Effects of nicotine exposure on murine mandibular development

Abstract

Nicotine is known to affect cell proliferation and differentiation, two processes vital to proper development of the mandible. The mandible, the lower jaw in mammals and fish, plays a crucial role in craniofacial development. Malformation of the jaw can precipitate a plethora of complications including disrupting development of the upper jaw, the palate, and or impeding airway function. The purpose of this study was to test the hypothesis that in utero nicotine exposure alters the development of the murine mandible in a dose dependent manner. To test this hypothesis, wild type C57BL6 mice were used to produce in utero nicotine exposed litters by adding nicotine to the drinking water of pregnant dams at concentrations of 0 μg/ml (control), 50 μg/ml (low), 100 μg/ml (medium), 200 μg/ml (high) throughout pregnancy to birth of litters mimicking clinically relevant nicotine exposures. Resultant pups revealed no significant differences in body weight however, cephalometric investigation revealed several dimensions affected by nicotine exposure including mandibular ramus height, mandibular body height, and molar length. Histological investigation of molars revealed an increase in proliferation and a decrease in apoptosis with nicotine exposure. These results demonstrate the direct effects of nicotine on the developing mandible outside the context of tobacco use, indicating that nicotine use including tobacco alternatives, cessation methods, and electronic nicotine delivering products may disrupt normal growth and development of the craniofacial complex.

Fig 1. Mandible landmarks and measurement schematics.

A) Cephalometric landmarks on murine mandible used for measurements. Dark lines between points indicate measures used in this analysis. Schematic modified from Klingenberg et al. 2004. B) Representative hematoxylin and eosin stained molars with lengths measured indicated with black lines, area of interest surrounding molars indicated in yellow, and molars 1–3 identified.

Fig 2. In Utero nicotine exposure model and cephalometric analysis of mandible.

A) Nicotine metabolite cotinine is present at levels approximating active smoking in the blood serum of dams pretreated for 3 weeks with nicotine in drinking water. B) Representative lateral digital X-Rays of 0 μg/ml (control), 50 μg/ml (low), 100 μg/ml (medium), or 200 μg/ml (high) nicotine exposed post-natal day 15 mouse pups. C) In utero exposure to nicotine did not reduce weight of post-natal day 15 mouse pups as compared to control. Data categorized by sex with mean per exposure indicated with grey bar. Total mandibular length (D) did not vary with nicotine exposure, however, in utero exposure to high dose nicotine decreased the height of the ramus (E) significantly compared to low dose nicotine, perhaps indicating a dose dependent effect. Neither angular process height (F) nor condylar length (G) varied significantly with exposure. Molar length was significantly reduced between low and medium dose nicotine (H).The height of the body of the mandible did not vary in the mid or posterior measures but was reduced with low dose nicotine (50 μg/ml compared control) anteriorly (I). Data categorized by litter with mean for males per exposure indicated with black bar and mean for females per exposure indicated with white bar. *p<0.05 for differences between doses. No significant differences were identified between sexes.

Fig 3. Histological analysis of mandibular molars.

A) Representative hematoxylin and eosin stained sections of mandibular molars from individuals exposed to 0 μg/ml (control), 50 μg/ml (low), 100 μg/ml (medium), or 200 μg/ml (high) nicotine in utero. Molars 1–3 are marked M1, M2, M3 with tongue above for orientation. B) Histomorphometric analysis of molar length for M1, M2, M3 indicate no change with exposure. C) Length of erupted molars 1 and 2 together is reduced with the highest nicotine dose compared to the low dose however, the total molar length does not vary (D). n = 4 per exposure Scale Bar = 500 μm. *p<0.05.

Fig 4. Cell proliferation and death in mandibular molars.

A-B) Representative histological images of mandibular molars stained for Proliferating Cell Nuclear Antigen (PCNA, A) and Active Caspase 3 (Caspase, B) from individuals exposed to 0 μg/ml (control), 50 μg/ml (low), 100 μg/ml (medium), or 200 μg/ml (high) nicotine in utero. Black horizontal arrows indicate positive staining and white vertical arrows indicate negative cells for each target. C) Quantification of percent of positive staining within the area of interest surrounding the molars (Fig 2B) indicates increasing proliferation with nicotine exposure. D) Apoptosis as indicated by positive caspase staining was reduced in the area of interest surrounding the molars in individuals exposed to high dose nicotine as compared to control and all other nicotine doses. n = 4 per exposure Scale Bar = 500 μm. *p<0.05 **p<0.01.