offspring were measured weekly starting from the age of 1 week old using a scale (TE1502S; Denver Instrument; Bohemia, NY). Non-fasting BGLs were measured starting from the age of 3 weeks old using a Prodigy Autocode Blood Glucose Meter (Charlotte, NC) by nicking the tail of each mouse to allow for collection of a small sample of venous blood. Thereafter, weekly BWs and non-fasting BGLs were measured and recorded. Mice with non-fasting BGLs higher than 250 mg/dL for two consecutive weeks were considered diabetic (Guo et al. 2014). If non-fasting BGLs were 600 mg/dL or higher for two consecutive weeks, the mice were euthanized humanely using CO2 asphyxiation followed by dislocation of the cervical vertebrae. All remaining mice were euthanized using this method at the end of the study. Female offspring were euthanized at 22 weeks of age and male offspring at 25 weeks of age. Females were euthanized earlier due to high incidence of T1D in the control group. According to Taconic Biosciences (https://www.taconic.com/mouse-model/nod), NOD mice diabetes occurrence should reach 50% incidence in both sexes at 13–15 weeks of age. Our studies extended beyond that time point to ensure maximal diabetes incidence was reached. Nonetheless, males in our control and treatment groups did not reach above 30% T1D incidence.

2.2.3. Glucose tolerance and insulin tolerance tests in offspring

At 17 weeks of age, both the glucose tolerance (GTT) and insulin tolerance tests (ITT) were performed on offspring. For the GTT (Susiarjo et al. 2015), mice were fasted overnight (approximately 16 hours), and then had their BWs and BGLs measured. Based on their respective weights, each mouse was injected intraperitoneally with 2 g/kg BW of glucose (Sigma). BGLs were measured 15, 30, 60, and 120 minutes after injection. For ITT (Cui et al. 2015), baseline BWs and non-fasting BGLs were obtained, followed by an intraperitoneal injection of 1.5 IU/kg BW insulin (Sigma). BGLs were then measured at 15, 30, 60, and 120 minutes after injection.

2.2.4. Behavior tests in offspring

Four behavioral tests were conducted to evaluate cognitive functions: Y-maze test for assessing working memory, tail suspension test for the evaluation of depression-related behaviors, open field test for measuring locomotion and anxiety-like behavior, and novel object test for short-term memory. A locomotor activity test was performed to verify that differences in behaviors were not due to physical impediment. At 3 weeks of age, the Y-maze, open field, and novel object tests were performed. At 12 weeks of age, behavior tests were repeated, with the addition of the tail suspension test.

Y-maze test

The Y-maze is an apparatus consisting of three arms of equal lengths that converge into a “Y” shape. The purpose of this test is to assess working memory by observing if the mouse can remember which arm it has already explored. In theory, mice should enter each arm without repeating a previous arm. A mouse was placed in one arm of the maze and recorded for 10 minutes. Distance traveled in each arm, number of entries into each arm, number of spontaneous alternations, sequential order of arm entries, and total number of arm entries were analyzed using ANYmaze (Stoeling, Wood Dale, IL). A spontaneous alternation is defined by the occurrence of a mouse entering a different arm of the maze in each of three consecutive arm entries (Miedel et al. 2017). It is calculated using the equation:

%SpontaneousAlternations=#spontaneousalternationstotalnumberofarmenteries−2*100
Tail suspension test

The tail suspension test was used to evaluate depression-related behaviors in mice (Can et al. 2012). In brief, mice were hung by their tails with medical tape for six minutes, and the amount of time mobile/immobile was calculated. Mice that spend more time mobile were considered to have less depression-related behavior.

Open field test

The open field test measures anxiety-like behavior and locomotion. In our studies, a 25×25 cm apparatus was used for the 3-week-old mice. When retested at 12 weeks, a 45×45 cm apparatus was used to allow mice more space to wander. Mice were placed in the center of the apparatus and allowed to wander freely for ten minutes. Distance traveled, time spent in the corners, time spent in the edges, and time spent in the center were measured. The percentage of time spent in the center, corners, and periphery, along with the distance traveled, were calculated using ANYmaze (Stoeling, Wood Dale, IL). Mice that are anxious would spend more time in the corners and edges than in the center.

