Developmental benzo[a]pyrene exposure alters stress hormones, neurotransmitters and behavioral responses of mice dependent on Cyp1 genotype

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Foster , Mickayla Kowalski , Amanda Honaker , Katelyn M. Clough , Alexandria Easton , Kevin Berling , Duong Pham , Annika White , Kayla Wypasek , View ORCID Profile Christine Perdan Curran doi: https://doi.org/10.1101/2025.08.13.670196 Jade Perry a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Taylor Easybuck a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mackenzie Feltner a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Emma G. Foster a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mickayla Kowalski a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amanda Honaker a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katelyn M. Clough a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexandria Easton a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kevin Berling a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Duong Pham a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Annika White a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kayla Wypasek a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christine Perdan Curran a Department of Biological Sciences, Northern Kentucky University , 100 Nunn Drive, Highland Heights, Kentucky, 41099, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christine Perdan Curran For correspondence: curranc1{at}nku.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) produced during combustion processes and when grilling foods. Epidemiological studies indicate exposure to PAHs during pregnancy lead to learning and memory deficits as well as behavioral problems that persist into adolescence. Studies in rodents and zebrafish have frequently reported anxiolytic effects of BaP exposure in adult animals and in developmental studies. We conducted sequential experiments of Cyp1a1(−/−) and Cyp1b1(−/−) knockout mice compared with wild type C57BL/6J mice to determine if genotype changes the response to developmental BaP exposure. We treated pregnant dams from gestational day 10 to postnatal day 25 (P25) with BaP in corn oil-soaked cereal or the corn oil vehicle and tested one male and one female offspring beginning at P60. We found increased exploratory behavior in the elevated zero maze for Cyp1a1(−/−) knockout mice, but no significant differences in Cyp1b1(−/−) knockouts. In contrast, Cyp1b1(−/−) knockout mice buried fewer marbles in a second test of anxiety-like behavior. There were no significant differences when Cyp1a1(−/−) knockout mice were tested. BaP decreased immobility time in Cyp1a1(−/−) knockouts in the forced swim test, but increased immobility time in wild type and Cyp1b1(−/−) knockout mice. We measured plasma corticosterone levels at baseline and following the forced swim test and monoamine neurotransmitters at the end of behavioral testing. BaP treatment increased corticosterone in wild type mice, but decreased it in Cyp1a1(−/−) knockout mice. Both BaP-exposed and corn oil control Cyp1b1(−/−) knockout mice had higher corticosterone levels compared with wild type mice. Dopamine and serotonin signaling were altered in the hypothalamus dependent on genotype, treatment and sex. Together, these data suggest that both CYP1A1 and CYP1B1 have a normal role in brain functioning or development, and that CYP1 genotype alters the response to developmental BaP exposure in behavioral and biochemical tests related to stress, anxiety and depression. Highlights BaP-exposed Cyp1a1(−/−) mice had lower corticosterone and decreased immobility in the forced swim test BaP exposure increased FST immobility in wild type and Cyp1b1(−/−) mice Cyp1a1(−/−) and Cyp1b1(−/−) knockout mice showed less anxiety-like behavior Developmental BaP exposure altered corticosterone levels dependent on Cyp1 genotype Genotype, treatment and sex all impacted neurotransmitter levels in the hypothalamus Genetic differences in CYP enzymes altered susceptibility to developmental BaP exposure 1. Introduction Benzo[a]pyrene (BaP) is a widespread pollutant and known carcinogen produced from multiple combustion processes. BaP ranks in the top ten on the United States’ list of priority pollutants (ASTDR 2022). Human exposures are widespread from vehicle exhaust, fossil fuel-burning power plants, wildfire and tobacco smoke, and grilled food ( ATSDR 1995 ). In recent years, evidence has been accumulating that developmental exposure to BaP and other polycyclic aromatic hydrocarbons (PAHs) can alter normal brain