[Effects of prenatal nicotine exposure on enamel formation of offspring mice]

Objective: To investigate the effects of nicotine on the morphology, structure of offspring's dental germ, enamel organ and other dental tissues and the further potential epigenetic mechanisms by establishing prenatal nicotine exposure mouse model. Methods: Ten C57BL/6 pregnant mice were randomly divided into control group (physiological saline subcutaneous injection) and prenatal nicotine exposure (PNE) group (nicotine subcutaneous injection) by using a random number table. Postnatal day 0 (P0), postnatal day 14 (P14) and postnatal day 25 (P25) offspring mice were collected for subsequent experiments. The offspring mice were divided into offspring control group and offspring PNE group according to the maternal group respectively. Weights of P0 and P25 offspring mice were recorded. Micro-CT, scanning electron microscope (SEM) and Vickers hardness test were performed to analyze the related parameters of hard tissues including alveolar bones and mandibular incisors. Total RNAs were extracted from mandible tissues and the third generation of dental epithelial stem cells (DESC) in P25 mice. The relative expression levels of osteogenic and ameloblastic differentiation related genes were measured by real-time quantitative PCR (RT-qPCR). Immunohistochemical stainings of paraffin sections were then performed to observe the distribution and expression level of proliferating cell nuclear antigen (Pcna), amelogenin (Amelx), histone H3 trimethylated at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (Ezh2). Cell counting kit-8 (CCK-8) assays were used to detect the cell viabilities of DESCs after administrations of different concentrations of nicotine (0.01, 0.1, 1 mmol/L) and GSK126 (an inhibitor of histone methyltransferase Ezh2). Results: Compared with the control group, pregnant mice in PNE group were more likely to have adverse pregnancy outcomes, such as significantly lower offspring body weight [P0: offspring control (1.20±0.04) g, offspring PNE (0.99±0.02) g, P<0.001; P25: offspring control (15.26±1.70) g, offspring PNE (9.65±1.32) g, P<0.001] and increased stillbirths rate [offspring control (0), offspring PNE (46.40±9.30) %, P<0.001]. At P14 and P25, the distance parameters between the enamel mineralized deposits of mandibular incisors and the mesial surface of the first molar in offspring PNE group [P14: (-1 349±45) μm; P25: (-1 192±147) μm] was significantly decreased compared with the control group [P14: (-506±380) μm, P25: (504±198) μm] (P<0.05, P<0.001). The enamel column and enamel column stroma of incisors in offspring PNE group were blurred, arranged loosely and disorderly than those in the control group, while the microhardness of incisor enamel in offspring PNE group [(245.7±18.4) MPa] was significantly lower compared to the control group [(371.9±28.7) MPa] (P<0.001). HE staining showed disordered pre-ameloblast (Pre-Am) arrangement and delayed mineralization deposition point in offspring PNE group compared with the control group, while the length of transit-amplifying cell (TA) and Pre-Am region were prolonged as well. Immunohistochemical staining results displayed that the overall Pcna (P<0.05), H3K27me3 (P<0.01), Ezh2 (P<0.01) expression of labial cervical loop (LaCL) in PNE group were increased, while the positive signal of Amelx in ameloblast cytoplasm was impaired. In vitro, the addition of 1 mmol/L nicotine could significantly upregulate the expression level of Pcna (P<0.01) and downregulate the expression levels of B lymphoma Mo-MLV insertion region 1 (P<0.05), leucine rich repeats and immunoglobulin like domains 1 (P<0.05), Amelx (P<0.01). In addition, 1 mmol/L nicotine could also significantly enhance the proliferation activity of DESCs (P<0.001). Addition of 10 μmol/L GSK126, could rescue the proliferation activation effect of 1 mmol/L nicotine on DESCs. Conclusions: PNE may delay the process of enamel formation and lineage differentiation, leading to the abnormal proliferation of DESCs and changes of epigenetic modification state in H3K27me3, which affect the development of enamel in offspring mice,suggesting PNE might be one of risk environmental factor for tooth development.