Novel object test

The novel object test has been used to assess short-term memory, which is conducted in 3 intervals (Wang et al. 2016). During the first interval, the habituation period, mice were placed in the same apparatus as the open field test and allowed to freely explore it for ten minutes. The purpose of this procedure was to allow the mice to get familiarized with the apparatus. This interval also served as our open field test. Twenty-four hours following the habituation period, mice were placed in the center of the same apparatus. Inside the apparatus were two identical objects (familiar objects) placed in adjacent corners, far enough from the corners to allow mice to examine the object from all sides. To prevent moving of the objects, mounting putty was placed on the bottom. Mice were allowed to explore the familiar objects for ten minutes (this is often referred to as the learning period). The final interval tests short-term memory. Approximately 1.5 hours following the learning period, mice were placed in the same apparatus, however one of the objects had been replaced with a new object (novel object). Again, mice were allowed to explore the apparatus and objects freely for ten minutes. The time spent exploring each object was calculated, and the object bias index scores were calculated for the novel object using the equation:

Index=TimespentexploringthenovelobjectTimespentexploringfamilarobject+Timespentexploringnovelobject

A 25 × 25 cm apparatus was used for 3 weeks old mice and a 45 × 45 cm was used when mice reached 12 weeks of age to allow mice more space to wander. The familiar objects were dice for the 3 weeks old mice and wooden blocks for the 12-week-old mice. The novel object was a marble for 3-week-old mice and a ping pong ball for 12-week-old mice.

2.3. Statistics

The rates of diabetes development and total diabetes incidence over time were analyzed with Likelihood ratio and Logrank test, respectively. For all other data sets (e.g., behavior tests, weekly non-fasting BGLs, and weekly BWs in mice, average lifespan and time spent laying, total number of progeny, number of body bends and number of head thrashes in C. elegans), Dunnett’s test (VH as the reference group) was used for homogeneous data and Wilcoxon test for non-homogeneous data, determined by unequal variances analysis using Bartlett’s test. A group was considered statistically significant if p < 0.05. JMP Pro 13 (SAS Inc., Cary, NC) and GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA) were used for statistical analysis and data visualization.

3. Results

3.1. C. elegans

3.1.1. Recovery study in C. elegans following a single 48-h exposure to BPS

Developmental BPS exposure in the F0 generation for 48 h produced a significant decrease in the average lifespan in a concentration-related manner (Figure 2A). However, such single BPS exposure in F0 worms at 0.1 μM did not produce significant effects on the average lifespans in the subsequent generations (F1 and F2) when compared to the control (Figure 2B). There was also a significant increase of lifespan in both F1 and F2 worms when compared to F0 worms at 0.1 μM BPS. Taken together, this suggests a recoverable effect. Similar to the reversible effect observed at 0.1 μM BPS, worms treated with 1.0 μM BPS in the F0 generation did not exhibit significant effects in the subsequent generations when compared to the control (Figure 2C). Additionally, there were significant increases in the average lifespan in both F1 and F2 generations when compared to F0 or each other. Worms exposed to 5.0 μM BPS during the F0 generation had a significant effect in the F1, but not the F2 generation, when compared to the control with regard to lifespan (Figure 2D). In addition, both the F1 and F2 generations exhibited a significant increase in lifespan when compared to F0, also supporting a reversible effect. For the 10.0 μM BPS exposure (Figure 2E), similar observations were made as the 5.0 μM BPS treatment except that the F2 generation continuously had a decreased lifespan when compared to the control. However, there was still some recovery when compared to the F1 generation.

Figure 2.

Figure 2.

