Prenatal smoke exposure induces persistent Cyp2a5 methylation and increases nicotine metabolism in the liver of neonatal and adult male offspring

Abstract

Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5 . We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life.

Postnatal Smoke Exposure Further Increases the Hepatic Nicotine Metabolism in Prenatally Smoke Exposed Male Offspring and Is Linked with Aberrant Cyp2a5 Methylation

“…It is well known that smoke exposure effects on DNA methylation and gene expression are sex-dependent, as described by our research group [ 18 , 33 , 34 ] and others [ 28 , 29 , 30 , 35 , 36 ]. Similarly, we found that the distinct effects of PreSE and/ or PostSE on Cyp2a5 methylation, mRNA levels and the nicotine conversion rate were different in male and female offspring.…”

“…Gene expression analysis in whole liver mRNA isolates was done via qPCR using qPCR MasterMix Plus (Eurogentec, Seraing, Belgium) with commercially available primers for Cyp2a4/5 (Mm00487248_m1, TaqMan ® Gene Expression Assay, Applied Biosystems, Foster City, CA, USA), Dnmt1 , Dnmt3a and Dnmt3b (Invitrogen, The Netherlands), and normalized to the housekeeping gene Gapdh (Mm99999915_m1, TaqMan ® Gene Expression Assay, Applied Biosystems, Foster City, CA, USA). Detection of amplification reactions was performed using the LightCycler ® 480 System (Roche Diagnostics GmbH, Mannheim, Germany), as previously described [ 18 ].…”

“…To ensure that the nicotine conversion was linear with time, and aldehyde oxidase availability was not rate-limiting, a 45-min incubation time was selected from the tested experimental durations (15, 30, 45 and 60 min), and 1mg/mL was selected as the optimal final concentration of cytosolic protein from a tested range between 0.25, 0.5, 1, 2, 3, 4, 6, and 8 mg/mL. These optimizations of nicotine-to-cotinine conversion were originally examined in the liver from PreSE embryonic and neonatal mice [ 18 ]. For the assay, a normalized 0.5 mg/mL liver microsomal protein and 80 µL cytosolic fraction with a protein content of 1.0 mg/mL were added to 80 µL of a freshly prepared nicotine containing solution, which is a mix of 4.4 µL S(-)-nicotine (≥99%, N3876, Sigma-Aldrich, St.Louis, MO, USA), 1.5 mL demineralized water and 28.5 mL of 100 mM sodium phosphate buffer pH 7.4.…”

“…The used amplification and sequencing primers used in this study are listed in Table 2. Microsomes were obtained from homogenized livers of 20-week-old mice to measure nicotine metabolism, as previously described [5,18]. Briefly, the homogenized liver tissue was centrifuged at 9000× g for 20 min at 4 • C in phosphate buffer with EDTA.…”

“…Prenatal programming is now thought to be one of the missing links to set the stage for metabolic disease development, albeit that the underlying mechanisms are not yet fully described. Previously, our studies have demonstrated that PreSE increased cotinine conversion rate, which was accompanied by aberrant methylation profiles and deregulated Cyp2a5 mRNA expression in livers from fetal, neonatal and adult offspring [ 18 ]. In this study, we asked the question whether postnatal smoking (PostSE) would further enhance the effects of PreSE, as it has been shown that children of mothers who smoke during pregnancy will most likely be exposed to cigarette smoke while growing up [ 3 , 19 ] and have a higher chance to start smoking during adolescence [ 20 ].…”