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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Neurotoxicology. 2021 Jul 14;86:78–84. doi: 10.1016/j.neuro.2021.07.003

The Use of Tocofersolan as a Rescue Agent in Larval Zebrafish Exposed to Benzo[a]pyrene in Early Development

Zade Holloway 1, Andrew Hawkey 1, Helina Asrat 1, Nidhi Boinapally 1, Edward D Levin 1
PMCID: PMC8440378  NIHMSID: NIHMS1726138  PMID: 34273383

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants created by incomplete combustion. Benzo(a)pyrene (BaP), the prototypic PAH, is known to exert toxicity through oxidative stress which is thought to occur through inhibition of antioxidant scavenging systems. The use of agents that reduce oxidative stress may be a valuable route for ameliorating the adverse effects of PAHs on neural development and behavior. This study was conducted to determine if tocofersolan (a synthetic water-soluble analog of vitamin E) supplementation can prevent or reduce neurobehavioral deficits in zebrafish embryos exposed to BaP during early development. Newly hatched zebrafish were assessed on locomotor activity and light responsivity. Zebrafish embryos were exposed to vehicle (DMSO), tocofersolan (0.3 μM - 3 μM), and/or BaP (5 μM) from 5-120 hours post-fertilization. This concentration range was below the threshold for producing overt dysmorphogenesis or decreased survival. One day after the end of exposure the larval fish were tested for locomotor activity under alternating light and dark 10 min periods, BaP (5 μM) was found to cause locomotor hypoactivity in larval fish. Co-exposure of tocofersolan (1 μM) restored control-like locomotor function. Based on the findings of this study, this model can be expanded to assess the outcome of vitamin E supplementation on other potential environmental neurotoxicants, and lead to determination if this rescue persists into adulthood.

Keywords: Zebrafish, Benzo[a]pyrene, Tocofersolan, Vitamin E, Locomotion

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants created through incomplete combustion. Human exposure can occur through inhalation of vehicle emissions, smoking cigarettes, cooking, and even breathing air from indoor fireplaces (Lawal, 2017; Zhang et al., 2015). Understanding the risk of substances containing PAHs is of great importance due to the many observations of developmental toxicity induced by these compounds, such as the prototypic PAH benzo[a]pyrene (BaP) (Hakura, 2013; Hecht, 2002; Moorthy et al., 2015). Specifically, these toxicants have the potential to developmentally disrupt behavior, learning, and memory, which makes them a prime area of concern in human exposure scenarios (Das et al., 2016; Maciel et al., 2014; Saunders et al., 2001).

BaP, among other PAHs, is known to exert its carcinogenic effects by inducing oxidative stress, specifically by producing reactive oxygen species that are detrimental to gene expression, cell signaling, and can lead to tumor growth and cell death (Christmann et al., 2016; Zhao et al., 2018; Zuo et al., 2014). Interestingly, evidence has recently been found that neurotoxicity induced by BaP may occur at lower concentrations and earlier in the lifespan than the concentrations required for carcinogenicity (Bostrom et al., 2002; Chepelev et al., 2015). Due to its lipophilic nature, BaP can readily cross the blood-brain barrier (BBB) allowing for direct access to the central nervous system (Grova et al., 2008), and one method of neurobehavioral toxicity is thought to occur due to inhibition of antioxidant scavenging systems in the brain (Saunders et al., 2006). Regardless of the mechanism of action, the adverse effects of BaP on neural systems, motor skills, and cognitive performance in humans and animal models are an important area of concern.

