All experimental procedures were in accordance with National Institutes of Health guidelines (NIH Publication No. 85–23, revised 1996) and approved by the Institutional Animal Care and Use Committee at Texas A&M University. Timed-pregnant Sprague Dawley rats purchased from Charles River (Wilmington, MA) were housed in a temperature-controlled room (23°C), with a 12:12-h light–dark cycle. Animals were randomly assigned to pair-fed control (Control; n = 5 dams), pair-fed Juice without Nicotine (Juice; n = 5 dams), or Juice with Nicotine (Nicotine; n = 4 dams) groups on gestational day (GD) 5. All animals were weighed prior to the start of treatments. Control and Juice animals were yoked with Nicotine animals of similar weight for the duration of the study. Nicotine group animals received an ad libitum diet and daily feed intakes were measured. Control and Juice group animals were fed the same amount of diet consumed by Nicotine group animals. All animals had free access to water.

The e-cig vaping system and exposure paradigm were described previously (Orzabal et al., 2019). The vaping chamber system, custom-engineered for thee-cig vaping protocol, comprised a Sense Herakles Sub-Ohm Tank (Sense Technology, San Francisco, CA). Precision-controlled aerosol release technology with a programmable atomizer and airflow control was used to establish the e-cig vaping paradigm. The constant airflow rate of 2.5 L/min through the vaping chambers was controlled with approximately 42 mL of e-cig vapor puff, for 1 s in 20 s intervals. The animals underwent vaping exposure 3 h/day, 5 days/week during pregnancy, from GD 5 to GD 20. The vaping exposure paradigm utilized in this study has been shown to effectively modulate nicotine yield during vaping treatments (Talih et al., 2015).

The e-cig vaping liquid (juice) was compounded with a composition of 80:20 propylene glycol (Fisher, Hampton, NH) to glycerol (Acros Organics, Fair Lawn, NJ), similar to e-cig liquids available on the market. In the present study, we selected a ratio similar to that utilized by human e-cig users to get a “throat hit” sensation, and that has a higher amount of nicotine in the vapor as documented (Li et al., 2016; Ooi et al., 2019; Orzabal et al., 2019, 2022). For the Nicotine group, animals were first acclimatized to lower doses of 5% (50 mg/mL) nicotine added in the same e-cig liquid (juice) during GD 5–8, then treated with higher doses of 10% (100 mg/mL) nicotine during GD 9–20. The 10% nicotine concentration was based on the average nicotine concentration found in e-cigs on the vaping market (Matta et al., 2007; Goniewicz et al., 2013). As described previously, to obtain physiologically relevant levels of serum nicotine and account for differences in human and rodent nicotine metabolism, we previously evaluated the serum nicotine concentrations of dams over a 24 h period (Matta et al., 2007; Orzabal et al., 2022) and reported that the e-cig vaping paradigm utilized in this study produces serum nicotine levels (median peak serum nicotine =27.7 ng/mL), comparable to moderate/high level human smokers (Russell et al., 1980; Benowitz et al., 1983; Benowitz and Jacob, 1984; Lawson et al., 1998; Moyer et al., 2002; Wagener et al., 2017; Wagner et al., 2017; Orzabal et al., 2019). The Nicotine group was utilized to test the effects of vaping e-cig liquid with nicotine. The Juice group was tested for the effects of vaping nicotine-free e-cig liquid (juice). The Control group animals were set in identical conditions to animals in the Juice and Nicotine groups, but with only room air passing through the vaping chambers. We previously reported the chemical composition of the aerosols produced by our vaping system via mass spectrometry that showed several hazardous aldehyde species and other chemicals in addition to propylene glycol and glycerol (Orzabal et al., 2019, 2022). As reported previously (Orzabal et al., 2022), these aerosols resemble the chemical profile of aerosols derived from human e-cig vaping devices (Goniewicz et al., 2013, 2014; Hahn et al., 2014; Talih et al., 2015).

