In Utero Exposure to Maternal Electronic Nicotine Delivery System use Demonstrate Alterations to Craniofacial Development

Introduction

Usage of Electronic Nicotine Delivery Systems (ENDS) is an ever-emerging public health crisis, with health implications on most organ systems.^1–11 The ENDS industry has grown since introduction in the 2000s to become a multi-billion-dollar industry and is still growing. User demographics continue to be low among adults over the age of 25 (∼3–4%), but are increasing at alarming rates in middle school (10.5%), high school (27.5%), and young adults aged 18–24 (7–8%).^10–18 These demographics include naïve users who never used cigarettes, calling into question the ENDS industries’ stance that electronic cigarettes have a great potential as a smoking cessation product,^19–21 a position not wholly shared by the public health community.^22–27 As there continues to be disagreement in the field concerning the potential safety hazards and long-term health outcomes of ENDS,^28–30 the fact remains that use of these products continues to rise. Furthermore, these products have now been implicated in acute lung injury,^{7,22,25,31–39} and potentially irreversible impacts on vascular health.^40,41 Calls to regulate ENDS products has driven legislation to increase age of purchase and decrease availability of flavored ENDS products, ¹⁵ as well as attempts to ban manufacturing and sale. As there is continued use and popularity of ENDS, especially among youth, there is a critical and pressing need for study of the health hazards associated with these products.

With most ENDS use occurring in the naïve populations of adolescents and young adults, the potential dangers of in utero exposures and maternal health cannot be ignored.^42–53 There is a wide range of estimates of ENDS use in pregnancy with reports suggesting rates of ∼1–4%.^54–57 The common components of most ENDS, nicotine and the carrier (most common are propylene glycol and vegetable glycerin, or PG/VG), have already been studied for their effects on health and should give cause for caution.^58–60 Although the cigarette has been studied extensively for health outcomes there is a paucity of data for nicotine replacement therapies and now emerging ENDS for effects on the developing organ systems.

As with many disease states, altered growth and development of the craniofacial tissues is due to a number of factors that is best described by gene-environment interactions with teratogens influencing development by decreasing the threshold for penetrance. ⁶¹ Many in utero teratogenic exposures that influence craniofacial development have been studied.^62–71 Of particular interest now are nicotine delivery devices to garner a better understanding of the effects of early exposures to this psychoactive drug on development.^66,72,73 Nicotine is the common component of cigarettes, many nicotine replacement therapies, and the emerging ENDS. As research has determined nicotine alone can elicit growth alterations,^62–71 work is needed to understand if ENDS exposures in utero will have similar effects. These data may prove necessary to inform public health policies and regulations surrounding ENDS to provide more substantial evidence concerning ENDS as a “safer” alternative to traditional tobacco use.

This preliminary report seeks to establish a model for the study of ENDS components on craniofacial development. Overall efforts are needed to understand how the formulation of the common carrier components nicotine and PG/VG affect craniofacial development with these emerging technologies. Our hypothesis is that ENDS exposure will alter craniofacial growth in a murine experimental model.

Methods

8 week old sexually mature male and female mice (C57BL6 (Jackson Laboratories 000664)) were used in this study, initially as 9 dams and 9 males to allow for pair mating. After an initial 1-week acclimation to the environment (no exposure) the animals were subjected to the described exposure regimen below. All female dams were exposed the next day after pairing with a male (∼E0). Exposures were facilitated by use of the Scireq inExpose system (EMKA Technologies, Montreal QC, Canada, https://www.scireq.com/inexpose). This real time exposure control system allows for regulation of nicotine concentration, puffs/minute, and time of exposure. Exposure modalities implemented here used a 1 puff/minute, 4 h/day, five days a week, throughout gestation exposure approach. Human user behaviors for electronic cigarettes are largely unexplored or have been characterized as variable.^74–77 Our group has previously published this regimen allowing for nicotine levels to reach that of an active nicotine product use.^78,79 Further, cotinine levels were collected from the urine of adult female mice previously to establish these parameters using an ELISA kit (Mybiosource MBS580061). Exposures used here were filtered air control, vehicle control 50/50 PG/VG or EC Vehicle, and PG/VG vehicle with 20 mg/ml nicotine added, or EC Nicotine. Murine gestation varies between 19–21 days. Fetal loss was not noted in our model but some post-natal loss of whole litters did occur allowing for re-enrollment of the female dam. Male breeders were removed upon obvious sign of pregnancy of the dam (approximately 7–10 days after pairing). Exposures were continued until birth of litter at PN1, when exposures ceased.