development with persistent, adverse effects during childhood and adolescence ( Perera et al. 2012 , 2018 ; Margolis et al 2016 , 2021 ). In addition to lowered IQ at school age ( Vishnevetsky et al. 2015 ), Perera et al. (2012) reported a positive association between prenatal PAH exposure and multiple behavioral outcomes including anxiety, depression and attention deficits in 6-7-year-old children. Those impairments and poor inhibitory control were recently associated with persistent problems in academic performance in adolescents ( Margolis et al. 2021 ). In a meta-analysis of 16 epidemiological studies on human PAH exposure, Zhen et al. (2023) concluded that prenatal PAH exposure increased the risk of attention deficits nearly 3-fold and that PAH exposure increased the risk of depression in adults. Numerous animal studies have confirmed that BaP exposure is strongly associated with not only learning and memory deficits, but persistent changes in behavior. Grova et al. (2008) used a range of doses from 0-200 mg/kg/day (i.p.) in adult female Balb/c mice for 10 days and found increased exploratory behavior in the hole board test and elevated plus maze at the 20 and 200 mg/kg/day dose. This study partially replicated their findings of an anxiolytic effect of high-dose BaP in young adult Balb/c female mice tested in the Y-maze ( Grova et al. 2007 ). Similar anxiolytic effects were seen in female Swiss Albino mice dosed orally with 0.02 and 0.2 mg/kg/day BaP for 17 days ( Bouayed et al. 2012 ). The BaP-treated mice showed similar behavior in the tail suspension test to the positive control animals treated with an anti-depressant and had higher levels of serotonin in the hippocampus and hypothalamus. Using a far different dosing approach, Das et al. (2017) directly injected 0.2 µg/kg of BaP into the cisterna magna of postnatal day 5 (P5) male Wistar rat pups prior to behavioral testing at P30. Similarly, these researchers reported anxiolytic effects with BaP-treated pups spending significantly more time in the open arms of the elevated plus maze and more time in the light zone in the light-dark text. Aparna et al. (2020) reported anxiolytic effects in adult zebrafish exposed to 0.2 mg/L of BaP with decreased time spent in the bottom during the novel tank diving test. Hamilton et al. (2021) found anxiolytic effects in zebrafish acutely dosed with 10 μM and 100 μM BaP. It’s important to note that none of these studies considered sex as a biological variable, which is a critical factor we addressed in our studies. We previously demonstrated that both high-affinity Ahr b Cyp1a2(−/−) and poor-affinity Ahr d Cyp1a2(−/−) knockout mice were uniquely susceptible to developmental BaP neurotoxicity when tested as young adults ( Honaker et al. 2022 ). Our earlier studies focused primarily on endpoints related to learning, memory and motor function. Our current studies were designed to determine if genetic differences exacerbate the neurotoxicity of developmental BaP exposure and its effects on measures of anxiety, depression and stress. All CYP1 enzymes have a potential role in BaP metabolism, but we focused these studies on Cyp1a1(−/−) and Cyp1b1(−/−) knockout mice based on earlier work demonstrating significant differences in BaP metabolism and immunotoxicity ( Uno et al. 2004 , 2006 ) and recent findings in humans that genetic differences in CYP1A1 increased the concentration of PAHs that crossed the placenta and accumulated in cord blood ( Dong et al. 2018 ). 2. Methods 2.1 Animals All animal experiments were conducted under protocols approved by the Northern Kentucky University Institutional Animal Care and Use Committee (IACUC) and follow the ARRIVE Guidelines ( Percie du Sert et al. 2020 ; Kilkenny et al. 2010 ). C57BL/6J (wild type for all genotypes) mice were purchased from The Jackson Laboratory (Bar Harbor, ME), Cyp1a1(−/−) ( Dalton et al. 2000 ) and Cyp1b1(−/−) ( Buters et al. 1999 ) mouse lines were used for the current study. The knockout lines were routinely back-crossed to C57BL/6J mice to avoid confounding by genetic drift. Genotypes were confirmed through PCR genotyping from tail snips at weaning and after sacrifice. 2.1.1 Animal husbandry Animal housing and care followed the Guide for the Care and Use of Laboratory Animals (8th edition). All mice were maintained in the Northern Kentucky University vivarium. Mice were housed in polysulfone shoebox cages with corncob bedding and one cotton nestlet as enrichment. Up to 4 adult mice were housed together, grouped by sex, genotype, and treatment. Water and Lab Diet 5015 chow (18.9% protein, 11% fat, 32.6% starch) were provided ad libitum and lighting was set to a 12 h: 12 h light-dark cycle. Cages were changed weekly, and mice were checked daily for any possible health concerns. 