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Lifespan (A-E) and fertility (F-J) assays for C. elegans following a single 48-h exposure to BPS during the developmental stage. Shown are average lifespans for F0 worms at 0–10 μM BPS (A), and for F0, F1 and F2 worms at 0.1 μM (B), 1.0 μM (C), 5.0 μM (D) and 10.0 μM (E), respectively. N=350–500. For panel B, the data for F0 and F1 lifespan were non-homogeneous, and they were compared using the Wilcoxon test. Also shown are the numbers of offspring hatched per worm for F0 at 0–10 μM BPS (F), and for F0, F1 and F2 worms at 0.1 μM (G), 1.0 μM (H), 5.0 μM (I) and 10.0 μM (J), respectively. N=30–50. The values are presented as mean ± SEM. a = significant compared to the control, b = significant compared to F0, and c = significant compared to F1. p < 0.05. F0, F1 and F2 were described in Figure 1.

In terms of fertility, there was a significant decrease in the number of offspring hatched following BPS treatment at 1.0, 5.0, and 10.0 μM for the F0 generation (Figure 2F). However, there were no significant differences in the number of offspring hatched in any generation (F0, F1, or F2) of worms exposed to 0.1 μM BPS when compared to the control or each other (Figure 2G). Worms treated with 1.0 μM BPS in F0 had no significant changes in the number of offspring hatched in either F1 or F2 generation when compared to the control. In contrast, the number of offspring hatched was significantly increased in both the F1 and F2 generations when compared to F0 (Figure 2H). These observations suggested an ability to recover. Worms exposed to 5.0 μM BPS during F0 had a decrease in offspring hatched in the F1 generation when compared to the control (Figure 2I), suggesting a continued effect. However, when the F2 generation was compared to the control, there was no difference in the number of offspring hatched, supporting an ability to recover. In support of this notion, there was a significant increase in the number of offspring hatched when the F1 and F2 generations were compared to F0. Worms exposed to 10.0 μM BPS during F0 had a significant decrease in the number of offspring hatched in F0 and F1 when compared to the control (Figure 2J), consistent with a continued effect. In contrast, the F2 generation exhibited no significant difference in the number of offspring hatched when compared to the control, suggesting reduced toxicity and an ability to recover. In agreement with this observation, the F2 generation worms had a significant increase in offspring hatched when compared to F0.

Both the embryonic lethality and number of offspring hatched per day were not significantly different among the treatment groups or generations (data not shown). In addition, there were no differences in the age of worms when they started laying eggs for any concentrations of BPS tested (0–10 μM) or in any of the generations (data not shown). In contrast, F0 worms exposed to 1.0, 5.0 or 10.0 μM BPS had a decrease in the number of days spent laying eggs when compared to the control (Figure 3A), suggesting reproductive toxicity. However, none of the subsequent generations exhibited a decrease in the number of days spent laying eggs at any BPS concentrations tested (data not shown), suggesting a reversible effect and ability to recover.

Figure 3.

Figure 3.

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Days spent laying and behavioral assays in F0 C. elegans following developmental exposure to BPS for 48 hours. Shown are number of days spent laying (A), and the number of head thrashes (B) and body bends (C). The values are presented as mean ± SEM. a= significance compared to the control. N=30–70. For panel B, the data at 1 μM BPS were non-homogeneous and Wilcoxon test was thus used for comparison, although it was significant by Dunnett’s test when compared to control.

There were also behavioral changes in F0 worms following exposure of age-synchronized eggs to BPS for 48 hours. Worms exposed to 0.1 μM exhibited a significant increase in terms of head thrashes when compared to the control (Figure 3B). Additionally, there was a trend toward increased number of body bends in the 0.1 μM BPS group (Figure 3C; p=0.0877). However, there were significant decreases in the number of body bends in both the 5.0 and 10.0 μM BPS treatment groups (Figure 3C) when compared to the control, suggesting an impaired locomotion and toxicity at these concentrations.

3.1.2. C. elegans exposed to BPS for multiple generations

Exposure to BPS for 2 or 3 generations in worms had vastly different effects from those only exposed for one generation. Worms treated with BPS at 0.1 μM (Figure 4A) or 1.0 μM (Figure 4B) for two (F1) or three (F2) generations exhibited a significant reduction in average lifespan when compared to the control or F0, suggesting an accumulative effect. However, there were no significant differences in lifespan for either concentration when worms treated for two generations (F1) were compared to those treated for three generations (F2), suggesting a threshold effect. On the other hand, worms treated with BPS at 5.0 μM (Figure 4C) or 10.0 μM (Figure 4D) for two (F1) or three (F2) generations had significantly decreased lifespans when compared to the control or any other generations, consistent with an accumulative effect.

