Acute Exposure to Microplastics Induced Changes in Behavior and Inflammation in Young and Old Mice

Environmental pollutants have become quite ubiquitous over the past two centuries; of those, plastics, and in particular, microplastics (<5 mm), are among the most pervasive pollutants. Microplastics (MPs) have found their way into the air, water system, and food chain and are either purposely produced or are derived from the breakdown of larger plastic materials. Despite the societal advancements that plastics have allowed, the mismanagement of plastic waste has become a pressing global issue. Pioneering studies on MPs toxicity have shown that exposure to MPs induces oxidative stress, inflammation, and decreased cell viability in marine organisms. Current research suggests that these MPs are transported throughout the environment and can accumulate in human tissues; however, research on the health effects of MPs, especially in mammals, is still very limited. This has led our group to explore the biological and cognitive consequences of exposure to MPs in a rodent model. Following a three-week exposure to water treated with fluorescently-labeled pristine polystyrene MPs, young and old C57BL/6J mice were assessed using behavioral assays, such as open-field and light–dark preference, followed by tissue analyses using fluorescent immunohistochemistry, Western blot, and qPCR. Data from these assays suggest that short-term exposure to MPs induces both behavioral changes as well as alterations in immune markers in liver and brain tissues. Additionally, we noted that these changes differed depending on age, indicating a possible age-dependent effect. These findings suggest the need for further research to better understand the mechanisms by which microplastics may induce physiological and cognitive changes.


Introduction
Plastics, which are durable, low-cost, and rapidly produced [1], have contributed to some of the greatest recent advancements in society, including modern technology and medical advancements, such as single-use syringes and modern prosthetics [2,3]. These breakthroughs, in combination with many other daily uses for plastics, have led to an almost exponential increase in global plastic production over the past century that has now surpassed 400 million tons per year, with a projected increase to over 1 billion tons within the next~30 years [4]. This booming plastic production, however, has also led to a significant pollution problem as up to~70% of the world's plastic ends up in landfills or is mismanaged in the environment [4]. Plastics in the environment have been shown to leach harmful chemicals [5,6], can be ingested by marine organisms [7], and potentially serve as a transport method for invasive species such as viruses.
In addition to these direct harmful impacts, plastics exposed to environmental factors, such as UV radiation, oxidation, and physical abrasion, have been shown to result in the formation of microplastics (MPs) [8]. Microplastics (MPs) are also purposely produced for use in paints, detergents, and personal care products, such as toothpaste, sunscreen, and cosmetics [9,10]. Microplastics are defined as plastic particles less than 5 mm in diameter

Cell Viability and Cellular Uptake
In order to study the effect that MPs have in vivo, we first tested cellular uptake and viability in vitro after exposure to commercially available pristine fluorescent polystyrene particles (PS-MPs). U-2 OS cells were cultured and subsequently treated with 0.1 and 2 µm PS-MPs at concentrations ranging from 0.01 to 1000 µg/mL. Following exposure times of 24, 48, and 72 h, cell viability was assessed via MTT assay. After 48 and 72 h, PS-MPs of both sizes induced a dramatic decrease in cell viability, which became more exaggerated with increased concentrations ( Figure 1A). Microscopic analysis revealed internalization of PS-MPs in U-2 OS cells as early as after 24 h of exposure ( Figure 1B).