On GD 21, a day before parturition, animals—were deeply anesthetized with isoflurane in the anesthesia chamber and sacrificed by decapitation and fetuses were carefully separated. Two male fetuses from each dam were randomly selected from a similar position in the uterine horn. One pup per dam was used for immunoblotting while the other pup was used for immunohistochemistry. During normal development, male and female hippocampal development differ significantly and brain development in humans and animal is directly regulated by sex hormones (Yagi and Galea, 2019; Mu et al., 2020). To accurately identify hippocampal-specific alterations induced by prenatal e-cig aerosol exposure without the confounding factor of sex, only male offspring were assessed in these experiments. Fetal whole brains from the male fetuses were extracted under a dissection microscope via craniotomy and serially washed in cold phosphate buffered saline (PBS). One of the fetal brains was dissected, meninges were removed, and hippocampi were micro-dissected in ice-cold HEPES buffer. Individual samples were then flash frozen and stored at −80°C until immunoblot analyses. For immunohistochemistry, the other whole fetal brain was embedded in optimal cutting temperature compound (OCT) (Sakura Finetek, Torrance, CA) and cryopreserved for immunofluorescence.

Hippocampal tissues were lysed in ice-cold RIPA buffer, pH 7.5 (Cell Signaling Technologies, Danvers, MA) with a protease inhibitor cocktail (cOmplete Mini; Roche Applied Biosciences, Germany). The lysates were centrifuged at 10,000 xg for 10 min at 4°C and supernatants were collected. Total protein concentrations were determined using the Bradford method and a BCA assay kit (Thermo Scientific, Rockford, IL). The protein samples (20 μg) were separated on 4 ~ 20% Tris–HEPES polyacrylamide gels (Bio-Rad, Hercules, CA) by electrophoresis. The separated proteins were transferred onto PVDF membranes (Immobilon-P; Millipore, Bellerica, MA). The membranes were blocked with 5% BSA in 0.01 M PBS (pH 7.4) and 0.05% Tween-20 (TPBS), for 1 h at room temperature. Subsequently, the membranes were probed with one of the following primary antibodies (Cell signaling, Danvers, MA), overnight at 4°C: Phospho-mTOR (Ser2448) (1:1,000; #2971); mTOR (1:1,000; #4517); Phospho-4E-BP1 (Thr37/46) (1:1,000; #2855); 4E-BP1 (1:1,000; #9644); Phospho-p70S6K (Thr389) (1:1,000; #9234); P70S6K (1:1,000; #9202); DEPTOR (1:1,000; #11816); RAPTOR (1:1,000; #2280); RICTOR (1:1,000; #2114); Phospho-PKCα (Thr638/641) (1:1,000; #9375); PKCα (1:1,000; #2056); and β-actin (1:5,000; #4967). The membranes were then incubated with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. The immune complexes were detected by chemiluminescent substrate (Thermo Scientific, Rockford, IL). Densitometry analysis was performed with AzureSpot software (Azure Biosystems, Dublin, CA).

Fetal brains were sectioned at 8 μm in a coronal plane using a Leica CM1860 cryostat (Leica Biosystems, Buffalo Grove, IL). Sections were fixed with ice-cold methanol (30 min, −20°C), rinsed in PBS, incubated in 10% normal serum (60 min), followed by incubation with Phospho-mTOR (Ser2448) (1:250; #2971) primary antibody overnight at 4°C in a humidified chamber. The sections were further incubated with goat anti-rabbit IgG secondary antibody (Alexa Fluor 488, #A11008; Invitrogen, Carlsbad, CA), for 60 min at room temperature. Nuclei were stained with DAPI (ProLong Gold antifade, #P36931; Invitrogen, Carlsbad, CA). Digital images were captured using an Olympus BX63 stereomicroscope equipped with U-HGLGPS fluorescent light source, ORCA-Flash 4.0 LT digital camera (Hamamatsu, Japan), and Olympus cellSens Dimension 1.16 software (Olympus, Japan).