Litters were allowed to age until postnatal day 15 when sacrifice occurred for the study of cephalometric parameters. After sacrifice, pups were weighed and skulls were fixed for Microcomputed Tomography (MicroCT) analyses. Methodology followed^{64–69,71–73} using a Skyscan 1176 (Bruker Kartuizerseg 3B, 2550 Kontich, Belgium) scanner to garner 3D reconstructions for analysis. Using Analyze Pro software (Analyze Direct, Overland Park, KY), renderings were oriented and subject to cephalometric landmarking and measure of cranial, facial, and cranial base dimensions.^{63–66,68–71} Linear and data were compared by treatment (Filtered Air control, EC Vehicle and EC Nicotine).

Statistical analysis was conducted using SPSS 28.0 (IBM, Armonk, NY). Data were screened for normality and homogeneity of variance. If these assumptions were met analyses followed using a one-way ANOVA with post-hoc Bonferonni analyses. If assumptions were violated a non-parametric approach as utilized using Kruskal Wallis with post-hoc Bonferonni analyses. Differences were considered significant if p < 0.05.

Results

Initial screening suggests female C57BL6 adults exhibited cotinine levels equivalent to clinical levels observed after active nicotine use^66,72,73 (Figure 1A). Of the 9 pregnant dams enrolled in the study, 8 netted litters resulted in n = 27 total pups at post natal day 15 for cephalometric analyses. Data demonstrated a significant decrease in weight for pups that were exposed to EC nicotine in utero (Figure 1B). Data, however, was within normal published range for age (Jackson Laboratories).

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Figure 1.

A. Cotinine levels in ENDS-exposed mice. Female mice exposed to filtered air (FA), E-cigarette Vehicle (PG/VG) and EC Vehicle with nicotine added (20 mg/mL nicotine). Note detectable levels of cotinine in the nicotine exposed animals equivalent to active nicotine user. B. Weight of resulting C57BL6 postnatal day 15 offspring in exposed literature segregated by sex. Note decrease in the EC Nicotine group but within normal range for age ** p < 0.01*** p = 0.001, **** p < 0.001. Bars represent mean+/- standard error.

Calvarial cephalometric measures did not demonstrate any differences by exposure modality in this sample data set (Figure 2). Specifically, there were no significance differences in cranial length, cranial height, cranial width, or cranial index. However, our data did suggest several segregating significant differences for facial measures in the EC nicotine in utero exposed group (Figure 3). Specifically posterior and mid facial width demonstrated decreased width or a narrowing for EC nicotine compared to filtered air controls. Facial length also demonstrated a significant difference with EC nicotine having shorter lengths compared to filtered air control. Measures of the palate also demonstrated a decreased palatal width in the EC vehicle group and a shortening of the palate and cranial base as measured from the spheno-occipital synchondrosis to the incisor interface with the premaxilla for EC nicotine.

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Figure 2.

A. Denotes mean maximum cranial width, or cephalometric point CW indicated the widest part of the cranium. Note no significant differences. B. Denotes mean maximum cranial length, or cephalometric point of the posterior parietal bone to nasion. Note no significant differences. C. Denotes mean maximum cranial height, or cephalometric point measured from opisthion to the frontoparietal suture. Note no significant differences. D. Denotes mean cranial Index, a reference of cranial width to cranial length. Note no significant differences. Bars represent mean+/- standard error.

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Figure 3.

A. Denotes mean anterior facial width measured across the anterior junctions of the zygomatic arch and the maxillary process. Note no significant differences. B. Denotes mean mid facial width measured across the midpoints of the zygomatic arches. Note a statistically significant decrease form EC nicotine exposed pups compared to control. C. Denotes posterior facial width measured across the posterior junction of the zygomatic arches and temporal bones. Note a statistically significant decrease form EC nicotine exposed pups compared to control. D. Denotes mean facial length measured from the spheno-ethmoidal junction to rhinion. Note a statistically significant decrease form EC nicotine exposed pups compared to control. E. Denotes width of the palate measured between the first and second molars bilaterally. Note a statistically significant decrease in measures for the EC vehicle group compared to control. F. Denoted length of the palate and cranial base as measured from the spheno-occipital synchondrosis to the interface of the incisors with the premaxilla. Note significant decrease in length observed for the EC nicotine exposed pups compared to control. * p < 0.05, ** p = 0.01. Bars represent mean+/- standard error.