Figure 4.

Figure 4.

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Lifespan (A-D) and fertility (E-H) assays for C. elegans exposed to BPS for one to three generations. Shown are average lifespan for worms treated with BPS for one to three generations at 0.1 μM (A), 1.0 μM (B), 5.0 μM (C) and 10.0 μM (D). N=350–500. Also shown are the numbers of offspring hatched per worms following BPS treatment for one to three generations at 0.1 μM (E), 1.0 μM (F), 5.0 μM (G) and 10.0 μM (H). N=30–50. The values are presented as mean ± SEM. a = significant compared to the control, b = significant compared to F0, and c = significant compared to F1. p < 0.05. F0, F1 and F2 were described in Figure 1.

In terms of fertility, worms exposed to 0.1 μM for two (F1) or three (F2) generations had a decrease in the number of offspring hatched when compared to the control or any other generations (Figure 4E), suggesting an accumulative effect. Worms treated with BPS at 1.0 μM for two (F1) or three (F2) generations exhibited a reduction in total offspring hatched when compared to the control (Figure 4F). However, when the number of offspring hatched among different generations were compared, only the difference between the F0 and F2 generations was significant, consistent with a threshold effect. Similarly, worms exposed to BPS at 5.0 μM for two (F1) or three (F2) generations had a significant decrease in offspring hatched when compared to the control or the F0 generation (Figure 4G), suggesting an accumulative effect. However, there was no significant difference in the number of offspring hatched between the F1 and F2 generations, consistent with a threshold effect. Worms treated with BPS at 10.0 μM for three generations exhibited a reduction in number of offspring hatched when compared to the control and all other generations (Figure 4H), suggesting an accumulative effect.

The age of C. elegans when started laying eggs, and the number of days spent laying after developmental exposure to BPS (0–10 μM) for one to three generations were also assessed. Worms exposed to 0.1 μM BPS for two generations (F1) exhibited a significant increase in their age when worms started laying eggs in comparison to the control (Figure 5A). Similarly, worms treated with 5.0 μM for two or three generations exhibited an increase in the average age when they started laying eggs (Figure 5B). This increase in average age when they started laying eggs could signify a possible delay in maturation. In contrast, the number of days spent laying eggs was decreased in worms exposed to 1.0 μM BPS for one (F0) and two (F1), but not for three (F2), generations (Figure 5C). There was also a decrease in the number of days spent laying eggs in the worms exposed to 5.0 μM BPS for three (F2) generations when compared to the control (Figure 5D). However, there were no significant differences in the age of C. elegans when they started laying eggs and the number of days spent laying at other concentrations (data not shown). Taken together, this reduction in the number of days spent laying suggests a reduction in fertility.

Figure 5.

Figure 5.

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The age of C. elegans when they started laying eggs (A, C) and the number of days spent laying (B, D) following developmental exposure to BPS for one to three generations. C. elegans were treated for one (F0), two (F1), or three (F2) generations at 0.1 μM (A), 1.0 μM (B), and 5.0 μM (C, D). The values are presented as mean ± SEM. a = significant compared to the control. p< 0.05. N=30–50.

3.2. Gestational NODEF Study

There were no significant differences in BWs and non-fasting blood BGLs in female offspring at the weaning time (Figure 6A and C). In addition, there were no significant differences in weekly non-fasting BGLs between BPS exposed and the control female offspring at any time points (Figure 6C). However, there was a significant increase in the body weights of the BPS-treated female offspring when compared to the control at 18 weeks of age (Figure 6A). For male offspring, there were significant decreases in both BW (Figure 6B) and non-fasting BGLs (Figure 6D) at the time of weaning following BPS exposure when compared to the control. In addition, male offspring exposed to BPS had significantly lower non-fasting BGLs at the age of 12 weeks when compared to the control (Figure 6D). However, there were no significant differences in diabetes incidence at any time points between the groups in either the female (Figure 6E) or male pups (Figure 6F). Neither the GTT nor ITT were significant at any time points during the 2-hour assay for either the female or male offspring (data not shown). In terms of organ weights (absolute or relative weights), there were no significant differences in either the female or male offspring when compared to the control (data not shown).