Research in neurophysiology has shown us that sub-chronic exposure to BaP in adult rats can impair behavioral performance and also modulate levels of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in the hippocampus (Kempadoo et al., 2016; Whitlock et al., 2006), a site well-characterized for its involvement in memory, spatial learning, and long-term potentiation (Xia et al., 2011). When rats are exposed prenatally, offspring have been shown to have reduced long-term potentiation and diminished cortical activity (McCallister et al., 2008; Wormley et al., 2004). Even low BaP exposure concentrations that are not overtly toxic can act directly on developing neuronal cells, resulting in excess cell numbers at the expense of normal emergence of dopamine and acetylcholine neurotransmitter phenotypes in vitro (Slotkin et al., 2013; Slotkin and Seidler, 2009). Research in humans follow a similar trend with exposure leading to impaired cognitive performance and in some cases alterations in DA, 5-HT, NE, and ACh (Perera et al., 2009). A more recent study expands the scope of affected systems, revealing that increased exposure to BaP in school children is significantly linked to a decrease in volume of the caudate nucleus, a subsystem of the basal ganglia (Mortamais et al., 2017). This finding is seminal considering the basal ganglia contains circuitry that forms interconnecting feedback networks with the limbic system and frontal cortex; these pathways are termed the motor, motivational, and associative corticostriatal loops, or basal ganglia loops (McHaffie et al., 2005; Seger, 2009). Thus, BaP and other PAHs have the potential to disrupt many major forms of neuronal communication, producing an overall negative effect on brain function that may extend to aberrant development in the offspring.

Recent work supported that developmental exposure to BaP can impact behavior, learning, and memory in adult and embryonic zebrafish (Knecht et al., 2017), and the use of oxidant scavengers like N-acetylcysteine (NAC) have been shown to have promising effects on rescuing organisms from developmental deformities caused by oxidative stress (Arnold, 2016). Vitamin E (α-tocopherol) is a free radical scavenger that protects cells from oxidative stress and displays antioxidant activities in vitro and in vivo (Ingold et al., 1987; Traber and Atkinson, 2007). In rats that had experienced traumatic brain injury (TBI), vitamin E was found to reverse the cognitive deficits and reduced levels of BDNF seen in the untreated TBI group, and animals who received supplementation also exhibited a lower level of oxidized proteins compared with untreated TBI and sham animals (Wu et al., 2010). In another study with aging mice, supplementation of vitamin E led to an increase neuromuscular function and exploratory behavior (Navarro et al., 2005). The use of vitamin E as a treatment to blunt the neurobehavioral toxicity of environmental neurotoxicity has a promising therapeutic potential that could mitigate or reverse deficiencies caused by PAHs during development.

In the current study we chose a water-soluble derivative of the natural D isomer of α-tocopherol as a rescue agent, tocofersolan, in which polyethylene glycol-1000 is esterified to D-α-tocopherol via a succinic acid bridge (Thebaut et al., 2016). Tocofersolan has been used as a replacement therapy for certain syndromes in which natural vitamin E cannot be readily be absorbed, and has been shown to have a higher bioavailabilty when compared to other vitamin E formulations (Jacquemin et al., 2009). The current study sought to determine if tocofersolan supplementation can prevent or reduce neurobehavioral deficits in zebrafish embryos exposed to moderate and high concentrations of BaP during early development. Larval zebrafish were assessed for locomotor activity and light responsivity. By using this model, we can determine if the antioxidant properties of tocofersolan offer preventative benefits in the presence of potent neurotoxicant exposure, and establish a proper dosing regimen for supplementation.

2. Materials and Methods

2.1. Fish housing and husbandry

All the experiments were conducted using a local colony of AB* wild-type strain of zebrafish as parents. They were maintained, and bred in the Levin Lab in the Department of Psychiatry and Behavioral Sciences at Duke University. The experimental procedures were approved by the Duke University Institutional Animal Care and Use Committee. Adult zebrafish were held in mixed (females and males) groups at a density of approximately 5 fish/l in 3- or 10-l tanks kept on a recirculating flowing water system (Aquatic Habitats, Inc., Apopka, Florida; Aquatic Enterprises, Inc., Bridgewater, MA, USA). System water was a mixture of sea salt (Instant Ocean, 0.5 parts per thousand) and buffer (Seachem Neutral Regulator, 125 mg/l) in deionized water. Water chemistry, salinity, and temperature were monitored weekly. Illumination was set to 14:10h light:dark cycle and water temperature was kept at 28±1C. The fish were fed 3 times daily; morning and afternoon with brine shrimp (Artemia salina) hatched in-house over 24h (eggs from Brine Shrimp Direct, Ogden, UT, USA); and noon feeding with a mixture of solid pellet food containing; TetraMIN Tropical Flakes (Blacksburg, VA, USA); GEMMA Micro 300 micropellets (Skretting USA, Tooele, UT, USA); Zebrafish Complete Diet (Ziegler Bros., Inc., Gardners, PA, USA). Embryo collection, and embryo and larval rearing were conducted as described in our previous experiment (Glazer et al., 2018). Briefly, fertilized eggs were obtained by group breeding using in-tank inserts. Immediately after collection, the embryos were quickly bleached and transferred to large plastic Petri dishes for sorting, then glass Petri dishes for rearing and chemical exposures. At 0- to 6-day post-fertilization (dpf), the embryos were kept in an incubator at a temperature range of 28–29±1°C, and a 14:10 h light:dark cycle lights on at 7 AM.