In Vivo Exposure and Behavioral Studies
Next, we tested the effect of exposure to MPs in young (4-month-old, n = 40) and old (21-month-old, n = 40) C57BL/6J female mice. Animals were divided into four exposure groups (n = 10 per group): normal drinking water (control), 0.0025 mg/mL (low-dose), 0.025 mg/mL (medium-dose), and 0.125 mg/mL (high-dose) water treated with a 1:1 mixture of 0.1 and 2 µm PS-MPs ( Figure 2A). All mice were exposed to the appropriate dose of PS-MPs for 3 weeks via water delivery. To ensure that sedimentation of the PS-MPs would not drastically alter concentration throughout the day, each dosage was tested hourly for 10 h, followed by a measurement at 24 h. No significant changes in concentration were found throughout the 24 h period ( Figure 2B). We also monitored water consumption and body weights and did not find any alterations in either parameter (Supplementary Figure S1A-D).
At the end of the 3-week-long exposure, behavioral testing began. During the openfield test, mice were allowed to explore a low-lit chamber for 90 min with spontaneous movements monitored in the x-, y-, and z-directions. Several parameters to measure behavioral performance were recorded, including distance traveled, rearing activity, and duration in the center. Surprisingly, we found that acute exposure to PS-MPs induced an increase in distance traveled, which was more pronounced in older animals ( Figure 3A-D). Similarly, both young and old PS-MP-exposed mice reared significantly more in the openfield, as compared to age-matched controls ( Figure 3E-H). Young PS-MP-exposed mice did not spend more time in the center of the chamber overall ( Figure 3J), but both lowand high-dose groups spent more time in the center when analyzed as a function of time ( Figure 3I). Low-and medium-dose older animals also showed an increased duration in the center ( Figure 3K,L).

In Vivo Exposure and Behavioral Studies
Next, we tested the effect of exposure to MPs in young (4-month-old, n = 40) and old (21-month-old, n = 40) C57BL/6J female mice. Animals were divided into four exposure groups (n = 10 per group): normal drinking water (control), 0.0025 mg/mL (low-dose), ture of 0.1 and 2 μm PS-MPs ( Figure 2A). All mice were exposed to the appropriate dose of PS-MPs for 3 weeks via water delivery. To ensure that sedimentation of the PS-MPs would not drastically alter concentration throughout the day, each dosage was tested hourly for 10 h, followed by a measurement at 24 h. No significant changes in concentration were found throughout the 24 h period ( Figure 2B). We also monitored water consumption and body weights and did not find any alterations in either parameter (Supplementary Figure S1A-D).

Figure 2.
In vivo study design and PS-MPs delivery system concentration curve. (A) In vivo study design for short-term (3 weeks) exposure to PS-MPs in young (4 month-old) and old (21 month-old) female C57BL/6J mice (n = 10 per group). Schematic was created with BioRender.com (accessed on 2 December 2021). (B) Concentration of each dose (low, medium, high) of PS-MPs was measured hourly for 10 h without resuspension and again at 24 h without any resuspension.
At the end of the 3-week-long exposure, behavioral testing began. During the openfield test, mice were allowed to explore a low-lit chamber for 90 min with spontaneous movements monitored in the x-, y-, and z-directions. Several parameters to measure behavioral performance were recorded, including distance traveled, rearing activity, and duration in the center. Surprisingly, we found that acute exposure to PS-MPs induced an increase in distance traveled, which was more pronounced in older animals ( Figure 3A-D). Similarly, both young and old PS-MP-exposed mice reared significantly more in the open-field, as compared to age-matched controls ( Figure 3E-H). Young PS-MP-exposed mice did not spend more time in the center of the chamber overall ( Figure 3J), but both low-and high-dose groups spent more time in the center when analyzed as a function of time ( Figure 3I). Low-and medium-dose older animals also showed an increased duration in the center ( Figure 3K,L).  Effects of exposure to PS-MPs on locomotion in young and old mice. Spontaneous locomotor activity of 4-and 21-month-old female C57BL/6J mice (n = 10 per group) exposed to low (blue), medium (green), and high (red) doses of PS-MPs, as compared to control mice (gray). Both young and old mice exposed to PS-MPs showed marked increases in (A-D) distance traveled, (E-H) rearing activity, and (I-L) duration in center. Significances were determined by unpaired t-test, one-way ANOVA (B,D,F,H,J,L), or two-way ANOVA with post hoc analysis (A,C,E,G,I,K) and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and α p < 0.10 as trending.
In the light/dark preference test, mice were again placed in a chamber, now divided into light and dark zones, with movements monitored in the x-, y-, and z-directions. Parameters recorded for this assay included duration in zone, distance traveled, and rearing activity. PS-MP-exposure did not affect the duration spent in light and dark zones in either age group ( Figure 4A,B). This assay did, however, confirm the increased distance traveled ( Figure 4C,D) and rearing activity ( Figure 4E,F) revealed in the open-field test ( Figure 3A- Effects of exposure to PS-MPs on locomotion in young and old mice. Spontaneous locomotor activity of 4-and 21-month-old female C57BL/6J mice (n = 10 per group) exposed to low (blue), medium (green), and high (red) doses of PS-MPs, as compared to control mice (gray). Both young and old mice exposed to PS-MPs showed marked increases in (A-D) distance traveled, (E-H) rearing activity, and (I-L) duration in center. Significances were determined by unpaired t-test, one-way ANOVA (B,D,F,H,J,L), or two-way ANOVA with post hoc analysis (A,C,E,G,I,K) and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and α p < 0.10 as trending.
In the light/dark preference test, mice were again placed in a chamber, now divided into light and dark zones, with movements monitored in the x-, y-, and z-directions. Parameters recorded for this assay included duration in zone, distance traveled, and rearing activity. PS-MP-exposure did not affect the duration spent in light and dark zones in either age group ( Figure 4A,B). This assay did, however, confirm the increased distance traveled ( Figure 4C,D) and rearing activity ( Figure 4E,F) revealed in the open-field test ( Figure 3A-H). These findings are again more pronounced in older animals. Effects of exposure to PS-MPs on light-dark preference in young and old mice. Exploratory behavior during the light-dark (solid and transparent bars, respectively) preference assay of 4-and 21-month-old female C57BL/6J mice (n = 10 per group) exposed to low (blue), medium (green), and high (red) doses of PS-MPs, as compared to control mice (gray). Both young and old mice exposed to PS-MPs showed striking increases in (C,D) distance traveled and (E,F) rearing activity. (A,B) No alterations were found for duration in light/dark zone. Significances were determined by unpaired t-test with * p < 0.05, and α p < 0.10 as trending.

