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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Neurotoxicology. 2022 Jan 10;89:41–54. doi: 10.1016/j.neuro.2022.01.005

Figure 3.

Figure 3.

Nicotine exposure evokes upregulation of epigenetic mechanisms required for POA masculinization in neonates. A) Pie chart outlining genomic location distribution of the top 285 masculinizing differentially methylated regions (DMRs) between male and female POA. Differential methylation was calculated using publicly available WGBS data obtained from neonatal male, female, and estradiol-masculinized female POA (Nugent et al., 2015). B) Prior to targeted validation of DMRs, animals were developmentally exposed to either vehicle (DMSO) or nicotine (2mg/kg/day) throughout the window for POA sexualization (GD18-PND10). At PND0 and PND1, male and female pups were subcutaneously injected with either vehicle (sesame oil) or estradiol to induce masculinization. At PND4, POA was isolated in male and female pups, and co-extracted for DNA and RNA. C) Validation of range and linearity of bisulfite pyrosequencing assays designed for four of the top 285 DMRs. Bisulfite-treated rat DNA methylation standards serially prepared at 0%, 25%, 50%, 75%, and 100% methylation were used to correlate the input versus measured methylation values for the following DMRs: Dlgap1, Kcnn3, Nkain3, and Tab1. Data generated was analyzed using Pearson’s correlation (r). Dlgap1: r=0.9808, p=0.0032; Kcnn3: r=0.9945, p=0.0005; Nkain3 r=0.9977, p=0.0001; Tab1: r=0.9987, p<0.0001. N = 3 replicates for each serial DNA standard. D) Average percent DNA methylation across each DMR measured in control and treated PND4 POA. DNA methylation at the Nkain3 DMR was significantly hypermethylated in vehicle control females compared to all other groups (two-way ANOVA p=0.0061, F=4.111, sex*treatment p=0.0097). DNA methylation at the Tab1 DMR was hypermethylated in vehicle control females compared to nicotine-treated females (a-priori t-test, p=0.0410). Dlgap1: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 6 male, n = 7 female. Kcnn3: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 6 male, n = 7 female. Nkain3: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 6 male, n = 7 female. Tab1: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 6 male, n = 7 female. E) Expression of methylation-dependent masculinizing genes (MDMGs=Cyp19a1, Ebf2, Ebf3, and Nr2f2) and Dnmts (Dnmt1 and Dnmt3a) in control and treated PND4 POA. For each gene, expression was measured across multiple cohorts as fold change relative to the vehicle control male within each cohort, and subsequently normalized as percent of the control males across combined cohorts. Although there was no significant main effect of sex, Ebf2 expression was significantly higher in nicotine treated animals compared to vehicle controls (two-way ANOVA between nicotine and vehicle control groups p=0.1146, F=2.340, sex main effect p=0.8612, treatment main effect p=0.0259, sex*treatment p=0.6165). Cyp19a1: (Vehicle) Control n = 6 male, n = 5 female; Estradiol n = 5 male, n = 4 female; Nicotine n = 7 male, n = 6 female. Ebf2: (Vehicle) Control n = 4 male, n = 6 female; Estradiol n = 4 male, n = 3 female; Nicotine n = 4 male, n = 5 female. Ebf3: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 6 male, n = 6 female. Nr2f2: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 7 male, n = 7 female. Dnmt1: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 7 male, n = 7 female. Dnmt3a: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 7 male, n = 6 female. F) Heat map representing sex differences in normalized fold change values across MDMGs and Dnmts. Expression represented as the ratio of male to female normalized fold change values. M:F expression of Nr2f2 was higher in vehicle controls compared to nicotine treated animals (a-priori t-test, p=0.0499). Cyp19a1: (Vehicle) Control N=5 litters; Estradiol N=4 litters; Nicotine N=6 litters. Ebf2: (Vehicle) Control N=4 litters; Estradiol N=3 litters; Nicotine N=4 litters. Ebf3: (Vehicle) Control N=5 litters; Estradiol N=5 litters; Nicotine N=6 litters. Nr2f2: (Vehicle) Control N=6 litters; Estradiol N=5 litters; Nicotine N=7 litters. Dnmt1: (Vehicle) Control N=6 litters; Estradiol N=5 litters; Nicotine N=7 litters. Dnmt3a: (Vehicle) Control N=6 litters; Estradiol N=5 litters; Nicotine N=5 litters. G) Heat map representing treatment differences in normalized fold change values across MDMGs and Dnmts. MDMG expression was significantly increased in the POA of nicotine-treated neonates as compared to vehicle controls (two-way MANOVA p=0.0716, F=2.909, treatment main effect p=0.0113, sex main effect p=0.9752, sex*treatment p=0.8749). Cyp19a1: (Vehicle) Control n = 6 male, n = 5 female; Estradiol n = 5 male, n = 4 female; Nicotine n = 7 male, n = 6 female. Ebf2: (Vehicle) Control n = 4 male, n = 6 female; Estradiol n = 4 male, n = 3 female; Nicotine n = 4 male, n = 5 female. Ebf3: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 6 male, n = 6 female. Nr2f2: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 7 male, n = 7 female. Dnmt1: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 7 male, n = 7 female. Dnmt3a: (Vehicle) Control n = 6 male, n = 6 female; Estradiol n = 5 male, n = 5 female; Nicotine n = 7 male, n = 6 female. C-G) *P< 0.05, **P< 0.01, ***P < 0.001 Student’s t-test post hoc analysis compared to vehicle-treated females following significant two-way ANOVA. # P<0.05 a-priori t-test compared to vehicle-treated females. ## P< 0.05 treatment as main effect between vehicle control and nicotine-treated groups following two-way ANOVA. ### P<0.05 treatment as main effect between vehicle control and nicotine-treated groups in a repeated MANOVA. N indicates the number of litters. Error bars represent standard error of the mean.