2.1.2 Breeding Female and male mice of the same genotype were mated over a 4-day breeding cycle. The day a vaginal plug was found was considered gestational day 0.5 (G0.5), and the female mouse was removed from the breeding cage. To control for maternal care, litters were balanced at six pups per dam by either culling or cross-fostering pups of the same genotype and treatment group. 2.2 Benzo[a]pyrene treatments Benzo[a]pyrene (> 96% purity; Millipore-Sigma) was dissolved in corn oil. Beginning at G10, pregnant dams were randomly assigned to treatment groups. The BaP group was given 10 mg/kg/day BaP dissolved in corn oil while the control group received an equivalent volume of the corn oil vehicle. To avoid the stress of daily injections or gavage, dams ingested the treatments on small pieces of cereal (Cap’n Crunch TM peanut butter). Treatment of dams continued until P25 when the pups were weaned, so all exposures to the offspring were in utero and via lactation. 2.3 Behavioral testing At P25, one male and one female pup from each litter were randomly selected for behavioral testing beginning at P60. Animals from all treatment groups were tested together in overlapping cohorts to avoid confounding by seasonal differences in behavior. Only one test was conducted per day within a 4 h time period to avoid confounding by circadian rhythms. Experiments were conducted under reduced lighting with no investigators present, and all investigators were blinded to treatment for animal handling and scoring. For all tests, 15-20 litters per group were used with a single trial for each animal. When multiple animals were tested simultaneously, only animals of the same sex were present in the room. Experiments are described in the order they were conducted. We tested and analyzed each knockout line separately; therefore, results are presented sequentially and separately. 2.3.1 Elevated zero maze As previously described ( Brown-Villalona et al. 2020 ), a circular maze (105 cm diameter) with two open and two closed quadrants was used to assess anxiety-like behavior and hyperactivity. Mice were placed in a closed quadrant and behavior was recorded for 5 min. The videos were then scored using ODlog TM for the following: latency to first leave the closed quadrant, head dips over the sides of the maze, zone crossings from one closed quadrant to another, and total time spent in the open quadrants. 2.3.2 Marble burying Mice were placed in a polysulfone shoebox cage filled with 5 cm of woodchip bedding and 15 identical marbles evenly placed across the bedding in a 3 x 5 grouping using a template. The mice were left alone for 20 min before being removed. The number of marbles buried at least 2/3 were recorded as buried, and pictures of each cage were taken to ensure accurate scoring. 2.3.3 Forced swim test Mice were placed in 10 cm opaque cylinders filled with water (25±1°C), and their behavior was recorded for 6 min. Videos were scored using ODLog TM for the following: latency to start floating and the total time spent floating, which was used to calculate the percent time immobile. 2.4 Corticosterone assays Blood was collected from the saphenous vein of each animal at baseline and 5 min after the forced swim test, centrifuged, and the plasma stored at −80°C until analysis. We used an ELISA kit (Alpco Diagnostics) to quantify corticosterone levels following the vendor’s protocol. 2.5 Neurotransmitter analysis Animals were sacrificed by decapitation following behavioral testing ∼P120. Brain regions were dissected, snap frozen and stored at −80°C until analysis. Methods were modified from Gutierrez et al. (2018) . Tissues were weighed and homogenized in 25 volumes of 0.2N perchloric acid with a glass Dounce homogenizer and centrifuged. Monoamine neurotransmitters dopamine and serotonin and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5HIAA), were quantified using high-performance liquid chromatography (Waters Alliance e2695) with electrochemical detection (Waters 2465). Using an autosampler, 10 µl of supernatant was injected onto a Waters XBridge C18 column (3.5 µm, 4.6 x 150 mm) with isocratic MD-TM mobile phase (Thermo Fisher Scientific) and a constant flow rate of 0.5 mL/min. Using Empower software (Waters, Inc. Milford, MA), chromatograms were integrated and analyzed with neurotransmitter concentrations calculated based on standard calibration curves. Data from the hypothalamus analysis are presented here. 2.6 Statistical analysis IBM SPSS statistical software (Version 28.0) was used to analyze all data for main effects of genotype, treatment and sex as well as their interactions. When differences were found, post-hoc analyses were done using corrections for multiple comparisons. We considered significance when P < 0.05. Data are presented as least square means ± the standard error of the mean (SEM). Behavioral tests included a minimum of 15 litters per group. Biochemical assays included a minimum of 8 litters per group. These group sizes were consistent with our prior experience using highly inbred mouse lines. 