2.2. Chemicals

Dimethyl Sulfoxide Reagent Plus, ≥99.5% (DMSO; CAS No. 67-68-5, Lot No. SHBG9650V) and Benzoapyrene (BaP; CAS No. 50-32-8, Lot No. SLBP4938V) were purchased from Sigma-Aldrich (St Louis, MO, USA). Tocofersolan (CAS No. 9002-96-4, Lot No. 19575) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

2.3. Developmental Exposure

Zebrafish embryos were exposed from approximately 5-h post-fertilization (hpf) until 5 dpf to either 0.1% DMSO alone or one of the following individual chemicals and concentrations in 0.1% DMSO: Tocofersolan (0.3, 1, 3 μM); and/or BaP (5 μM). The concentration of 5 μM of BaP is a commonly used concentration in toxicity studies and is regarded as an environmentally relevant concentration (Zhu et al., 2014). Pilot studies showed that this concentration caused reliable decreases in activity without increased malformations or decreased fertility. Stock solutions were 20 μM, and working solutions were prepared at 1000x final concentration in 100% DMSO by conducting serial dilutions from the stock solutions. Before the start of exposure, a lab member who was not involved in the study would replace the solution names with letters, thus blinding the experimenters to the identity of the treatments for the full duration of the exposure and behavioral testing. The control groups were exposed to the same concentration of DMSO used in the experimental groups. Three separate sets of exposures were conducted; Set 1 consisted of tocofersolan with a DMSO control; Set 2 consisted of BaP with a DMSO control; Set 3 consisted of a co-exposure of BaP and tocofersolan with a DMSO control. At 5 hpf, embryos were sorted under a dissecting microscope, discarding unfertilized or abnormally developing embryos, then randomly and evenly distributed into glass Petri dishes (inner diameter 9 cm; depth 2 cm) at a density of up to 40 embryos per dish with 1ml system water per embryo, and immediately exposed to the above detailed treatments. The exposure solutions were replaced every 24 h. Each day, the embryos were examined and dead or malformed individuals were recorded and removed. At 5 dpf, the embryos were rinsed twice with system water without toxicant or treatment exposure, transferred to clean glass Petri dishes with system water, and placed in the incubator until larval activity testing at 6 dpf.

2.4. Larval Photomotor Response

At 6 dpf, larvae were placed into 96-well plates with 0.5 ml glass well inserts filled with system water and tested for locomotor activity in response to alternating light conditions. Exposure conditions were all represented within each plate and across multiple plates. Plates were then returned to the incubator for an hour before being placed into a DanioVision lightbox controlled by the EthoVision XT tracking software (version 11.5, Noldus, Wageningen, The Netherlands). Locomotor activity was tracked during a paradigm in which an initial 10-min acclimation period in the dark (0% illumination) was followed by 2 cycles of 10 min at 100% illumination (5000 lx) and 10 min at 0% illumination. An infrared camera tracked larval locomotion across the 50-min trial. All larval testing was conducted during the diurnal light phase between 10 AM and 5 PM. Exposure groups were generated at 40 subjects per replicate petri dish and 24 subjects were randomly selected from each group for locomotor activity testing. Locomotor activity was recorded at a rate of 30 frames per second and a track smoothing protocol was applied based on 10 samples before and after every sample point in order to exclude slight movements that might introduce noise to the calculations. Total distance moved is reported in cm per minute or cm per 10 min.