Bioaccumulation of MPs
To determine if MPs in drinking water are absorbed and are able to translocate and accumulate in tissues after exposure, samples of liver, kidney, gastrointestinal tract, lung, spleen, heart, and brain tissues from both young and old exposed mice were cryosectioned and counterstained with DAPI. Unexpectedly, we detected PS-MPs within intracellular compartments of every tissue examined ( Figures 5 and 6). We also observed PS-MPs in urine and fecal matter.

Assessing Impact of MPs on Immune Markers
Upon observing entry of PS-MPs into the brain, we performed fluorescent immunohistochemistry of GFAP (glial fibrillary acidic protein), a marker of glial cells, which include activated astrocytes. We found decreased GFAP expression in brains from both young and old mice exposed to PS-MPs, as compared to age-matched controls ( Figure 7A). A Western blot of brain lysates confirmed decreased GFAP expression ( Figure 7B), and quantification revealed that this reduction was significant in young mice exposed to PS-MPs, as compared with age-matched controls ( Figure 7C). Upon observing entry of PS-MPs into the brain, we performed fluorescent immunohistochemistry of GFAP (glial fibrillary acidic protein), a marker of glial cells, which include activated astrocytes. We found decreased GFAP expression in brains from both young and old mice exposed to PS-MPs, as compared to age-matched controls ( Figure  7A). A Western blot of brain lysates confirmed decreased GFAP expression ( Figure 7B), and quantification revealed that this reduction was significant in young mice exposed to PS-MPs, as compared with age-matched controls ( Figure 7C). Alterations of immune markers in liver tissues from PS-MP-exposed mice were also examined. qPCR analysis revealed a ~2-fold increase in mRNA expression of inflammatory cytokine TNF-α (tumor necrosis factor) in liver from young and old mice exposed to PS-MPs ( Figure 8A). Calcium-binding proteins S100a8 and S100a9 (calgranulins), which mediate inflammatory responses, were also assessed. Young PS-MP-exposed mice showed little change in S100a8 and moderate increases in S100a9 mRNA expression. Old mice, however, exhibited higher levels of both calgranulins, as compared to young controls, and a ~3-fold increase in S100a8 and a ~4.5-fold increase in S100a9 mRNA expression in old mice exposed to PS-MPs, as compared to age-matched controls ( Figure 8B,C). Alterations of immune markers in liver tissues from PS-MP-exposed mice were also examined. qPCR analysis revealed a~2-fold increase in mRNA expression of inflammatory cytokine TNF-α (tumor necrosis factor) in liver from young and old mice exposed to PS-MPs ( Figure 8A). Calcium-binding proteins S100a8 and S100a9 (calgranulins), which mediate inflammatory responses, were also assessed. Young PS-MP-exposed mice showed little change in S100a8 and moderate increases in S100a9 mRNA expression. Old mice, however, exhibited higher levels of both calgranulins, as compared to young controls, and a~3-fold increase in S100a8 and a~4.5-fold increase in S100a9 mRNA expression in old mice exposed to PS-MPs, as compared to age-matched controls ( Figure 8B,C). mediators S100a8 and S100a9 in liver tissue. (A) qPCR analysis showed approximately 2-fold creases in mRNA expression of TNF-alpha in both young and old PS-MP-exposed mice, as compar to age-matched controls. (B,C) Further analyses also showed significant increases in S100a8 a S100a9 mRNA expression in old mice exposed to PS-MPs, as compared to controls. Significanc were determined by unpaired t-test with α p < 0.10, * p < 0.05, and ** p < 0.01.