Immunoblotting data for the expression levels of different proteins were analyzed utilizing one-way ANOVA with a post hoc analysis of uncorrected Fisher’s LSD. Normality was assessed using Shapiro–Wilk normality test, and dependent measurements passed normality prior to performing ANOVA. All data are presented as mean ± SEM. Significance was established a priori at p < 0.05.

Our data showed that e-cig vaping during gestation altered the mTOR system. Although little is known about the effects of e-cig exposure on the mTOR system in the developing brain, there are reports documenting e-cig effects on other organ systems (Orzabal et al., 2019, 2022; Orzabal and Ramadoss, 2019). The mTOR signaling system is intricately associated with fetal and neonatal brain development and is essential for growth, survival, and differentiation of neuronal stem and progenitor cells (Saxton and Sabatini, 2017). Normal development of brain size is also controlled by mTOR system and mice lacking mTOR exhibit smaller brain size with decreased neurons and neuronal progenitor cells (Kim, 2015). Although studies on brain mTOR in the context of e-cigarette vaping are limited, others have reported effects of e-cig vaping on mTOR in other organs systems. For instance, one study using pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and patients with pulmonary arterial hypertension (PAH) showed differential susceptibility of PAH group to e-cig bubbling and demonstrated pharmacologic induction of autophagy via direct inhibition of mTORC1 and indirect activation of mTORC2 with rapamycin reversing the e-cig-induced downstream effects on endothelial dysfunction and AKT3 signaling (Liu et al., 2023). We had previously reported that e-cig vaping during gestation results in an increase in the concentration of the branch chain amino acids (leucine, isoleucine, valine) in the rat fetal lungs (Orzabal et al., 2021), and others have shown that the increases in branch chain amino acids results in activation of mTORC1, which in turn modulates the production of reactive oxygen species and leads to an inflammatory phenotype (Zhenyukh et al., 2018). Although the current study did not assess relationship between mTOR system and brain structural and functional alterations following e-cig vaping during pregnancy, others have utilized female CD1 pregnant mice and demonstrated that prenatal e-cig vaping decreases brain glucose utilization and worsens the outcome in offspring hypoxic–ischemic brain injury, but did not specifically assess the mTOR pathway (Sifat et al., 2020). Outside the e-cig research area, mutations of mTOR have been associated with a spectrum of brain overgrowth phenotypes (Mirzaa et al., 2016).

To determine if gestational chronic nicotine exposure dysregulates mTORC1 signaling, we analyzed the abundance levels of downstream molecules within mTORC1 signaling pathway. The phosphorylation level of 4EBP1 (P-4EBP1) and total 4EBP1 expression were both significantly higher in the fetal hippocampi from the Nicotine group. Hippocampal phosphorylation of p70S6K (P-p70S6K) and Total p70S6K increased in the Nicotine group compared to the Control group. To determine if gestational chronic nicotine exposure dysregulates the downstream of mTORC2 signaling pathway, we analyzed the protein kinase C alpha (PKCα). The phosphorylation level of PKCα (P-PKCα) was significantly lower in the fetal hippocampi from the Nicotine group. mTOR makes up the catalytic subunit of two complexes, mTORC1 and mTOR2, each of which have distinctive substrates and functions, and unique accessory proteins and sensitivity to rapamycin (Yang et al., 2013; Zarogoulidis et al., 2014; Liu and Sabatini, 2020). mTORC1 plays a major role in convergence of multiple cellular signals to sense the availability of nutrients, growth factors, and energy to stimulate cellular growth and catabolic activity during stress (Liu and Sabatini, 2020). In contrast, little is known about mTORC2 although it is possible that mTORC2 may have some regulating effect on mTORC1 system (Liu and Sabatini, 2020; Szwed et al., 2021). mTORC1 directly promotes protein synthesis by ultimately phosphorylating the Eukaryotic Translation Initiation Factor 4E-binding Proteins (4E-BPs), as well as Ribosomal Protein S6 Kinase 1 (S6K1; Carabulea et al., 2023). Others have shown that nicotine significantly reduced hyperalgesia in mice that received acute/repeated rapamycin injections, and nicotine reversed the outcomes of rapamycin on the phosphorylation of 4E-BP1 and S6K (Li S. et al., 2019; Li Y. et al., 2019). Outside the e-cig field, secondhand smoke during pregnancy showed decreased activation of the mTOR family of proteins in mice placenta, and these may be explained by differences between smoking and vaping and also the tissue being studied, i.e., placenta vs. the brain in the current study (Mejia et al., 2017). In another study, the human brain microvascular endothelial cell line was cultured, and nicotine treatment significantly activated the PI3K/Akt/mTOR pathway (Wu et al., 2020). Overall, this is the first report to demonstrate that the nutrient-sensing mTORC1 and the mTORC2 pathways are both altered by e-cig vaping during development.

