Sex-specific neurobehavioral and prefrontal cortex gene expression alterations following developmental acetaminophen exposure in mice

Abstract

Acetaminophen (N-acetyl-p-aminophenol (APAP), also known as paracetamol) is one of the most common medications used by the general population, including pregnant people. Although many human observational and animal model studies have shown associations between prenatal and early postnatal APAP exposure and attention deficit hyperactivity disorder, autism spectrum disorders, and altered neurodevelopment, the existing literature is limited. In particular, no mouse studies of prenatal APAP exposure have investigated offspring attention deficits in behavioral tasks specifically designed to measure attention, and no prior rodent studies have utilized 'omics' technologies, such as transcriptomics, for an untargeted exploration of potential mechanisms. We randomly assigned pregnant mice (starting embryonic day 4-10) to receive APAP (150 mg/kg/day) or vehicle control through postnatal day 14. We evaluated 111 mouse offspring in a battery of behavioral tests, including pup ultrasonic vocalizations, elevated plus-maze, open field test, CatWalk (gait), pre-pulse inhibition, and the automated 5-choice serial reaction time task. Prefrontal cortex was collected at birth from 24 pups for RNA sequencing. Developmental APAP treatment resulted in increased and hastened separation-induced pup vocalizations between postnatal days 2 and 11, as well as decreased ambulation and vertical rearings in the open field in male but not female adult offspring. APAP treatment was also associated with altered sex-specific prefrontal cortex gene expression relating to glutathione and cytochrome p450 metabolism, DNA damage, and the endocrine and immune systems. This study provides additional evidence for the neurodevelopmental harm of prenatal APAP exposure and generates hypotheses for underlying molecular pathways via RNA sequencing.

Keywords: ADHD; Acetaminophen; Autism; Behavior; Neurodevelopment; Paracetamol; Prenatal; RNA sequencing.

Fig. 1.

Pup Ultrasonic Vocalizations (USV). Among 4 control and 4 treatment dams, 6 pups per dam were removed one at a time from the dam and placed into an insulated chamber where USVs were recorded for 3 min by an ultrasonic microphone. Mean ± standard error shown for number of USVs recorded across 4 days of testing, stratified by sex (n = 48). Treatment by sex interaction P = 0.638. Significant post-hoc pairwise treatment by sex by day contrasts shown for repeated measures models (*P < 0.05 following Bonferroni correction).

Fig. 2.

Open Field Test (OFT). Mice were allowed to roam freely in a clear Plexiglas open field arena for 60 min. Mean ± standard error shown for total ambulatory movement stratified by sex (A, B: treatment by sex interaction P = 0.020 and 0.001, respectively), total number of rearings defined as the number of times the mouse disrupted two infrared beams by standing on its hind limbs (C, D; treatment P = 0.033 and 0.127, respectively), and (E, F) time spent in the center zone of the arena (E, F; treatment P = 0.378 and 0.174, respectively) (n = 111). Significant post-hoc pairwise treatment by sex by time bin contrasts shown for repeated measures models (^†P < 0.05; *P < 0.05 following Bonferroni correction).

Fig. 3.

CatWalk gait analysis. Mice completed three full crossings over an illuminated walled glass walkway. CatWalk XT software automatically measured 226 parameters related to mouse gait and locomotion via a high-speed camera underneath the walkway. Raw (A) and FDR-adjusted (B) P-values and beta estimates from separate linear regressions of treatment on each parameter shown. Red horizontal line at significance threshold of P = 0.05 (n = 90).

Fig. 4.

Elevated plus maze (EPM). P values presented for acetaminophen treatment effect from mixed effects models. Mice were placed in the central junction facing a closed arm and allowed to roam the maze for five-min. Individual data points and box and whiskers shown for the time spent in open arms (A, P = 0.958), closed arms (B, P = 0.469), the central junction (C, P = 0.753), and the ratio of time spent in the open versus closed arms (D, P = 0.788) (n = 104).

Fig. 5.

Pre-pulse inhibition (PPI). Mean ± standard error shown for PPI % inhibition, calculated separately for each pre-pulse dB level as: (response to 110 dB alone - response to 110 dB with pre-pulse) / response to 110 dB alone. The baseline response was defined as: (response to 110 dB alone - response to 68 dB alone) / response to 110 dB alone (n = 90). Treatment P = 0.525; all post-hoc pairwise treatment by pre-pulse dB level contrasts were non-significant.

Fig. 6.

5-Choice Serial Reaction Time Task (5CSRTT). Individual data (one point per mouse per 5CSRTT session) and box and whiskers shown for accuracy, omission, and premature response percent for 5CSRTT training (A-C, treatment P = 0.743, 0.738, and 0.910, respectively), variable stimulus duration probe (D-F, treatment P = 0.946, 0.803, and 0.229, respectively), variable delay probe (G-I, treatment P = 0.781, 0.484, and 0.722, respectively) and distraction probe (J-L, treatment P = 0.643, 0.848, and 0.663, respectively) (n = 16 mice, see methods for details).

Fig. 7.

Ensemble of Gene Set Enrichment Analysis (EGSEA). APAP treatment over control fold changes in top 20 enriched terms for (A) MSigDB hallmark gene sets and (B) KEGG pathways sorted from top to bottom by EGSEA median ranking score. Analysis repeated for all mice and stratified by sex (n = 24).