Discussion
As global plastic production continues to rapidly grow, leading to the ubiquito presence of microplastics, we set out to understand the potential harmful impacts of M in mammalian systems with a particular focus on age as a potential co-factor in adver exposure outcomes. To first establish in vitro toxicity of 0.1 and 2 μm PS-MPs, U-2 OS ce were exposed at concentrations ranging from 0.01 to 1000 μg/mL for exposure times of 2 48, and 72 h. Following these exposures, cell viability was assessed via an MTT assay (F ure 1A). Data collected from this assay showed that for both sizes of PS-MPs, cell viabil was significantly reduced, especially as concentration and exposure time increased. Thu suggesting that PS-MPs in this size range exhibit cytotoxicity. Additionally, PS-MPs we found to enter cells within 24 h of exposure and accumulated perinuclearly ( Figure 1B) With this in mind, an in vivo study was designed to determine the effects of the MPs in a rodent model (Figure 2A). Following a 3-week exposure to PS-MPs, C57BL mice were tested in a series of behavioral assays including the open-field and light-da preference tests. Both assays showed significant changes in parameters such as distan traveled, rearing activity, and duration in the center between the control and the expos groups for both old and young mice (Figures 3 and 4). Overall, these changes seemed be more pronounced in older animals, which may be due to age-related dysfunction e asperating the effects of the PS-MPs on behavioral performance ( Figures 3D,H,L a  4D,F). The behavioral changes exhibited by the young mice, however, suggest that ev without increased age as a co-variable, PS-MPs can induce altered behavior in roden after just 3 weeks of exposure ( Figures 3B,F,J and 4C,E).
To understand the physiological systems that may be contributing to these chang in behavior, we began by sectioning several major tissues including the brain, liver, ki ney, gastrointestinal tract, heart, spleen, and lungs to determine where these MPs may accumulating. Surprisingly, we detected the presence of PS-MPs in every tissue examin ( Figures 5 and 6), as well as in urine and feces. Given that in this study the MPs we delivered orally via drinking water, detection in tissues such as the gastrointestinal tra ( Figure 5), which is a major part of the digestive system, or in the liver and kidneys (Figu 5), which contribute to the detoxification of xenobiotics [35,36], was always probable. T detection of MPs in tissues such as the heart and lungs ( Figure 5), however, suggests th the PS-MPs are going beyond the digestive system and likely undergoing systemic circ lation. This is further supported by the detection of MPs in urine and in the brain ( Figu   Figure 8. PS-MPs altered mRNA expression of inflammatory cytokine TNF-alpha and inflammatorymediators S100a8 and S100a9 in liver tissue. (A) qPCR analysis showed approximately 2-fold increases in mRNA expression of TNF-alpha in both young and old PS-MP-exposed mice, as compared to age-matched controls. (B,C) Further analyses also showed significant increases in S100a8 and S100a9 mRNA expression in old mice exposed to PS-MPs, as compared to controls. Significances were determined by unpaired t-test with α p < 0.10, * p < 0.05, and ** p < 0.01.