Discussion/Conclusions

In this study, experiments were designed to establish a model for study of ENDS exposures to the developing embryo and begin to interrogate the effects on post-natal craniofacial growth. Preliminary analyses suggest ENDS modalities can be appropriately modeled for both adult health outcomes and an efficacious model to study in utero exposures and effects on developing systems. Data also suggest when in utero ENDS exposures are interrogated in the post-natal developing mouse, several segregating effects are noted. Firstly, some segregating decrease in post-natal weight was found, though in normal range for this model. This may suggest some developmental somatic delay and will require further study. Specific to the craniofacial skeleton, no segregating differences were observed grossly for the general parameters of cranial length, height, and width. This result is informative to several ends. Firstly, it suggests from a correlative perspective calvarial volume, approximated by these measures,^68,80,81 is not being greatly affected and thus overall neural expansion is not likely impacted. Further study will be necessary to confirm brain volumes are consistent. Secondly, it suggests from a craniofacial growth perspective that the calvaria might not be the target of altered growth due to the ENDS exposures modeled here.

Segregating differences were observed for several facial morphology parameters based on exposure modality. Specifically facial widths and facial lengths were observed to be decreased suggested facial narrowing and facial shortening due to ENDS exposures with nicotine. This is a significant result as the consequences of delayed or altered facial growth are many for the fields of oral health. Work will be continued to determine if there is a typical facies associated with nicotine exposures and more specifically ENDS exposures that occur during development. These results are also consistent with published literature that has modeled nicotine exposures in utero.^66,72,73 As there is a tight coordination between cranial base growth and morphology and the developing face,^82–90 continued work should focus on this anatomical area, specifically the synchondroses, as a potential target of ENDS exposures, as well as the developing nasal capsule^91–96 important to facial expansion.

Palatal morphology was also investigated here and suggests effects that were potentially unexpected. The carrier exposure alone was demonstrated to result in narrower palates. This suggests a need for continued study of carrier component exposures to understand the independent effects of product exposuers that include propylene glycol and/or vegetable glycine. A reduction in palatal and cranial base length was also observed here for the EC nicotine group. This is not unexpected given the reduced facial length observed as well as the fact that cranial base development has an intricate relationship with outgrowth of the face and palate. It is noted we did not observe any clefting of the murine palate or observe a phenotype that in when abstracted to human populations data suggest predisposes a population or individual to a cleft (wider palates or brachycephaly in general). ⁹⁷ However, another commonly cited craniofacial feature with altered growth is the narrow palate,^98,99 often arched, which is more akin to the phenotype observed here in our exposed murine model. We also note the segregating changes to the face, specifically narrower dimension has been previously shown by our group to occur in murine models exposed to nicotine in utero.^66,72,73 This further confirms previous work that has implicated nicotine exposure modalities in altered palatal development and morphology.^100–105

Future studies will be designed to capitalize on this established model of in utero ENDS exposure in several pertinent directions. ENDS chemistry is a logical target and is the focus of ongoing efforts. Commercial products of ENDS show markedly different chemistries varying nicotine concentration amounts, and the varier materials necessary to allow for vaporization and palatability. ¹⁰⁶ Research has suggested a synergy between nicotine and PG/VG in these products where nicotine concentrations are often increased with decreasing amount of PG materials. However, as nicotine is an alkaline, an acid is then often added, most often benzoic acid, to alter pH. Due to the varied possible permutations that chemistry was not studied here. The system proposed here is flexible for any “homemade” exposures and can be extended to commercial products as well. However, with the legality of commercial product production and marketing currently in question, that is not our current focus. Regimen of exposure was also chosen here for consistency, but it is noted that user variability very much plays into what effects may be observed. As ENDS products continue to develop, user habits will likely become clearer from behavioral and epidemiological studies.

Overall, this preliminary report suggests the in utero exposure to ENDS products can affect craniofacial development. Data suggest the most likely target of these effects in the developing face and palate. These data suggest a need for further investigation into these merging products.