Direct Effects of Nicotine Exposure on Murine Calvaria and Calvarial Cells

Abstract

Despite the link between adverse birth outcomes due to pre- and peri-natal nicotine exposure, research suggests 11% of US women continue to smoke or use alternative nicotine products throughout pregnancy. Maternal smoking has been linked to incidence of craniofacial anomalies. We hypothesized that pre-natal nicotine exposure may directly alter craniofacial development independent of the other effects of cigarette smoking. To test this hypothesis, we administered pregnant C57BL6 mice drinking water supplemented with 0, 50, 100 or 200 μg/ml nicotine throughout pregnancy. On postnatal day 15 pups were sacrificed and skulls underwent micro-computed tomography (µCT) and histological analyses. Specific nicotinic acetylcholine receptors, α3, α7, β2, β4 were identified within the calvarial growth sites (sutures) and centers (synchondroses). Exposing murine calvarial suture derived cells and isotype cells to relevant circulating nicotine levels alone and in combination with nicotinic receptor agonist and antagonists resulted in cell specific effects. Most notably, nicotine exposure increased proliferation in calvarial cells, an effect that was modified by receptor agonist and antagonist treatment. Currently it is unclear what component(s) of cigarette smoke is causative in birth defects, however these data indicate that nicotine alone is capable of disrupting growth and development of murine calvaria.

Effects of nicotine exposure on murine mandibular development

“…The observed reduction in cotinine in the highest dose of nicotine (200 μg/ml) may indicate less consumption of water in those individuals, however animals were monitored for dehydration daily throughout the pretreatment, breeding, and pregnancy and no indication of dehydration was observed. This reduction in cotinine may also be related to variable metabolism of nicotine[17–19, 27]. Unlike in the human population, we did not observe a reduction in weight of the nicotine exposed animals 15 days post-natal, indicating that if there was a reduction in birth weight associated with in utero nicotine, it was regained quickly after birth in this murine model [3, 28].…”

“…Pups from 2 or 3 litters resulting from the pre-treated paired breeding ( Table 1 ) were grown for 15 days, the age at which reliable radiographic images can be procured for cephalometric analysis, then sacrificed using carbon dioxide asphyxiation with secondary cervical spine dislocation [17, 22, 23]. All collected skulls were fixed with 4% paraformaldehyde, then switched to 70% Ethanol and bisected along the sagittal suture.…”

A variant in IL6ST with a selective IL-11 signaling defect in human and mouse

“…6 Environmental factors may additionally influence phenotype penetrance. In humans multiparity, macrosomia, 30,31 in utero exposure to nicotine 32,33 , and alcohol 34 have been identified as potential risk factors for the development of craniosynostosis. It will be interesting to see whether changes in the microbiota could also contribute to the variable penetrance of skeletal abnormalities in the context of impaired IL-11 signaling.…”

Ion Channel Contributions to Morphological Development: Insights From the Role of Kir2.1 in Bone Development

“…Both the cardiovascular and musculoskeletal systems are reliant on ion channels for proper function and development (Barchi, 1997;Rahm et al, 2018). Recent work shows that nicotine alone can increase proliferation of murine calvarial cells providing a possible mechanism for the increased observation of craniosynostosis in nicotine-exposed fetuses (Durham et al, 2019). They also show that several nicotinic acetylcholine receptor subunits (α3, α7, β2, β4) are present in the calvarial sutures and synchondroses.…”

“…They also show that several nicotinic acetylcholine receptor subunits (α3, α7, β2, β4) are present in the calvarial sutures and synchondroses. Nicotine exposure additionally affects other bone structures, such as the mandible, in which a reduction in mandibular ramus height, mandibular body height, and molar length can be observed (Durham et al, 2019). Additionally, e-cigarette aerosol exposure in Xenopus has recently been shown to cause craniofacial defects including midface hypoplasia and median facial clefts.…”