Discussion
As global plastic production continues to rapidly grow, leading to the ubiquitous presence of microplastics, we set out to understand the potential harmful impacts of MPs in mammalian systems with a particular focus on age as a potential co-factor in adverse exposure outcomes. To first establish in vitro toxicity of 0.1 and 2 µm PS-MPs, U-2 OS cells were exposed at concentrations ranging from 0.01 to 1000 µg/mL for exposure times of 24, 48, and 72 h. Following these exposures, cell viability was assessed via an MTT assay ( Figure 1A). Data collected from this assay showed that for both sizes of PS-MPs, cell viability was significantly reduced, especially as concentration and exposure time increased. Thus, suggesting that PS-MPs in this size range exhibit cytotoxicity. Additionally, PS-MPs were found to enter cells within 24 h of exposure and accumulated perinuclearly ( Figure 1B).
With this in mind, an in vivo study was designed to determine the effects of these MPs in a rodent model (Figure 2A). Following a 3-week exposure to PS-MPs, C57BL/6J mice were tested in a series of behavioral assays including the open-field and light-dark preference tests. Both assays showed significant changes in parameters such as distance traveled, rearing activity, and duration in the center between the control and the exposed groups for both old and young mice (Figures 3 and 4). Overall, these changes seemed to be more pronounced in older animals, which may be due to age-related dysfunction exasperating the effects of the PS-MPs on behavioral performance ( Figure 3D,H,L and Figure 4D,F). The behavioral changes exhibited by the young mice, however, suggest that even without increased age as a co-variable, PS-MPs can induce altered behavior in rodents after just 3 weeks of exposure ( Figures 3B,F,J and 4C,E).
To understand the physiological systems that may be contributing to these changes in behavior, we began by sectioning several major tissues including the brain, liver, kidney, gastrointestinal tract, heart, spleen, and lungs to determine where these MPs may be accumulating. Surprisingly, we detected the presence of PS-MPs in every tissue examined ( Figures 5 and 6), as well as in urine and feces. Given that in this study the MPs were delivered orally via drinking water, detection in tissues such as the gastrointestinal tract ( Figure 5), which is a major part of the digestive system, or in the liver and kidneys ( Figure 5), which contribute to the detoxification of xenobiotics [35,36], was always probable. The detection of MPs in tissues such as the heart and lungs ( Figure 5), however, suggests that the PS-MPs are going beyond the digestive system and likely undergoing systemic circulation. This is further supported by the detection of MPs in urine and in the brain ( Figure 6), which additionally demonstrates that the PS-MPs can pass the blood-brain barrier (BBB).
Given the ability of MPs to pass the BBB, an immediate concern was the potential for these xenobiotics to trigger neuroinflammation. GFAP (glial fibrillary acidic protein), a major intermediate filament protein found in mature astrocytes that is involved in many cell processes such as autophagy, neurotransmitter uptake, and astrocyte development [37], can be used to measure the expression of activated astrocytes and is a commonly used marker in neuroinflammatory studies [38]. Astrocytes are typically activated in response to neural stress or injury [39], and because of this, an increase in GFAP expression is often associated with an increase in neuroinflammation. Fluorescent immunohistochemical staining for GFAP in PS-MP-exposed mouse brain, however, showed a slight decrease in expression for older animals and a more pronounced decrease in young mice exposed to PS-MPs, as compared to age-matched controls ( Figure 7A). These results were confirmed with a Western blot analysis ( Figure 7B,C). Although these results are not typical of an inflammatory response, they are consistent with previous studies that suggest that GFAP expression might decrease in early stages of certain diseases, such as Alzheimer's disease (AD) [40,41], or in younger patients with disorders such as Major Depressive Disorder (MDD) [42]. These studies indicate that early pathology/early onset of disease may be characterized by astrocyte atrophy (as opposed to astrocyte hypertrophy later on), which may result in decreased GFAP expression. Although these mechanisms are still not well understood, our results suggest that exposure to PS-MPs results in a comparable agedependent pattern.
