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.

KEYWORDS:

• Prenatal smoke exposure
• epigenetics
• cotinine
• CYP2A5
• nicotine dependence
• sex difference
• mouse model

Author’s contributions

K.L, K.M and M.H designed the study, and K.M, K.L, L.V, W.K and M.R performed experiments. H.D was involved in the in vitro nicotine metabolism experiment, and I.G contributed to the interpretation of the in vitro nicotine metabolism result. K.L drafted the manuscript, which is finalized by T.P and M.H. All authors were involved in writing and had final approval of the submitted version.

Disclosure statement

The authors declare that they have no competing interests.

Ethical approval and consent to participate

The experimental setup was approved by the local committee on animal experimentation (DEC6589 B & C; University of Groningen, Groningen, The Netherlands) and governmental and international guidelines on animal experimentation.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by grants from the Lung Foundation Netherlands (LF3.2.11.013 to M.N. Hylkema) and a Fellowship from the Mongolian State Training Fund and UMCG (to K. Lkhagvadorj).