Figure 6.

Figure 6.

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Body weights (BWs), glucose levels, and diabetes incidence in offspring following developmental BPS exposure. Shown are weekly BWs for female (A) and male (B) offspring, weekly non-fasting blood glucose levels for female (C) and male (D) offspring, and diabetes incidence for female (E) and male (F) offspring. A mouse with a blood glucose level ≥250 mg/dL was considered diabetic. VHM, vehicle males. VHF, vehicle females. N = 4–6. The values are presented as mean ± SEM. *, p< 0.05.

At the time of weaning, behavior tests (open field, novel object, and Y-maze) were performed, and there were no significant differences between the control and BPS treatment group in these assays (Supplementary Figures 1–2). In contrast, when mice were retested at 12 weeks of age, there was a significant decrease in spontaneous alternations in the BPS exposed female offspring when compared to the control (Figure 7A), suggesting an impairment in working memory. In contrast, no such change was found in male offspring (Figure 7B). In terms of the open field test, there were small increases in the distance traveled by the female offspring (Figure 7C; p=0.079), but not the male offspring (Figure 7D), following BPS treatment when compared to the control. This could indicate some increases in anxiety-like behaviors. When BPS-treated animals were compared to the controls, a nonsignificant decrease in time spent in the center in male offspring (p=0.076; Figure 7F), but not in female offspring (Figure 7E), was observed, also indicating an increase in anxiety-like behaviors in male offspring. Because there were no statistical differences between sexes for the parameters measured, both sexes were combined for further statistical analysis. It was found that BPS-treated offspring spent significantly less time in the center than the control mice (Figure 7G). Overall, the results of the open field test suggested that male offspring might be more affected than female offspring, with males exhibiting signs of increased anxiety-like behaviors.

Figure 7.

Figure 7.

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Y-maze and open-field behavioral tests in NODEF offspring at 12 weeks of age. Shown are % spontaneous alternation during the Y-maze test in female (A) and male (B) offspring, the distance traveled during the open-field test for female (C) and male (D) offspring, the time spent in each section during the open-field test for female (E) and male (F) offspring, and the time spent in each section during the open-field test with both sexes combined (G). VHM, vehicle males. VHF, vehicle females. N = 5–6. The values are presented as mean ± SEM. *, p< 0.05.

Similar to the open field test, there were no significant changes in the distance traveled between groups during the novel object test at the 12-week age in either male or female offspring (Data not shown). However, BPS-exposed females had a significantly decreased novel object index score when compared to the control group (Figure 8A). The BPS-treated males also had a decreased novel object index score (Figure 8B), although it did not reach the level of statistical significance (p=0.076). In addition, when both sexes were combined, BPS exposed offspring had a significantly decreased novel object index score when compared to the control (Figure 8C). Overall, these observations suggest that BPS exposure leads to an impairment in memory and object recognition, with females being impacted to a greater extent. Furthermore, the tail suspension test showed a significant increase in time spent immobile in the exposed male offspring (Figure 8E), suggesting an increase in depression-related behaviors. In contrast, there was no difference in time spent immobile in the exposed female offspring when compared to the control (Figure 8D).

Figure 8.

Figure 8.

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Behavioral tests in NODEF offspring at 12 weeks of age. Shown are the novel object index during the novel object test for female (A), male (B) offspring and both sexes combined (C), and the time spent immobile for female (D) and male (E) offspring during the tail suspension test. VHM, vehicle males. VHF, vehicle females, VH, vehicle. N = 4–6. The values are presented as mean ± SEM. *, p< 0.05.