2.5. Data Analysis

All statistical analyses were performed with IBM SPSS Statistics 24. A mixed design analysis of variance with BaP exposure and tocofersolon treatment as between subject factors and illumination condition as the repeated measures was used for all behavioral tests. Locomotor activity of individual fish was used as the statistical unit for larval testing. All of the treatment conditions were represented in each plate. As recommended, interactions less than p < 0.10 were followed up with tests of the simple main effects of BaP exposure and tocofersolon reversal of those effects (Snedecor, 1967). In final analyses p < 0.05 (two-tailed) was always set as threshold for statistical significance.

3. Results

3.1. Experiment 1: Tocofersolan 0.3 μM and BaP 5 μM

The main effect of BaP on distanced traveled was significant (F(1,75) = 9.95, p < 0.005). The main effect of lighting condition was significant (F(1,75) = 29.86, p<0.0005) and the BaP x lighting condition was significant (F(1,75) = 4.57, p < 0.05). Analyses of the simple main effects of BaP in light and dark conditions shows that BaP treatment caused a significant degree of hypoactivity in the dark condition (p < 0.0005), but not in the light condition (p = 0.08) (Fig. 1). The post-hoc analysis revealed that larval fish in the BaP group (83.0 ± 4.1 cm) were significantly hypoactive in the dark condition compared to the DMSO controls (105.9 ± 4.4 cm). No significant effect of this concentration of tocofersolan was seen.

Figure 1.

Figure 1.

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Photomotor response assay for tocofersolan (0.3 μM) and benzo[a]pyrene (5 μM) (mean ± sem).

3.2. Experiment 2: Tocofersolan 1 μM and BaP 5 μM

Figure 2 shows the results from the study of BaP interactions with the 1 μM tocofersolan treatment. The main effect of BaP was significant (F(1,166) = 28.96, p < 0.0005). The main effect of lighting condition was significant (F(1,166) = 110.70, p<0.0005). Also, the BaP x tocofersolan condition was significant (F(1,166) = 7.54, p < 0.01). The three-way interaction of BaP x tocofersolan x lighting condition (F(1,166) = 3.26, p = 0.07) prompted tests of the simple main effects. Averaged across lighting conditions, zebrafish exposed to BaP alone were significantly (p < 0.0005) hypoactive (83.5 ± 4.4 cm) relative to controls (112.9 ± 3.8 cm). Tocofersolan (1 μM) significantly (p < 0.05) attenuated this hypoactivity (94.6 ± 3.4 cm), even though by itself the 1 μM tocofersolan concentration did not increase activity (104.6 ± 3.1 cm). In the dark condition, BaP alone (93.8 ±5.9 cm) caused a significant (p < 0.0005) hypoactivity relative to control (126.4 ± 4.6 cm), which 1 μM of tocofersolan (112.0 ± 4.7 cm) significantly (p < 0.01) attenuated, even though this concentration of tocofersolan did not by itself increase activity (116.4 ± 4.6 cm). In the light condition BaP exposure (73.3 ±4.2 cm) also produced significant (p < 0.0005) hypoactivity relative to controls (99.5 ± 4.8 cm), an effect that was not significantly attenuated by tocofersolan (77.2 ± 3.7 cm). tocofersolan alone (92.8 ± 4.0 cm) did not significantly change activity in the light condition.

Figure 2.

Figure 2.

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Photomotor response assay for tocofersolan (1 μM) and benzo[a]pyrene (5 μM) (mean ± sem).

3.3. Experiment 3: Tocofersolan 3 μM and BaP 5 μM

Figure 3 shows the results from the 3 μM tocofersolan treatment of BaP exposed zebrafish. The main effect of BaP was significant (F(1,88) = 11.26, p < 0.005). The main effect of lighting condition was significant (F(1,88) = 104.72, p < 0.0005). The interaction of BaP and tocofersolan did not prompt tests of the simple main effects (p = 0.10). The control zebrafish had average locomotor scores of 105.5 ± 4.3 cm, the BaP only exposed zebrafish 87.7 ± 3.1 cm, the tocofersolan only exposed zebrafish 98.7 ± 2.8 cm and the zebrafish with combined exposure 92.6 ± 3.7 cm.

Figure 3.

Figure 3.

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Photomotor response assay for tocofersolan (3 μM) and benzo[a]pyrene (5 μM) (mean ± sem).