To investigate the effect of gestational chronic nicotine exposure on mTOR complexes, we analyzed hippocampal levels of DEPTOR, RAPTOR, and RICTOR. The DEPTOR expression level was not affected by prenatal chronic exposure to e-Cig nicotine. The expression levels of RAPTOR and RICTOR were significantly higher in the Nicotine group compared to those of Control. Distinctly, the RAPTOR expression level increased over 10-fold in the Nicotine group compared to Control. RAPTOR is essential for intracellular localization of mTORC1 and has a role in recruiting substrates by binding to respective mTOR signaling motifs and the core of mTORC2 is instead defined by the scaffold protein, RICTOR (Carabulea et al., 2023). There is an overall lack of information available about the effects of e-cig on RICTOR, RAPTOR, and DEPTOR in any organ system, including the brain, especially in development. This study fills a major gap in the literature and provides critical information of brain hippocampal effects of e-cig vaping.

e-liquids are not standardized by manufacturers (Smith et al., 2020). VG (40 CFR 180.950) and PG (21 CFR 184.1666) are both generally regarded as safe for oral ingestion as liquids by the FDA and EPA (GRAS), whereas aerosolized VG/PG has not been classified as safe(Qu et al., 2018; Berkelhamer et al., 2019; Frazier et al., 2021). Currently, the proportion of VG:PG is chosen based on personal preferences for the sensory profiles VG and PG contribute. Flavor chemicals are a major component of e-cig refill fluids (Behar et al., 2018; Hua et al., 2019; Krusemann et al., 2020), but the current study did not use flavorings. The data showed that at least as far as mTOR in fetal hippocampus is concerned, neither VG nor PG played a role, and the alterations were nicotine dependent. Nicotine in vapor form has been reported to have a range of effects on reproductive function, fetal and child development such as low birth weight, increased blood pressure, and increase in inflammatory phenotype in the newborn (Suzuki et al., 1980; Aubert et al., 1987; Mohsenzadeh et al., 2014). In contrast to our data, one study that examined effects of dihydroxyacetone, a combustion production of propylene glycol and glycerol in e-cig vapor on human hepatocellular carcinoma cell line, showed slight increases in p-mTOR, while mTOR, RICTOR and RAPTOR displayed fluctuating levels (Hernandez et al., 2022); these differences could be due to the levels of dihydroxyacetone in an in vitro setting.

The effect of e-cig vaping on the hippocampal mTOR signaling in the context of pregnancy and development is largely unknown. We utilized an established gestational e-cig vaping paradigm to delineate how vaping impacts the developmental hippocampal mTOR signaling system. Based on our data, we conclude that gestational e-cig vaping exposure alters mTORC1 and mTORC2 signaling, their activity indices, and related downstream pathways in the developing hippocampus. Although we report deficits in fetal body weight and length, we did not examine neuroanatomic and structural deficit; future studies are warranted to assess the structural, functional, and behavioral deficits that arise out of developmental e-cig vaping exposure and if and how they relate to mTOR signaling in hippocampus. These studies may in turn facilitate development of clear targeted pharmacological and nutraceutical therapeutic strategies for any developmental deficits related to hippocampal functions of learning and memory during development.