BACKGROUND Perfluorohexane sulfonate (PFHxS) is a frequently detected per- and polyfluoroalkyl substance in most populations, including in individuals who are pregnant, a period critical for early life development. Despite epidemiological evidence of exposure, developmental toxicity, particularly at realistic human exposures, remains understudied. OBJECTIVES We evaluated the effect of gestational exposure to human-relevant body burden of PFHxS on fetal and placental development and explored mechanisms of action combining alternative splicing (AS) and gene expression (GE) analyses. METHODS Pregnant ICR mice were exposed to 0, 0.03, and 0.3μg/kg/day  from gestational day 7 to day 17 via oral gavage. Upon euthanasia, PFHxS distribution was measured using liquid chromatography-tandem mass spectrometry. Maternal and fetal phenotypes were recorded, and histopathology was examined for placenta impairment. Multiomics was adopted by combining AS and GE analyses to unveil disruptions in mRNA quality and quantity. The key metabolite transporters were validated by quantitative real-time PCR (qRT-PCR) for quantification and three-dimensional (3D) structural simulation by AlphaFold2. Targeted metabolomics based on liquid chromatography-tandem mass spectrometry was used to detect amino acid and amides levels in the placenta. RESULTS Pups developmentally exposed to PFHxS exhibited signs of intrauterine growth restriction (IUGR), characterized by smaller fetal weight and body length (p<0.01) compared to control mice. PFHxS concentration in maternal plasma was 5.01±0.54 ng/mL. PFHxS trans-placenta distribution suggested dose-dependent transfer through placental barrier. Histopathology of placenta of exposed dams showed placental dysplasia, manifested with an attenuated labyrinthine layer area and deescalated blood sinus counts and placental vascular development index marker CD34. Combined GE and AS analyses pinpointed differences in genes associated with key biological processes of placental development, proliferation, metabolism, and transport in placenta of exposed dams compared to that of control dams. Further detection of placental key transporter gene expression, protein structure simulation, and amino acid and amide metabolites levels suggested that PFHxS exposure during pregnancy led to impairment of placental amino acid transportation. DISCUSSION The findings from this study suggest that exposure to human-relevant very-low-dose PFHxS during pregnancy in mice caused IUGR, likely via downregulating of placental amino acid transporters, thereby impairing placental amino acid transportation, resulting in impairment of placental development. Our findings confirm epidemiological findings and call for future attention on the health risk of this persistent yet ubiquitous chemical in the early developmental stage and provide a new approach for understanding gene expression from both quantitative and qualitative omics approaches in toxicological studies. https://doi.org/10.1289/EHP13217.

中文翻译：

---

结合选择性剪接和基因表达分析，评估妊娠期暴露于全氟己烷磺酸盐对小鼠胎盘发育的影响。

背景技术全氟己烷磺酸盐(PFHxS)是一种在大多数人群中经常检测到的全氟烷基和多氟烷基物质，包括在怀孕的个体中，怀孕是生命早期发育的关键时期。尽管有暴露的流行病学证据，但发育毒性，特别是在现实的人类暴露中，仍然没有得到充分研究。目的 我们评估了妊娠期暴露于人体相关的 PFHxS 负荷对胎儿和胎盘发育的影响，并结合选择性剪接 (AS) 和基因表达 (GE) 分析探索了作用机制。方法 怀孕的ICR小鼠从妊娠第7天到第17天通过口服灌胃暴露于0、0.03和0.3μg/kg/天。安乐死后，使用液相色谱-串联质谱法测量 PFHxS 分布。记录母体和胎儿表型，并检查组织病理学是否有胎盘损伤。多组学通过结合 AS 和 GE 分析来揭示 mRNA 质量和数量的破坏。关键代谢物转运蛋白通过实时定量 PCR (qRT-PCR) 进行定量验证，并通过 AlphaFold2 进行三维 (3D) 结构模拟。基于液相色谱-串联质谱的靶向代谢组学用于检测胎盘中的氨基酸和酰胺水平。结果 发育过程中暴露于 PFHxS 的幼鼠表现出宫内生长受限 (IUGR) 的迹象，其特征是与对照小鼠相比，胎儿体重和体长较小 (p<0.01)。母体血浆中的 PFHxS 浓度为 5.01±0.54 ng/mL。PFHxS 经胎盘的分布表明通过胎盘屏障的转移呈剂量依赖性。暴露母鼠胎盘的组织病理学显示胎盘发育不良，表现为迷路层面积变薄、血窦计数和胎盘血管发育指数标记物 CD34 下降。GE 和 AS 联合分析确定了与对照母鼠相比，暴露母鼠胎盘中与胎盘发育、增殖、代谢和运输等关键生物过程相关的基因的差异。进一步检测胎盘关键转运蛋白基因表达、蛋白质结构模拟以及氨基酸和酰胺代谢物水平表明，妊娠期间PFHxS暴露导致胎盘氨基酸转运受损。讨论 本研究的结果表明，小鼠在怀孕期间接触人类相关的极低剂量全氟己烷磺酸会导致 IUGR，可能是通过下调胎盘氨基酸转运蛋白，从而损害胎盘氨基酸转运，导致胎盘发育受损。我们的研究结果证实了流行病学发现，并呼吁未来关注这种持久而普遍存在的化学物质在早期发育阶段的健康风险，并为毒理学研究中从定量和定性组学方法理解基因表达提供了一种新方法。