3.4. Combined Analysis

All of the studies are summarized in figure 4. The overall analysis showed significant main effects of both BaP and tocofersolan. BaP (5 μM) exposure significantly (F(1,337) = 24.78, p < 0.0005) decreased larval locomotor activity. There was a significant main effect of tocofersolan (F(3,337) = 2.80, p < 0.05) increasing locomotor activity compared to the BaP group. There was an interaction between BaP and tocofersolon (F(3,337) = 2.36, p = 0.08) that prompted tests of the simple main effects of the different concentrations of tocofersolon on the BaP-induced hypoactivity. These comparisons showed that BaP when given alone caused a significant degree of hypoactivity (p < 0.0005) relative to control. This hypoactivity was significantly (p < 0.01) attenuated by the 1 μM tocofersolon concentration. The 3 μM tocofersolon concentration did not quite (p = 0.09) cause a significant attenuation of the BaP-induced hypoactivity. The lowest tocofersolon concentration tested, 0.3 μM, did not show any sign of attenuating the BaP-induced hypoactivity.

Figure 4.

Figure 4.

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Tocofersolan concentration-effect (0.3, 1 and 3 μM) attenuation of BaP (5 μM) effects on larval activity (mean ± sem).

4. Discussion

Zebrafish are becoming an advantageous species for assessing potential risks of environmental hazards to the public and have proven their efficacy as a model organism to help identify neurotoxicants and neuroprotectants (Bailey et al., 2013; Eimon and Rubinstein, 2009; Parng et al., 2006). More importantly, zebrafish can be used to detect neurotoxicity at chemical exposure levels lower than those that produce overt signs of toxicity, such as developmental malformations or mortality. Additionally, it is important to note that although environmental PAH exposures typically occur in complex mixtures, BaP is highly toxic and one of the most prominent PAHs found in the environment (Gerger and Weber, 2015). The concentration of BaP used in this experiment, 5 μM, is a commonly used concentration in toxicity studies and is regarded as an environmentally relevant concentration (Zhu et al., 2014). The current study was designed to determine if supplementation of an antioxidant which is a vitamin E derivative, tocofersolan, could prevent or reverse deficits caused by BaP in larval zebrafish.

The larval photomotor response (LPR) assay was used to measure changes in swimming patterns, such as habituation and overall distance moved. Unexposed larval zebrafish typically exhibit lower swim movement in the light condition compared to the dark condition following light-dark transitions (MacPhail et al., 2009). In all three of the current experiments, larval exposure to 5 μM BaP consistently led to reduced locomotor activity in the dark condition compared to controls which were exposed to the same concentration of DMSO so that possible effects of DMSO alone were accounted for in the control group. This is consistent with other studies which have shown developmental and acute BaP exposure in mice is associated with impaired motor activity and cognitive performance (Chen et al., 2012; Saunders et al., 2006). As stated, BaP is thought to exert damage to cellular components through oxidative stress and inflammatory processes (Costa et al., 2010; Khattab et al., 2021; Shi et al., 2021). If the balance between reactive oxygen species (ROS) and antioxidant defenses is disrupted, oxidative stress and cellular damage can occur. For example, experimental enhancement or suppression of ROS during embryogenesis has been shown to alter embryonic development and growth, indicating a specific level of ROS is required to regulate a normal progression of embryonic development (Dennery, 2010; Lin et al., 2020). Oxidative stress occurs following PAH exposure, such as BaP, because these toxicants undergo metabolization by cytochrome P450 (CYP) enzymes, which leads to the production of ROS that can cause damage to DNA (Briedé et al., 2004; Lin et al., 2018). Further, oxidative stress and inflammatory processes are known products of aryl hydrocarbon receptor (AHR) activation and are commonly associated with overt toxicity (Dalton et al., 2002). Interestingly, 5uM BaP has been shown to be sufficient to increase AHR expression and induce oxidative stress through production of ROS (Tsuji et al., 2011; Zhu et al., 2014). Ligand binding to AHRs not only leads to oxidative damage, but also other forms of cellular dysfunctions. Through activation of AHRs, BaP metabolism advances the production of more noxious metabolites, which are known to cause DNA strand breaks, DNA adducts, and mutations seen with in vitro and in animal models (Genies et al., 2013; Nebert et al., 2004). Treatment with an antioxidant, specifically vitamin E, in the presence of BaP exposure has been shown to significantly suppress BaP-induced ROS levels and decrease DNA adducts (Zhu et al., 2014).