Similar to our findings, results from Lee and colleagues [43] found that following either a 4-or 8-week exposure to 2 µm carboxyl-modified PS-MPs via an oral gavage, 6-week-old C57BL/6J mice exhibited accumulation of MPs in both liver and brain tissues, alterations in cognitive behavior, and modifications in immune markers in the brain. However, Lee et al. did not find any alterations in the open-field test in mice exposed to PS-MPs, in contrast to our study. This could be due to several differences between the setup of the two studies, such as age of the animals, method and length of delivery of MPs, PS-MPs having different surface chemistries, as well as Lee's study only using one size of MPs. Despite some discrepancies between the studies, it is evident that PS-MPs can travel to and exert detrimental effects on the brain after absorption. Further studies are needed to dissect the underlining molecular mechanisms of such an effect. One possibility proposed by Lee et al. is that neurotoxic effects of PS-MPs may depend on the vagal-pathway-dependent gutbrain axis; however, other mechanisms including the impairment of blood detoxification pathways in the liver cannot be excluded. Several studies have indeed suggested that the ability of a toxin to reach the brain may be in part due to liver dysfunction, since the liver is a major site of blood detoxification. If the liver is unable to properly function, this can lead to toxin build-up in the blood [44], which may ultimately reach the brain. Similarly, hepatic failure or injury may result in increased BBB permeability [45,46]. Thus, we investigated whether PS-MPs induced an inflammatory response in the liver from young and old mice in our study and found an approximately two-fold increase in the mRNA expression of inflammatory cytokine TNF-α in both young and old PS-MP-exposed mice, as compared to controls ( Figure 8A). Additionally, in older PS-MP-exposed mice, there was a 3-fold increase in S100a8 mRNA expression and a 4.5-fold increase in S100a9 mRNA expression ( Figure 8B,C). S100a8 and S100a9 are Ca 2+ -binding proteins that help mediate inflammatory responses. Increased expression of these genes indicate inflammation of the liver, which may play a role in allowing the MPs to enter the bloodstream and ultimately reach the brain and other major organs.
Overall, since human exposure to MPs is inevitable due to their persistence and pervasiveness in the environment, it is essential to better understand their toxicity to limit their impact on human health. In the present study, we have shown that 0.1 and 2 µm PS-MPs can reduce cell viability, translocate throughout the body, accumulate in tissues including brain tissue, markedly modify behavior in C57BL/6J mice after only 3 weeks of exposure, and significantly alter immune markers in both the liver and the brain. Additionally, the effects of exposure seem to be age-dependent. Research into the effects of exposure to MPs in mammals is still a very broad field with many variables worth pursuing. In this study, we chose to focus specifically on the effects of exposure to pristine polystyrene microplastics (PS-MPs) via drinking water in female C57BL/6J mice. There are still many questions that remain, including but not limited to how sex, MP delivery method, length of exposure to MPs, and MP surface chemistry impact exposure outcome. While these variations were not explored in this study, future work should examine these factors in order to understand the mechanisms by which MPs exert these effects and how these mechanisms are altered with age.