4. Discussion and Conclusion

This study demonstrated that developmental exposure to BPS resulted in neurotoxicity in adult NODEF mice, and possibly in C. elegans. Although C. elegans are an excellent research model, there are limitations, especially in examining neurological changes. Worm behaviors are subjective and difficult to quantitate, and the locomotion has no direct relationships with most neurological changes. Nonetheless, the behavioral changes in F0 worms following developmental BPS exposure for 48 hours were investigated along with the behavioral effects of mice treated with BPS for one generation gestationally. There was a significant decrease in the number of body bends in worms treated with 5.0 or 10.0 μM BPS, suggesting an impairment in locomotion and toxicity. Interestingly, worms exposed to 0.1–1.0 μM BPS exhibited increases in head thrashes with a statistical significance observed at the 0.1 μM concentration. Additionally, there was also a nonsignificant increase in the number of body bends in the 0.1 μM BPS group. Although increases in locomotive behaviors can be beneficial, roles for bioamine neurotransmitters such as dopamine and serotonin have been implicated in these locomotive activities (Mesce and Pierce-Shumomura 2010). Hyperactivity (e.g., attention-deficit hyperactivity disorder) may develop when the relationship between dopamine and serotonin is dysregulated (Oades, 2008). As BPA exposure disrupts neurotransmitters through the modulation of transaminase activity in the brain of rodents (Zalko et al 2016; Zhang et al 2019; Ogi et al 2015), it will be important to determine if BPS has similar effects as BPA on neurotransmission.

In developmental NODEF mouse study, it was found that gestational BPS exposure caused behavioral changes in offspring of both sexes at 12, but not at 3, weeks of age. Female offspring exhibited an impairment in both working and short-term memory, as shown by the lower novel object index in the novel object test and % spontaneous alternations in the Y-maze test, respectively. On the other hand, male offspring exhibited an increase in depression-like and anxiety-like behaviors, indicated by an increase in the time spent immobile during the tail suspension test. When the results of the open field tests for both sexes were combined, there was a significant decrease in the time spent in the center of the apparatus, also suggesting an increase in anxiety-like behavior. Consistent with our findings, developing zebrafish exposed to a low concentration of BPS (0.0068 μM) also exhibited an increase in anxiety-like behavior (Kinch et al. 2015). Moreover, our recent study in adult male NODEF mice suggested that adult BPS exposure decreased curiosity/desire to explore and short-term memory, and increased anxiety-like behavior in female mice (McDonough et al. 2021). In addition, developmental BPA exposure also caused increases in anxiety-like and depression-like behaviors (Ohtani et al. 2017) and an impairment in working and short-term memory with higher sensitivity in female mice (Tian et al. 2010). Taken together, it was concluded that in utero BPS exposure might induce increased anxiety-like and depression-like behaviors along with an impairment of working and short-term memory in mice. However, there also existed differences between male and female offspring in NODEF mice following developmental BPS exposure, suggesting a relation between sex and adverse effects exerting through different mechanisms (e.g., epigenetic).

There are only two developmental studies examining the effects of BPS on behaviors and the brain in rodents (Castro et al. 2015; LaPlante et al. 2017). However, neither of these studies utilized tests related to memory, depression-like behaviors, or anxiety-like behaviors. Therefore, further research is needed to determine the underlying mechanisms for our above observations (e.g., fetal basis of adult diseases). It has been reported that exposure to BPS induces precocious hypothalamic neurogenesis in embryonic zebrafish (Kinch et al. 2015). It is possible that BPS implores a similar mechanism in mice. In contrast, adult female zebrafish exposed to BPS at low (1 μg/L) and high (30 μg/L) concentrations exhibited differential effects. Exposure to the low concentration of BPS resulted in increased phosphorylation of the ERK and improved memory (Naderi et al. 2020). Conversely, the higher concentration impaired short-term memory and decreased ERK phosphorylation. (Naderi et al. 2020). This dysregulated CREB/ERK pathway could be another possible mechanism BPS used to cause neurotoxicity in mice. Additional possible mechanisms that BPS used to cause behavioral deficits include altering the expression of 5α-R isozymes and genes associated with serotonin systems (Castro et al. 2015). In this regard, it has been reported that BPS exposure led to a decrease of placental serotonin levels in mice (Mao et al. 2020) and the disruption of signal transmission between nerve cells in the brains of fish (Schirmer et al. 2021).