In the current study, we administered a water-soluble version of vitamin E, tocofersolan, simultaneously with BaP to determine if the locomotor deficits could be prevented or reversed. Although BaP caused significant hypoactivity in the dark condition and 0.3 μM (Figure 1) along with 3 μM tocofersolan (Fig. 3) did not lead to significant reversal in locomotor deficits, the co-exposure with 1 μM tocofersolan (Fig. 4) resulted in locomotor activity that was significantly higher than the 5 μM BaP alone group. This suggests that 1 μM tocofersolan did prevent or reverse detriments caused by BaP and strengthens the notion that agents which modulate oxidation may be a valuable route for ameliorating the effects of PAHs on neural development and behavior. BaP has been shown to increase oxidative stress by increasing reactive oxygen species (ROS) production (Banerjee et al., 2016). The current results are consistent with other studies that show the protective effects of vitamin E against oxidative stress and cytotoxicity induced by exposure to BaP (Perocco et al., 2000; Zhu et al., 2014). Oxidative stress has been shown to pose a threat to brain-derived neurotrophic factor (BDNF) function, which could disrupt cognitive function and synaptic plasticity throughout development leading to a host of downstream issues (Wu et al., 2004). Interestingly, supplementation of vitamin E has been shown to lead to an increase in neuromuscular function and exploratory behavior in mice, which may help to explain some of the effects seen in the current study (Navarro et al., 2005). Furthermore, vitamin E is vital to normal development and brain function in animals. Chronic deficiency of this vitamin was recently explored in adult zebrafish, finding that insufficiency can lead to cognitive and neurological dysfunction, likely due to increased oxidative stress in the brain (McDougall et al., 2017).

Our findings highlight the importance of examining the therapeutic effects of antioxidants on developmental neurotoxin exposure. However, results of the current study should be considered along with several important limitations. Although BaP is known to exert its cellular effects through oxidative stress, the current study only examined effects on motor activity and light/dark response in larval zebrafish. The direct cause-effect relationship of tocofersolan on the oxidative effect of BaP was not assessed, therefore it is hard to determine the specific mechanisms at play in this reversal of locomotor deficits caused by tocofersolan exposure. Additionally, this significant ameliorative effect occurred when zebrafish were exposed to tocofersolan at 1uM, but not at the higher 3uM concentration, suggesting that there is an optimal concentration for the rescue provided by tocofersolan. Therapeutic drug discovery approaches and pharmacology commonly assume that a monotonic concentration-response relationship will occur, yet the consistency of this is being increasingly challenged due to the network topology and dynamics of nodes within bio-molecular pathways (Van Wijk et al., 2015). There is growing evidence that bio-molecular pathways have the potential to exhibit a non-monotonic concentration-response relationship, which helps to explain the occurrence and functional significance of the drug interactions seen in the current study (Lagarde et al., 2015; Li et al., 2007). Too much or too little oxidative stress may be detrimental. Non-monotonic effects, while often unexpected, often suggest the presence of multiple mechanisms with different thresholds for perturbation. In the presence of multiple sensitive components of a pathway (or parallel pathways), a lower concentration which is insufficient to impact mechanisms with higher thresholds may have distinct rather than lesser effects when compared to a higher concentration. These findings emphasize the urgent need for more research dedicated to assessing the outcome of tocofersolan or vitamin E supplementation on other potential environmental neurotoxicants that induce oxidative stress, and additionally lead to determination of positive effects, if any, persisting into adulthood. This study lays the foundation for further research concerning the optimal antioxidant treatment regimen and the mechanisms for reversing the developmental neurotoxicity of BaP and other polycyclic aromatic hydrocarbons.

Highlights.

  • Benzo(a)pyrene (BaP) exerts neurotoxicity through oxidative stress.

  • Zebrafish were exposed to tocofersolan and/or BaP for 5 days post-fertilization.

  • BaP (5 μM) caused locomotor hypoactivity in larval fish.

  • Co-exposure of tocofersolan (1 μM) restored control-like locomotor function.

Acknowledgement

Supported by the Duke University Superfund Research Center (ES010356)

Footnotes

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