(M1230-04-28) Impact of In-utero Electronic Cigarette Exposure on Neonatal Neuroimmune and Mitochondrial Function

Purpose: Electronic nicotine delivery system (ENDS), also commonly known as cigarette (e-Cig) is considered as a safer alternative to tobacco smoke and therefore has become extremely popular among all age groups and sex. Around 15% of pregnant women are now using e-Cig in the US which keeps increasing at an alarming rate. Harmful effects of tobacco smoking during pregnancy are well documented for both pregnant and postnatal health. However, there is lack of preclinical and clinical studies exist to evaluate the long-term effects of prenatal e-Cig exposure on postnatal health. Our recently published article demonstrated that maternal e-Cig exposure is associated with disrupted postnatal blood-brain barrier (BBB) integrity and deteriorated motor, learning and memory function. In this study, we have evaluated the effect of maternal e-Cig exposure on neonatal/postnatal mitochondrial function, oxidative stress and neuroinflammation which may elucidate the potential harmful neurotoxic impact of maternal e-cig use and associated cerebrovascular dysfunction in postnatal brain and will help to identify potential therapeutics.

Methods: Pregnant CD1 mice were exposed to e‐Cig vapor (2.4% nicotine) from gestational day 5 (E5) till postnatal day 7 (PD7), and primary neuron was isolated from pups at E16/17. Cellular total reactive oxygen species (ROS) and mitochondrial superoxide were measured in primary neurons using probe CM-H₂DCFDA and Mitosox red, respectively. Mitochondrial function was assessed by Seahorse XF Cell Mitostress analysis. Level of pro-inflammatory cytokines was measured in primary neurons, PD7 and PD90 by RT-PCR and immunobead assay. Expression of anti-oxidative markers such as super oxide dismutase (SOD-1, SOD-2), heme oxygenase (HO-1), nuclear factor erythroid 2-related factor 2 (NRF2), NAD(P)H Quinone Dehydrogenase 1 NQO1) and proinflammatory modulator nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) were evaluated by Western blot in primary neuron, PD7 and PD90.

Results: In our study, significantly higher level of cellular total ROS (P < 0.05) and mitochondrial superoxide (P < 0.01) was observed in prenatally e-cig exposed primary neuron. We also observed significantly reduced expression of anti-oxidative markers, increased expression of proinflammatory modulator and cytokines in primary neuron and PD7 (P < 0.05) but not in PD90 postnatal brain.

Conclusion: Our findings suggest that prenatal e-Cig exposure induces some long-term neurotoxic effects on neonates by increasing cytokines level, oxidative stress and disrupting mitochondrial function which may alter fetal brain immune function that makes such offspring more vulnerable to brain insults such as cerebrovascular and neurodegenerative dysfunction. In future, we would like to identify the potential therapeutics to treat OS, neuroinflammation and improve mitochondrial function in prenatally e-cig exposed offspring.

References: 1. Archie, S.R., Sifat, A.E., Zhang, Y. et al. Maternal e-cigarette use can disrupt postnatal blood-brain barrier (BBB) integrity and deteriorates motor, learning and memory function: influence of sex and age. Fluids Barriers CNS 20, 17 (2023). https://doi.org/10.1186/s12987-023-00416-5

Acknowledgements:

Funding: NIH Grants R01DA049737 and R01DA02912

Study Design

Effect of maternal e-cig use on (A) Mitochondrial superoxide (B) Cellular total ROS (C) Mitochondrial respiration (D) Energy map in primary neurons. Control: oxygenated air and Treatment: Blu e-cig (2.4% nicotine); n=3, P< 0.05. In-utero e-cig use increased mitochondrial superoxide, total ROS and decreases mitochondrial respiration indicating mitochondrial dysfunction.

Effect of in-utero e-cig use on anti-oxidative markers (NRF2, NQO1, SOD-2 and HO-1) and pro-inflammatory modulator (NFKB) in primary neuron and postnatal brain at PD7; n=4, P < 0.05
Control: oxygenated air and Treatment: Blu e-cig (2.4% nicotine)