As for the reduced lifespan and reproductive toxicity in C. elegans following developmental exposure to BPS, several patterns of toxicity were observed, including threshold, accumulative and recoverable effects. Generational studies of exposed C. elegans showed that high concentrations of BPS can reduce average lifespan and offspring numbers for one generation after treatment (F1), exhibiting a continued generational effect. However, only worms exposed to 10 μM during the first generation exhibited a continued significant decrease in average lifespan when compared to the control in the third generation (F2). In addition, worms exposed for multiple generations exhibited an accumulative effect until threshold was reached. Similar accumulative and threshold effects were reported by Xiao et al. (2019) who continuously exposed C. elegans to 0–100 μM BPS. To the best of our knowledge, this is the first study to examine the generational effects of BPS in C. elegans only exposed for a single period, and to examine the impact of a chemical on the number of days spent laying or the age of worms when they started laying eggs. It is interesting to note that only the F1 generation showed an increased age when worms started laying eggs (Figure 5 panel A), while the F2 was comparable to F0 and control nematode. The exact underlying mechanisms were unclear but it was possible that BPS activated certain pathway in F1 to help the worms in BPS detoxification, prior to F2 generation.

In this study, developmental BPS exposure did not affect BGLs or diabetes incidence in exposed offspring. However, male pups exposed to BPS had significantly lower BWs and BGLs when weaned, suggesting a disturbed metabolism that was reversible. Similarly, gestational exposure to BPA decreased the incidence of T1D in female offspring (Xu, Huang, Nagy, et al. 2019). Although the exact underlying mechanisms are unknown, it should be noted that toxicants. e.g., dioxin (Lefever et al. 2012), might target liver to affect the gluconeogenesis. Further histological/immunohistochemical studies in mice are warranted. These studies suggest that both BPA and BPS have sex-related effects, and more importantly, that they exert different effects likely using different mechanisms. However, in the BPA study, a higher dose of BPA (300 μg/kg BW) was used while a lower dose (3 μg/kg BW) was used in this study. The low dose of 3 μg/kg BW is also comparable to the concentration of 1.5 μM in our worm studies. Additionally, it is a more environmentally relevant concentration. Due to endocrine-disrupting chemicals being known to create a bimodal dose-response curve, further research utilizing both high and low concentrations of BPA and BPS are needed to compare their differences. Overall, our studies suggest that in utero BPS exposure induces increased anxiety-like and depression-like behaviors, an impairment of working and short-term memory in mice, and reproductive toxicity and a reduced lifespan in C. elegans.

Supplementary Material

1

Highlights.

  • Worms developmentally exposed to BPS for one generation had reversible toxicity

  • Worms exposed to BPS for 2 or 3 generations had accumulative and threshold effects

  • Mice exposed to BPS gestationally had adverse effects in their behaviors

Acknowledgments

This work was supported in part by NIH R21ES24487, NIH R41DK121553, and USDA National Institute of Food and Agriculture [grant no. 2016-67021-24994/project accession no. 1009090]. We would like to thank Dr. Nikolay Filipov, Ms. Jessica Carpenter, and Pooja Patel for their technical help in analyzing behavioral data. We would also like to thank Dr. Lili Tang for providing N2 C. elegans. The authors greatly appreciate Dr. Steven D. Holladay (Department Head, Professor, Department of Veterinary Biomedical Sciences at the University of Georgia) for his critical comments.

Footnotes

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Credit author statement

Callie M. McDonough: Writing- Original draft preparation, Methodology, Investigation, Formal analysis, Visualization

Daniel J. Guo: Investigation, Methodology, Writing- Review and Editing

Tai L. Guo: Conceptualization, Supervision, Funding acquisition, Methodology, Writing- Review and Editing, Project administration

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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