3. Results 3.1 Elevated zero maze We found a main effect of genotype and a main effect of sex (F 1,184 = 4.69, P < 0.05) in the elevated zero maze test. Cyp1a1(−/−) knockout mice spent significantly more time in the open quadrants (F 1,184 = 24.12, P < 0.001) and completed significantly more zone crossings (F 1,184 = 11.54, P < 0.01) compared with Cyp1a1(+/+) wild type mice ( Figs. 1A-B ). Female mice spent significantly more time in the open compared with male mice regardless of treatment or genotype (data not shown). We did not find any effects of genotype or treatment when testing Cyp1b1(−/−) knockout mice, but we did replicate our findings of sex differences with female mice spending more time in the open (F 1,12 0 = 5.66, P < 0.05) and completing more zone crossings (F 1,120 = 14.27, P < 0.001) compared with male mice (data not shown). Download figure Open in new tab Fig. 1 Elevated zero maze. There was a main effect of genotype with Cyp1a1(−/−) knockout mice spending more time in the open (A) and completing more zone crossings (B) compared with Cyp1a1(+/+) wild type mice. ** P < 0.01, *** P 0.05) when comparing BaP-exposed and control Cyp1a1(−/−) knockout and Cyp1a1(+/+) wild type mice ( Fig. 2A ). In contrast, we found that Cyp1b1(−/−) knockout mice buried significantly fewer marbles (F 1,120 = 8.90, P < 0.01) than Cyp1b1(+/+) wild type mice ( Fig. 2B ). We note there were differences in the wild type (C57BL/6J) results across the two experiments. This might indicate a difference in how investigators interpreted what constitutes a 2/3 buried marble. Download figure Open in new tab Fig. 2 Marble burying. There were no significant differences in the first experiment comparing Cyp1a1(−/−) knockout and Cyp1a1(+/+) wild type mice (A). There was a main effect of genotype in the second experiment with Cyp1b1(−/−) knockout mice burying fewer marbles compared with wild type mice (B). ** P < 0.01 3.3 Forced swim test (FST) We found a significant gene x treatment interaction in the forced swim test (F 1,140 = 4.10, P < 0.05) when comparing Cyp1a1(−/−) knockout and Cyp1a1(+/+) wild type mice. BaP-exposed Cyp1a1(−/−) knockout mice spent less time immobile compared with their corn oil-treated controls whereas BaP-exposed Cyp1a1(+/+) mice floated more than their corn oil controls ( Fig. 3A ). We found significant main effects of genotype (F 1,120 = 4.93, P < 0.05) and treatment (F 1,120 = 5.82, P < 0.05) when comparing Cyp1b1(−/−) knockout and Cyp1b1(+/+) wild type mice. Cyp1b1(−/−) knockout mice spent significantly less time immobile compared with Cyp1b1(+/+) wild type mice, and BaP-exposed mice floated significantly more than corn oil-treated controls ( Fig. 3B ). Download figure Open in new tab Fig. 3 Forced swim test. There was a gene x treatment interaction in the first experiment with BaP-exposed Cyp1a1(+/+) wild type mice spending slightly more time immobile than their corn oil-treated controls whereas BaP-exposed Cyp1a1(−/−) knockout mice spent less time immobile compared with their corn oil-treated controls (A). In the second experiment, there was a main effect of genotype with Cyp1b1(−/−) knockout mice spending less time immobile than wild type mice (B). * P < 0.05, a = significantly different from wild type, b = significantly different from corn oil. 3.4 Plasma corticosterone levels We found a significant gene x treatment interaction for baseline corticosterone levels (F 1,32 = 4.25, P < 0.05) as well as for levels following the stress of the forced swim test (F 1,85 = 4.79, P < 0.05) when comparing Cyp1a1(−/−) knockout and Cyp1a1(+/+) wild type mice. Baseline levels were significantly higher in BaP-exposed Cyp1a1(+/+) wild type mice, but lower in BaP-exposed Cyp1a1(−/−) knockout mice compared with corn oil controls ( Fig. 4A ). There was a similar trend in corticosterone levels following the stress of the forced swim test ( Fig. 4B ). In contrast, we found corticosterone levels were significantly