Single-Cell Multiome Impact of Prenatal Heavy Metal Exposure on Early Airway Development

Prenatal exposure to cadmium (Cd) and arsenic (As) can severely impair fetal lung development, leading to lifelong adverse effects. As two of the most common and toxic heavy metals, Cd and As pose risks to many communities through food and water consumption. We have shown that prenatal coexposure to Cd and As at levels relevant to human intake inhibits branching morphogenesis, yet cell type-specific mechanisms remain elusive. Here, we examined early embryonic (Embryonic Day [E]12) lungs from mice exposed prenatally to either 0 (control) or 250 (treated) ppb of both Cd and As. Through single-cell multiome sequencing (single-cell transposase-accessible chromatin with high-throughput sequencing + single-cell RNA sequencing) and high-resolution metabolomics, we present a multifaceted landscape of Cd- and As-induced molecular and cellular disruption. We identified 19 cell states that exhibited state-specific changes in gene expression related to cell proliferation and differentiation. Velocity analysis integrating RNA splicing and chromatin kinetics showed profound disruptions in cell fate, particularly affecting differentiation of Sox2+ proximal progenitors and Wnt2+ mesenchymal progenitors. Gene regulatory network analysis pinpointed the diminished function of Gata6 and Gli2 as central to these disruptions, which was further confirmed by their reduced protein expression in exposed E12, E14.5, and E17 lungs. Additionally, metabolomic alterations in polyamine, tyrosine, and fatty acid biosynthesis correlated with changes in gene expression of catalytic enzymes. These findings demonstrate that Cd and As at levels relevant to human exposure impair early airway formation across multiple regulatory levels, including chromatin accessibility, transcription, and cell metabolism, and they provide insights into the factors central to cell resilience during this vulnerable stage of lung development.