Smoking during pregnancy causes double-strand DNA break damage to the placenta

Summary

Despite the adverse effects of smoking, many pregnancies are exposed to tobacco smoke. Recent studies have investigated whether smoking damages placental DNA by measuring DNA adducts. This study investigated whether a more severe lesion, double-strand DNA breaks, was also present in the tobacco smoking–exposed placenta. Term placentae from women who smoked during their entire pregnancies (n = 52), from those who had ceased smoking for at least 4 weeks before delivery (previous smokers, n = 34), and from nonsmoking women (n = 150) were examined using the DNA double-strand break marker phosphorylated γ H2AX. The extent of DNA damage was assessed according to cell type and additional markers were applied for cell fate (apoptosis and DNA repair), and function (human chorionic gonadotropin, human placental lactogen, and glucose transporter 1), to characterize the effect of the DNA damage on placental integrity. Marked phosphorylated γ H2AX–positive cells occurred in the villous syncytiotrophoblast and syncytial knot nuclei in placentae from smokers (P < .001). Phosphorylated γ H2AX foci did not colocalize with the DNA repair protein 53BP1, and damaged nuclei had a marked reduction in expression of human chorionic gonadotropin, human placental lactogen, and glucose transporter 1. Minimal DNA damage, similar to nonsmokers, was present in previous smokers including those that had ceased smoking for just over 4 weeks before delivery. In summary, smoking during pregnancy was associated with marked double-strand DNA break damage to the syncytiotrophoblast. We suggest that smoking cessation is important to prevent additional DNA damage and to facilitate DNA repair.

Introduction

Despite the risks, 13% to 25% of pregnancies are exposed to tobacco smoke [1], [2]. Tobacco smoking lowers birth weight, increases the risk of antepartum hemorrhage and abruption, and increases perinatal mortality and the risk of sudden infant death syndrome [3], [4], [5], [6]. Long-term effects from tobacco smoke exposure in utero include an increased risk for obesity and impaired lung function [7], [8], [9], [10], [11].

In many cases, the smoking-exposed placenta is normal by gross and by routine microscopic analysis [12], [13], [14], [15], [16]. Thus, a diagnostic report for a smoking-related placenta is unlikely to document changes because these require investigation and quantification beyond the constraints of routine diagnostic services. In some cases, the smoking-exposed placenta does demonstrate pathologic microscopic changes of chronic uteroplacental malperfusion including infarcts, villous hypoplasia, Tenny-Parker changes, and others [17]. Smoking induces DNA damage such as DNA adducts and the more severe double-strand DNA break lesion [18]. As early as 1986, it has been known that covalent DNA adducts can occur in the smoke-exposed placenta, but it is not known if double-strand DNA breaks also occur [19], [20], [21]. Double-strand breaks are hazardous leading to an increased risk of genetic instability, and as such, this type of DNA damage has been thoroughly studied in cancer [22], [23], [24].

Double-strand DNA breaks are detected by phosphorylated γ H2AX. As a component of the DNA repair response, the histone deacetylase γ H2AX becomes phosphorylated on serine 139 and accumulates at double-strand DNA break sites [25]. Phosphorylated γ H2AX staining (γ H2AX) is approximately 100 times more sensitive than other double-strand DNA break detection methods [18], [22], [25] and can be applied to paraffin-embedded tissues to examine the spatial distribution of double-strand DNA breaks [22], and γ H2AX foci form de novo upon DNA damage and disappear once the DNA break is repaired [18].

The premise for this study was that double-strand DNA breaks may contribute to the complications associated with smoking during pregnancy. This study addressed whether double-strand DNA breaks are present in the placentae from maternal smokers. Placentae from current smokers and nonsmokers were accessed using γ H2AX staining [18], [22]. Because of the severity of the double-strand break lesion, we also tested whether the DNA damage was associated with impaired cell function. The study was extended to test if smoking cessation before delivery could ameliorate DNA damage, by using placentae from women who ceased smoking up to 4 weeks before delivery.