higher in Cyp1b1(−/−) knockout mice (F 1,61 = 15.78, P < 0.001) after the forced swim test regardless of treatment ( Fig. 4C ). Download figure Open in new tab Download figure Open in new tab Fig. 4 Corticosterone levels in plasma. There was a significant gene x treatment interaction in the first experiment with BaP-treated Cyp1a1(−/−) knockout mice having lower levels of corticosterone compared with their corn oil-treated controls while BaP-treated Cyp1a1(+/+) wild type mice had significantly higher levels than corn oil controls at both baseline (A) and following the forced swim test (B). There was a main effect of genotype in the second experiment with Cyp1b1(−/−) knockout mice having higher levels of corticosterone following the forced swim test (C). * P < 0.05, *** P < 0.001 3.5 Hypothalamus neurotransmitter levels There was a significant gene x treatment x sex interaction for dopamine (F 1,181 = 4.18, P < 0.05) and its metabolite DOPAC (F 1,81 = 4.96, P < 0.05) in Cyp1a1 knockout and wild type mice. Corn oil-treated Cyp1a1(−/−) female mice had significantly higher dopamine levels than all other groups, and BaP-exposed male Cyp1a1(+/+) wild type mice had significantly reduced dopamine compared with females. ( Table 1 ). BaP-exposed male Cyp1a1(+/+) wild type mice and corn oil-treated Cyp1a1(−/−) male mice had significantly lower DOPAC levels compared with the corresponding females ( Table 2 ). View this table: View inline View popup Download powerpoint Table 1. Dopamine levels in the hypothalamus. View this table: View inline View popup Download powerpoint Table 2. DOPAC levels in hypothalamus. There were no significant differences in serotonin levels in the hypothalamus when comparing Cyp1a1 knockout and wild type mice; however, there were significant main effects of genotype (F 1,81 = 18.6, P < 0.001), treatment (F 1,81 = 5.44, P < 0.05) and sex (F 1,81 = 79.63, P < 0.001) for the serotonin metabolite 5-HIAA. Cyp1a1(−/−) knockout mice had significantly lower levels of 5-HIAA compared with Cyp1a1(+/+) wild type mice ( Fig. 5A ), and BaP-exposed mice had significantly lower levels of 5-HIAA compared with corn oil-treated mice ( Fig. 5B ). Female mice had significantly higher levels of 5-HIAA compared with male mice (data not shown). Download figure Open in new tab Download figure Open in new tab Fig. 5. Monoamine neurotransmitters in the hypothalamus. There was a main effect of genotype in the first experiment with significantly lower levels of the serotonin metabolite 5-HIAA in Cyp1a1(−/−) knockout mice (A). BaP exposure significantly lowered 5-HIAA levels in the first experiment (B). In the second experiment, the dopamine metabolite DOPAC (C) and serotonin levels (D) were both significantly lower in Cyp1b1(−/−) knockout mice compared with wild type mice. * P < 0.05, ** P < 0.01, *** P < 0.001 Genotype was highly significant in the hypothalamus of Cyp1b1(−/−) mice with lower levels of DOPAC (F 1,119 = 11.32, P < 0.01) ( Fig. 5C ) and a trend for significance for dopamine (F 1,119 = 3.66, P =0.058). Serotonin levels were also significantly lower in Cyp1b1(−/−) mice (F 1,119 = 8.15, P < 0.01) ( Fig. 5D ), but there was no difference in the levels of 5-HIAA (data not shown). There was no effect of BaP treatment for any neurotransmitter or metabolite in this second study. 4. Discussion Prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) has been linked to deficits in academic performance and behavior in highly exposed human populations ( Perera et al. 2012 , Vishnevetsky et al. 2015 , Margolis et al 2016 , 2021 ). We have completed sequential studies of Cyp1a1(−/−) and Cyp1b1(−/−) knockout mice exposed to the well-characterized PAH benzo[a]pyrene (BaP) during early brain development with behavioral testing conducted on young adults. Both knockout lines were back-crossed more than 8 generations onto a C57BL/6J (B6) background, and B6 mice were used as the wild type in all experiments. Multiple rodent studies indicated that BaP exposure in adult and neonatal animals produces an anxiolytic effect in commonly used tests of anxiety-like and depressive-like behavior ( Grova et al. 2007 , 2008 ; Bouayed et al. 2009; Das et al. 2017). The goals of our experiments were to determine if genetic differences altered the effects of developmental BaP exposure on adult behavior and to determine if males and females were differentially affected because prior studies did not include sex as a biological variable. We hypothesized that both knockout lines would be more susceptible due to reduced capacity to metabolize and clear BaP in the dams and