Transcriptional profiling to identify physical-chemical properties detrimental to nanomaterial-induced pulmonary response (part 3). Mus musculus

We examined the use of toxicogenomics data to 1) elucidate the underlying mechanisms of pulmonary responses following exposures to titanium dioxide (TiO2) nanoparticles (NPs) of different size and surface charge, and 2) determine if such responses are influenced by embedding particles in complex paint matrix. Adult C57BL/6 mice were exposed via single intratracheal instillation to three doses of free forms of NRCWE-030 (10.5 nm), NRCWE-025 (38 nm), NRCWE-001 (10 nm, neutral charge) NRCWE-002 (10 nm, positive charge) or to sanding dusts of paint consisting of NRCWE-030+NRCWE-025 or NRCWE-025 alone. Controls were exposed to dispersion medium without NPs. TiO2NPs were characterized for size, surface area, and agglomeration status in the exposure medium. Hyperspectral microscopy was performed to detect TiO2NPs in lungs and to determine the in vivo status of matrix-embedded TiO2NPs. Pulmonary gene expression profiles were generated using DNA microarrays on lung tissues collected at 24 h and 28 d post exposure time points. Bioinformatics tools were used to characterize size and surface property-pertinent effects. The data from 360 individual arrays were collapsed into 567 differentially expressed genes (False discovery rate adjusted P ≤ 0.05 and fold change ≥ 1.5 in any one condition). Unsupervised hierarchical clustering of the 567 genes revealed that TiO2NPs clustered mainly based on the post-exposure time points and then by the particle types. A meta analysis using pathway tools showed that the combination of smaller size and positive surface charge contributes to the potency of TiO2NPs. Although embedding in matrix dampens the overall toxicity of TiO2NPs, the smaller size positively influences the toxicity. Paint matrix itself did not induce any response. Finally, the observed differences in response reflected the differences in severity of the response and not the underlying mechanisms. Thus, transcriptional profiling is an effective tool to determine the important properties responsible for eliciting a response. Overall design: Female C57BL/6 mice of 5-7 weeks old were obtained from Taconic (Ry, Denmark) and were allowed to acclimatise for 1-3 weeks before the exposure. All mice were given food (Altromin no.1324, Christian Petersen, Denmark) and water ad libitum. Mice were grouped in polypropylene cages with sawdust bedding at controlled temperature 21±1°C, humidity 50 ±10 % in a 12 h-light: dark cycle. The experiments were approved by the Danish “Animal Experiments Inspectorate” and carried out following their guidelines for ethical conduct and care when using animals in research. Animals were exposed to 18, 54, or 162 ug of NRCWE-025 and NRCWE-030 dispersed in 2% serum in water. Each exposure group consisted of minimum of 5 animals. Controls were exposed to vehicle only. The data were compared to their respective controls within the group.

本研究利用毒理基因组学数据开展探究，旨在达成两项核心目标：其一，阐明不同尺寸与表面电荷的二氧化钛（TiO₂，titanium dioxide）纳米颗粒（nanoparticles, NPs）暴露后，肺部应答的潜在分子机制；其二，明确将颗粒嵌入复杂涂料基质后，是否会对上述肺部应答产生调控作用。

选用成年C57BL/6小鼠，通过单次气管滴注方式实施暴露处理。暴露组别涵盖：三种剂量梯度的游离型NRCWE-030（10.5 nm）、NRCWE-025（38 nm）、NRCWE-001（10 nm，中性电荷）及NRCWE-002（10 nm，正电荷），以及由NRCWE-030与NRCWE-025混合或单独NRCWE-025组成的涂料打磨粉尘组。对照组小鼠仅暴露于不含纳米颗粒的分散介质。

本研究对暴露介质中的TiO₂纳米颗粒开展表征分析，测定其尺寸、比表面积及团聚状态。采用高光谱显微镜检测肺部组织内的TiO₂纳米颗粒，并明确基质包埋型TiO₂纳米颗粒的体内分布与状态。于暴露后24小时及28天两个时间点采集肺组织，利用DNA微阵列（DNA microarray）技术构建肺部基因表达谱。借助生物信息学工具，解析与颗粒尺寸及表面特性相关的生物学效应。

将360张独立微阵列的原始数据整合筛选，最终得到567个差异表达基因（校正后错误发现率（false discovery rate, FDR）P≤0.05，任意实验条件下的倍数变化≥1.5）。对这567个差异基因开展无监督层次聚类分析，结果显示TiO₂纳米颗粒的聚类主要依据暴露后时间点，其次为颗粒类型。采用通路工具进行荟萃分析（meta-analysis）后发现，较小尺寸与正表面电荷的组合可增强TiO₂纳米颗粒的毒性效能。尽管基质包埋可降低TiO₂纳米颗粒的整体毒性，但较小的尺寸仍会正向提升其毒性水平。单纯的涂料基质不会引发任何肺部应答。最终观察到的应答差异仅反映了应答严重程度的不同，而非潜在分子机制的差异。由此可见，基因表达谱分析是筛选引发生物应答的关键颗粒特性的有效手段。

### 实验整体设计
选取5~7周龄的雌性C57BL/6小鼠，购自丹麦Ry市的Taconic公司，于暴露前适应性饲养1~3周。所有小鼠均可自由摄食（丹麦Christian Petersen公司生产的Altromin no.1324饲料）与饮水。小鼠被饲养于带有刨花垫料的聚丙烯笼盒中，饲养环境温度控制为21±1℃，相对湿度50±10%，采用12小时光照-12小时黑暗的循环光照制度。本实验已通过丹麦"动物实验监察局"的伦理审批，并严格遵循其关于动物实验的伦理操作与饲养护理指南开展。

小鼠通过单次气管滴注暴露于分散于2%血清水溶液中的受试颗粒，暴露剂量分别为18、54或162 μg。每个暴露组至少包含5只小鼠，对照组仅暴露于空白溶剂。各组数据均与其对应的对照组进行比较分析。