UC Riverside

Focus on tobacco-related disease concerns has shifted from cigarettes to other forms of tobacco use over the last 20 years. Due to their perception as “safer than a cigarette” by the general public, harm-reduction tobacco products (HRTPs) offer an appealing alternative for women struggling with nicotine addiction who find themselves pregnant. Some studies, however, suggest that HRTPs may increase risk of adverse pregnancy outcomes and hinder fetal skeletal development. To date, the mechanistic etiology of HRTP embryotoxicity is unreported. This thesis aims to address this knowledge gap by answering some of the questions surrounding how HRTPs molecularly and biochemically cause changes in the developing skeleton.

Early in vivo studies reported herein indicated that HRTP exposure directly targets early osteogenesis of the skull following in utero exposure of mouse embryos. To explore the molecular etiology of this outcome, the embryonic stem cell test (EST) protocol was adapted to an in vitro model of developmental osteogenesis using human pluripotent stem cells (hPSCs). Cultures were concurrently exposed to conventional sidestream cigarette smoke (CSC), harm-reduction sidestream cigarette smoke (HSC), or harm-reduction Snus smokeless tobacco extract (STE). While conventional and harm-reduction extracts both inhibited in vitro osteogenesis, only the HSC and STE harm-reduction extracts did so at sub-cytotoxic doses. Furthermore, inhibitory doses increased cellular levels of reactive oxygen species and reduced endogenous antioxidant enzyme activity. Molecular analysis found that CSC exposure incurred both DNA damage and a concurrent apoptotic response that was absent in cultures exposed to either HRTP extract. Biochemical exploration of HRTP impact on developing cultures found exclusive activation of survival kinase AKT and reduction of stress rescue kinase JNK in STE-exposed cultures. Concurrent treatment with an isoform-specific inhibitor of AKT or JNK activator rescued osteogenesis in STE cultures, implicating the specific misregulation of these kinases in poor osteogenic outcomes. Global proteomic analysis of AKT signaling targets also identified exclusive hyperphosphorylation of FOXO transcription factors—required for oxidative stress defense and adult bone homeostasis—in STE-treated cultures, marking FOXOs for nuclear exclusion. Collectively, our data suggest that HRTPs inhibit normal osteogenesis by disrupting the balance between embryonic osteogenesis, survival, and redox equilibrium mechanisms.