pups. We used complementary tests of anxiety-like behavior (elevated zero maze and marble burying), and both knockout lines showed less anxiety-like behavior than wild type mice regardless of BaP exposure. However, the results across tests were not consistent. Cyp1a1(−/−) knockout mice showed increased exploratory behavior in the zero maze ( Fig. 1A-B ), but no differences in the marble burying test ( Fig. 2A ). In contrast, Cyp1b1(−/−) mice were no different than wild type mice in the zero maze, but showed significantly less anxiety-like behavior in the marble burying test ( Fig. 2B ). We found no evidence of an anxiolytic effect of BaP, but we did find sex differences with females in both experiments demonstrating more exploratory behavior in the zero maze. Together, these findings suggest that the genotype and sex are more important than BaP treatment for outcomes related to anxiety-like behavior and that both CYP1 enzymes have some role in brain development or function. The forced swim test (FST) is commonly used to test depressive-like behavior in rodents and the effectiveness of antidepressant medications (Colletis et al. 2024, Vahid-Ansari et al. 2024 ). We found different trends in the two knockout lines of mice with BaP-exposed Cyp1a1(−/−) knockout mice having decreased immobility time compared to their corn oil controls ( Fig. 3A ). Cyp1b1(−/−) mice spent significantly less time immobile than wild type mice ( Fig. 3B ). Unexpectedly, we found that 10/mg/kg/day BaP treatment in dams increased immobility time in wild type and Cyp1b1(−/−) knockout offspring compared to their corn oil controls. This would be consistent with the findings of Zhang et al. (2016) who reported increased anxiety-like behavior in C57BL/6 adult male mice chronically dosed with 6.25 mg/kg/day BaP. In contrast, Bouayed et al. (2012) reported an anxiolytic effect of lower doses of BaP (0.02 and 0.2 mg/kg/day) in adult female Swiss albino mice in the tail suspension test, but no differences in the FST. This indicates that age, genetic background and dose all play a role in the behavioral outcomes. To help uncover neuroendocrine signaling that might underlie the behavioral differences, we measured corticosterone levels at baseline and 5 min following the FST and measured neurotransmitter levels in the hypothalamus at the end of behavioral testing. Once again, we found significant differences between the knockout lines and wild type mice as well as significant effects of BaP exposure. In our first experiment, BaP exposure increased corticosterone levels in wild type mice compared to corn oil controls, but decreased them in BaP-exposed Cyp1a1(−/−) compared to corn oil-treated knockouts. The pattern of a reduced stress response and reduced immobility in FST are consistent for Cyp1a1(−/−) knockout mice, but cannot explain the FST results for wild type mice. We found a strikingly different pattern in the Cyp1b1(−/−) knockout mice. Corticosterone levels were higher in both corn oil- and BaP-exposed knockouts compared with wild type mice, even though the knockouts spent less time immobile in the FST. This is the opposite pattern from the Cyp1a1(−/−) knockouts and suggests a different mechanism or a differential response to a stressful stimulus. Serotonin (5-HT) is a major monoamine neurotransmitter associated with changes in mood and behavior ( Tejeda-Martinez et al. 2024 ) that is metabolized to 5-HIAA. We found decreased serotonin turnover in Cyp1a1(−/−) knockout mice ( Fig. 5A ) and in mice exposed to BaP during early brain development ( Fig. 5B ) evidenced by significantly decreased levels of 5-HIAA in the hypothalamus. In contrast, Cyp1b1(−/−) knockout mice had lower levels of serotonin in the hypothalamus ( Fig. 5D ). It is now recognized that CYP enzymes have a role in tryptophan metabolism and that the presence of tryptophan metabolites can affect the level of CYP1enzymes in humans and other animals (Hadduch et al. 2023). Given the importance of tryptophan metabolism in multiple neurological disorders ( Marx et al. 2020 ) and the emerging understanding of the tryptophan-kynurenine pathway in the gut-brain axis ( Gheorghe et al. 2019 , Vazquez-Medina et al. 2024 ), it will be important to conduct further studies on the role of CYP1 enzymes in both the gut and in the brain to fully understand their roles and the impact of mutations that alter their metabolic capacities. Dopamine’s role in the hypothalamus is complex and intersects with numerous other neuroendocrine and neurotransmitter pathways (reviewed in Ugromov 2024). Dopaminergic signaling is central to normal