Trophoblasts are critical for a successful pregnancy and they mediate important steps such as implantation and delivery. Increased trophoblast DNA double strand breaks (DNA‐DSBs) is implicated in complicated pregnancies. Our laboratory showed a role for increased nuclear placental receptor for advanced glycation end‐products (nRAGE) in DNA‐DSB repair in trophoblasts. HMGB1 protein (nHMGB1) is a nuclear protein that acts as a chromatin‐binding factor involved the regulation of gene transcription that when released extracellularly, is involved in RAGE mediated inflammatory responses. We tested the hypothesis that nHMGB1 correlated with nRAGE availability during DNA‐DSB in trophoblasts. DSBs were induced in human trophoblasts with cigarette smoke extract (CSE). Assessment of HMBG1, Beclin1 (involved in phagocytosis) and Caspase 3 (pro‐apoptotic) were done by immunoblotting. Immunoprecipitation was used to quantify a RAGE and MNR11 complex. Invasion was measured in trophoblast cells in the presence or absence of an HMGB1 inhibitor (Glycyrrhizin; GLY). Decreased trophoblast nHMGB1 (1.8‐fold; p<0.02) was present during DNA‐DSB and this reduction was reversed (4.5‐fold; p<0.02) with GLY treatment. DNA‐DSBs induced a MRN11‐nRAGE complex (1.4‐fold; p<0.03) in trophoblasts and the complex was decreased (2.5‐fold; p<0.05) by GLY co‐treatment. CSE increased Beclin1 (1.9‐fold; p<0.02) and decreased Caspase 3 (2.2‐fold; p<0.02); however, GLY co‐treatment significantly decreased Beclin1 (9.0‐fold p<0.02) while increasing Caspase 3 (1.2‐fold; p<0.04). Compromised trophoblast invasion was improved (7.0‐fold; p<0.02) with GLY treatment. We conclude that CSE causes DNA‐DSBs in trophoblasts and HMGB1 plausibly regulates nRAGE availability at DNA‐DSBs. We further conclude that Beclin1 and Caspase 3 are regulated by HMGB1 inhibition and that HMGB1 targeting could be a factor in regulating trophoblast invasion during DNA‐DSBs. These studies provide a critical initial step in dissecting tobacco‐mediated placental deficiency.

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, and placental abnormalities, but studies investigating the inducibility of IUGR by secondhand smoke (SHS) are limited. The mTOR pathway regulates protein expression and cell growth. Disrupted mTOR signaling and apoptosis are associated with the development of several obstetric complications including IUGR. We tested the hypothesis that SHS exposure disrupts the mTOR pathway and apoptosis in mice. C57Bl6 mice were exposed to SHS for 4 days from day 14 to day 17 of gestation (dGA). At the time of necropsy (18 dGA) placental and fetal weights were recorded and tissues were immediately frozen for immunoblot analysis. Mice exposed to SHS demonstrated a significant reduction in fetal weight (7.35‐fold; p≤0.0001) and placental weight (1.13‐fold; p≤0.0001) compared to controls. Significant decreases were observed for placental activation of mTOR (2.1‐fold; p<0.03), p70 (1.9‐fold; p,0.03) and 4EBP1 (1.3‐fold (p<0.03) in IUGR placentas compared to controls. Increased placental active caspase 3 (1.1‐fold; p<0.04) and the antiapoptotic placental XIAP protein (2.2‐fold; p<0.03) were also detected during SHS treatment compared to controls. We conclude that decreased mTOR pathway is associated with lessened fetal and placental size during SHS induced IUGR. This decrease was associated with increased caspase 3 and that elevated XIAP protein may signify an attempted protective mechanism that counters increased apoptosis observed in the SHS‐induced IUGR placenta. These studies provide insight into tobacco‐mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.