Lung toxicity and gene expression changes in response to whole-body inhalation exposure to cellulose nanocrystal in rats

Human exposure to cellulose nanocrystal (CNC) is possible during the production and/or use of products containing CNC. The objectives of the current study were to determine the lung toxicity of CNC and the underlying molecular mechanisms of the toxicity. Rats were exposed to air or CNC (20 mg/m3, six hours/day, 14 d) by whole-body inhalation and lung toxicity and global gene expression profile were determined. Significant increases in lactate dehydrogenase activity, pro-inflammatory cytokine levels, phagocyte oxidant production, and macrophage and neutrophil counts were detected in the bronchoalveolar lavage cells or fluid from the CNC exposed rats. Mild lung histological changes, such as the accumulation of macrophages and neutrophils, were detected in the CNC exposed rats. Gene expression profiling by next generation sequencing identified 531 genes whose expressions were significantly different in the lungs of the CNC exposed rats, compared with the controls. Bioinformatic analysis of the lung gene expression data identified significant enrichment in several biological functions and canonical pathways including those related to inflammation (cellular movement, immune cell trafficking, inflammatory diseases and response, respiratory disease, complement system, acute phase response, leukocyte extravasation signaling, granulocyte and agranulocyte adhesion and diapedesis, IL-10 signaling, and phagosome formation and maturation) and oxidative stress (NRF2-mediated oxidative stress response, production of nitric oxide and reactive oxygen species in macrophages, and free radical scavenging). Our data demonstrated that inhalation exposure of rats to CNC resulted in lung toxicity mediated mainly through the induction of inflammation and oxidative stress.人类在生产或使用含纤维素纳米晶体（cellulose nanocrystal, CNC）的产品过程中，存在接触CNC的风险。本研究旨在明确CNC的肺部毒性及其潜在分子毒性机制。实验采用全身吸入暴露方式，将大鼠暴露于空气或浓度为20 mg/m³的CNC环境中（每日6小时，持续14天），随后检测其肺部毒性及全基因表达谱。检测结果显示，与对照组相比，CNC暴露组大鼠的支气管肺泡灌洗细胞及灌洗液中，乳酸脱氢酶活性、促炎细胞因子水平、吞噬细胞氧化产物生成量，以及巨噬细胞与中性粒细胞计数均显著升高。CNC暴露组大鼠还出现了轻度肺部组织学改变，具体表现为巨噬细胞与中性粒细胞在肺部的聚集。通过下一代测序开展基因表达谱分析，发现CNC暴露组大鼠肺部共有531个基因的表达水平与对照组存在显著差异。对肺部基因表达数据进行生物信息学分析后，鉴定出多个显著富集的生物学功能与经典通路，涵盖炎症相关通路（包括细胞迁移、免疫细胞趋化、炎症性疾病与应答、呼吸系统疾病、补体系统、急性期反应、白细胞渗出信号通路、粒细胞与非粒细胞黏附及迁移、白细胞介素10（interleukin-10, IL-10）信号通路，以及吞噬体形成与成熟）与氧化应激相关通路（包括核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2, NRF2）介导的氧化应激应答、巨噬细胞内一氧化氮与活性氧生成、自由基清除）。本研究数据证实，大鼠吸入暴露于CNC可诱发肺部毒性，该毒性主要通过诱导炎症反应与氧化应激介导。