circadian rhythms ( Mesgar et al. 2023 ), and stress can impair normal dopaminergic functioning in both rodents ( Ercan et al. 2023 ) and fish ( Gesto et al. 2008 ). We found decreased dopamine and its metabolite DOPAC in BaP-exposed wild type male mice ( Tables 1 - 2 ) and significantly decreased DOPAC in Cyp1b1(−/−) knockout mice ( Fig. 5C ). Further work is needed to determine whether these changes are related to dopamine synthesis or biodegradation; however, previous studies have implicated aryl hydrocarbon receptor agonists with dopaminergic dysfunction during brain development ( Tanida et al. 2014 ). The mechanism appears to be direct modulation of dopamine synthesis based on the studies of Akahoshi et al. (2009) who found dioxin (TCDD) increased expression of tyrosine hydroxylase mediated by binding of the aryl hydrocarbon receptor. Overproduction of dopamine can increase oxidative stress in neurons. Therefore, it’s plausible that developmental exposure to the AHR agonist BaP can act similarly in upregulating tyrosine hydroxylase ultimately leading to oxidative stress and neurotoxicity. Although new and novel mechanisms may play a role in differential responses to developmental BaP exposure, differential toxicokinetics must never be ignored. Adult mice with variation in the aryl hydrocarbon receptor and CYP1 enzymes have clear and striking differences in the metabolism and clearance of BaP ( Uno et al. 2004 , 2006 ). We found similar differences when measuring BaP and metabolite levels in wild type and Cyp1 knockout dams and their pups ( Feltner et al. 2023 ). Given recent human studies demonstrating that CYP1A1 polymorphisms can increase the concentration of PAHs crossing the placenta into cord blood ( Dong et al. 2018 ), it seems clear that the results from animal studies have translational value for humans. 5. Conclusion Exposure to BaP and other PAHs remains widespread through air, food and soil ( Bukowska et al. 2022 ) and will likely increase in coming years as climate change leads to more intense and frequent wildfires ( Ghetu et al. 2022 , Jain et al. 2024 ). We found that developmental BaP exposure altered corticosterone levels and neurotransmitter levels in the hypothalamus, but that genotype and sex were also key factors. Mutations in both CYP1A1 ( Frikha et al. 2024 ) and CYP1B1 ( Shahid et al. 2022 ) are well known in the human population; therefore, our work has potential translational value by identifying individuals at higher risk of neurobehavioral impairments following BaP exposure. Our multiple findings of behavioral changes in both knockout lines of mice regardless of treatment are also likely to lead to new insights into CYP1 enzymes and their roles in neuroendocrine homeostasis and neurotransmitter metabolism. 6. CRediT author statement Jade Perry: Conceptualization, Methodology, Investigation, Funding acquisition. Taylor Easybuck: Writing-Original draft, Data curation, Investigation, Visualization. Mackenzie Feltner : Investigation, Data curation, Supervision. Emma G. Foster: Investigation, Data curation, Supervision. Mickayla Kowalski: Investigation, Funding acquisition. Amanda Honaker : Investigation Angela Kyntchev : Conceptualization, Investigation, Katelyn Clough: Conceptualization, Investigation, Data curation, Allie Easton : Investigation. Kalyani Abbaraju : Investigation. Joseph Ashley : Investigation, Kevin Berling : Investigation . India Davis : Investigation. Duong Pham : Investigation. Annika White : Investigation, Visualization. Kayla Wypasek : Investigation. Christine Perdan Curran: Project administration, Funding acquisition, Analysis, Supervision, Writing-Original draft and Revision. 7. Data Availability Statement All data and detailed protocols are freely available upon request to the corresponding author. 9. Conflicts of Interest The authors have no conflicts to declare. 8. Acknowledgments We acknowledge support from multiple funding sources, including NIH grants R15ES030541, R15ES020053, P20GM103436, and NSF RSF-034-07, Society of Toxicology internship grants, and the following Northern Kentucky University sources: College of Arts and Sciences Collaborative Faculty-Student Project Awards, Faculty Development Project Grants, Center for Integrative Natural Sciences and Mathematics (CINSAM) UR-STEM Fellowships, Greaves and Herrmann Fellowships, and Student Undergraduate Research and Creative Activities awards. We thank Dr. Daniel W. Nebert of the University of Cincinnati Medical Center for the generous donation of Cyp1(−/−) knockout mice. 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