In utero tobacco exposure epigenetically modifies placental CYP1A1 expression

Abstract

The metabolic pathways used by higher-eukaryotic organisms to deal with potentially carcinogenic xenobiotic compounds from tobacco smoke have been well characterized. Carcinogenic compounds such as polycyclic aromatic hydrocarbons are metabolized sequentially in 2 phases: in phase I, CYP1A1 catalyzes conversion into harmful hydrophilic DNA adducts, whereas in phase II, GSTT1 enables excretion via conjugation into polar electrophiles. In an effort to understand susceptibility to in utero tobacco exposure, we previously characterized known metabolic functional polymorphisms and demonstrated that although deletion of fetal GSTT1 significantly modified birth weight in smokers, no polymorphism fully accounted for fetal growth restriction. Because smoking up-regulates CYP1A1 expression, we hypothesized that nonallelic (epigenetic) dysregulation of placental CYP1A1 expression via alterations in DNA methylation (meCpG) may further modify fetal growth. In the present article, we compared placental expression of multiple CYP family members among gravidae and observed significantly increased CYP1A1 expression among smokers relative to controls (4.4-fold, P < .05). To fully characterize CYP1A1 meCpG status, bisulfite modification and sequencing of the entire proximal 1-kilobase promoter (containing 59 CpG sites) were performed. CpG sites immediately proximal to the 5′-xenobiotic response element transcription factor binding element were significantly hypomethylated among smokers (55.6% vs 45.9% meCpG, P = .027), a finding that uniquely correlated with placental gene expression (r = 0.737, P = .007). Thus, in utero tobacco exposure significantly increases placental CYP1A1 expression in association with differential methylation at a critical xenobiotic response element.

Figure 1. CYP1A1 is significantly upregulated in the placenta from smokers compared with non-smokers

Panel 1A. Schematic of the metabolism of inhaled polycyclic aromatic hydrocarbons form cigarette smoke. PAHs are metabolized into reactive intermediates by the Phase I enzymes, such as CYP1A1. The reactive intermediates are metabolized into excretable substances by the Phase II enzymes, such as GSTT1. Thus, any combinatorial association of increased Phase I activity (e.g., increased expression of CYP1A1) in combination with decreased Phase II activity (e.g., decreased GSTT1 expression) may yield increased susceptibility to tobacco-related adverse outcomes. We have recently demonstrated that the relatively prevalent fetal GSTT1 deletion in modulates birth weight in response to in utero tobacco exposure. Panel 1B. CYP1A1 expression is significantly upregulated in the placenta from smokers compared with non-smokers (p=0.003). Placental expression of other CYP family members (CYP2A6, CYP2E1, and CYP1B1) as well as GSTT1 did not differ significantly between the two groups.

Figure 2. The XRE containing Region I of the CYP1A1 promoter is hypomethylated in placenta from smokers compared with non-smokers

Panel A. The four analyzed regions of the CYP1A1 promoter are depicted. Magenta lines represent CpG dinucleotides, and the red stars designate localization of the XREs. In total, among all four regions the precise methylation status of 59 CpG dinucleotides was determined in bisulfite-treated placental DNA from 19 nonsmokers and 15 smokers employing sequencing. Panel B. Methylation status of the 5 CpG sites in Region I, and 26 CpG sites in Region II. Two representative clones (from a minimum of four done in total) for each of 8 representative subjects are depicted. Open circles represent an unmethylated CpG, closed circles represent a methylated CpG site. Among smokers, 55.6% placental methylation within the XRE-containing Region I was observed, versus 45.9% among non-smokers (p=0.027). Region II is not differentially methylated between the two groups.

Figure 3. Plot of percent methylation of the promoter region in relation to relative expression of CYP1A1

Each point on the graph represents one subject, and includes both smokers and nonsmokers. The average methylation from a minimum of clones is plotted against the relative gene expression for each individual in order to provide direct estimates of the correlation between DNA methylation in a given region and expression of CYP1A1. Consistent with hypermethylation yielding gene silencing, increased methylation in the promoter of Region I, which contains a critical XRE, significantly and inversely correlates with gene expression (r =−0.737, p = 0.007). Consistent with our findings as presented in Tables II and III, there is no significant correlation between the degree of methylation at any CpG dinucleotide Regions II-IV and gene expression (data not shown).