Microplastic Particles
Dyed red aqueous pristine fluorescent polystyrene particles (PS-MPs; Thermo Scientific, Fremont, CA, USA) were obtained at diameters of 0.1 and 2 µm. MPs in this size range were selected because 0.1 µm represents the smallest end of the microplastics spectrum, bordering on nanoplastics, and 2 µm nears the upper limit of what may enter into cells [47,48]. MPs within this size range have recently been detected in human breastmilk [34], placentas [49], lungs [31], and on human hands and hair [50]. Prior to use, PS-MPs were centrifuged (Eppendorf 5424R, Hamburg, Germany) at 21,000× g at 4 • C for 1 h 45 min, and the supernatant was discarded. The PS-MPs were resuspended using sterile distilled water. This process was repeated 3 times to remove trace amounts of DMSO and sodium azide.

MTT Assay
Cell viability was assessed in vitro using an MTT assay. U-2 OS cells were seeded (Gibco, Waltham, MA, USA) in 24-well plates (Celltreat, Pepperell, MA, USA) and allowed 24 h to attach. Following this, cells were exposed to 0.01, 0. Biosystems, Wetzlar, Germany) was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.

Animals and Exposure
Both young (4-month-old, n = 40) and old (21-month-old, n = 40) female C57BL/6J mice were obtained from the National Institute of Aging (NIA) aged rodent colony (Charles River Laboratories, Kingston, NY or Raleigh, NC, USA). Female mice were selected based on their ability to be re-housed after the mice were delivered and acclimated in our animal facility in order to minimize weight discrepancies between treatment groups. All mice were acclimated for at least 2 weeks in our animal facility prior to testing. Within each age cohort, four exposure groups (n = 10 per group) were established to receive 1:1 ratios of 0.1 and 2 µm PS-MPs via drinking water: normal drinking water (control group), 0.0025 mg/mL (low-dose group), 0.025 mg/mL (medium-dose group), and 0.125 mg/mL (high-dose group). Drinking water was selected as the delivery vehicle over oral gavage in order to allow for continuous exposure as opposed to timed bursts, as well as to minimize external stress that may impact behavior performance. Water consumption and body weights were monitored throughout exposure to ensure comparable exposure between groups ( Figure S1). Water bottles were mixed every 10-12 h; mice were exposed for three weeks, during which time the drinking water was replaced as needed, i.e., every~10-12 days. Exposure dosages were selected according to previous studies [18]. All mice received a standard diet (Teklad Global Soy Protein-Free [Irradiated] type 2920X, Envigo, Indianapolis, IN, USA) and water ad libitum; they were group-housed based on how the mice were received from the source institution with up to 5 mice per ventilated cage with access to a small house and tissues for nesting. The mice were kept on a 12:12 light: dark cycle at 22 • C ± 1 and 30-70% humidity. Adequate measures were taken to minimize animal pain and discomfort. The investigation was conducted in accordance with the ethical standards and according to the Declaration of Helsinki and national and international guidelines and has been approved by the authors' institutional review board.

Behavior Experiments
The mice were acclimated in their home cage for 1 h in the testing room and were kept at 22 • C ± 1, 30-70% humidity, and~100 lux prior to testing. The testing room was in a neutral, quiet environment, and mice were tested between 9:00 and 17:00 (light phase) by the same researcher, with care taken to stagger the testing of mice from the different exposure and age cohorts. The mice were transported to and from the apparatus in a non-transparent plastic container cleaned with 70% ethanol after each use.

Open-Field Test (OF)
A multi-cage infrared-sensitive motion detection system (Fusion v6.5 SuperFlex, Omnitech Electronics, Columbus, OH, USA) was used to assess exploratory behavior and spontaneous locomotion. During this assay, the mice were placed in darkened transparent locomotor chambers (40 × 40 × 30 cm) equipped with a grid of infrared beams at floor level and 7.5 cm above floor level for 90 min while their movements were monitored in 5 min intervals in the x-, y-, and z-planes. All horizontal and vertical movements were recorded, and data were analyzed using the Fusion v6.5 software system. Locomotor boxes were cleaned with 70% ethanol after each test period.

Light-Dark Preference Test (LD)
To assess exploratory and anxiety-related behaviors, all mice were placed in locomotor chambers (40 × 40 × 30 cm) divided into light and dark zones that were equipped with a grid of infrared beams at floor level and 7.5 cm above floor level. All mice were tested for 30 min with their movements being monitored in the x-, y-, and z-planes using an infraredsensitive activity-monitoring cage system (Fusion v6.5 SuperFlex, Omnitech Electronics, Columbus, OH, USA). All movements were recorded, and data were analyzed using the Institutional Review Board Statement: The animal study protocol (#AN1920-020) was approved by the Institutional Review Board of University of Rhode Island, approved 26 June 2020.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data generated from this study are available upon request.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.