Effect of monomethylarsonous acid (MMAIII) on primary fibroblasts in vitro derived from Diversity Outbred (DO) mice aged 4-6 weeks

Genetically diverse cell populations derived from mice are powerful tools for studying gene-environment interactions. Using fibroblasts derived from Diversity Outbred (DO) mice, we performed a high-content screen (HCS) of cell morphology changes to the oxidative stress and DNA damage-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We show that fibroblast morphological changes in response to increasing MMAIII exposure can be modeled using dose-response modeling. We use dose-response model parameters (i.e., EC50) as traits to map cell morphology quantitative trait loci (QTL) in response to MMAIII exposure. We find that dose-response cmQTLs overlap many known genes associated with arsenic susceptibility, including Abcc4 and Txnrd1, and also represent novel targets, including Xrcc2. Moreover, we show that the cmQTL effects are reproducible and how mouse genetics can be leveraged in a systems toxicological approach to find candidate genes and support experimental validation. In summary, our work demonstrates that cmQTLs reflect the key molecular events that are known to occur during acute arsenic exposure, which are adaptable across chemicals and cell culture models. Overall design: To assess transcription changes in genetically diverse muring fibroblasts exposed to MMAIII, we selected 16 fibroblasts line from DO mice based on their haplotype at mouse chromosome 10:82.9 Mbp (GRCm38). We performed gene expression analysis obtained from RNA-seq of these 16 fibroblast lines in unexposed and 0.75 uM MMAIII exposed culture conditions. These dtaa were used for differential expression analysis and gene set enrichment analyss, in addition to supporting gene candidates within cmQTL intervals based on overall expression and differences in expression following MMAIII exposure.

源自小鼠的遗传多样性细胞群体，是研究基因-环境交互作用的有力工具。本研究利用来自多样性远交小鼠（Diversity Outbred, DO）的成纤维细胞，针对氧化应激与DNA损伤诱导剂——砷代谢产物一甲基亚砷酸（monomethylarsonous acid, MMAIII），开展了细胞形态变化的高内涵筛选（high-content screen, HCS）。结果显示，成纤维细胞对MMAIII暴露剂量升高产生的形态学变化，可通过剂量反应建模进行拟合。我们以剂量反应模型参数（即半最大效应浓度EC50）作为性状，对MMAIII暴露下的细胞形态学数量性状位点（quantitative trait loci, QTL）进行定位。研究发现，剂量反应型细胞形态学QTL（cmQTLs）与诸多已知的砷易感相关基因存在重叠，包括Abcc4与Txnrd1，同时也涵盖了Xrcc2等全新候选靶点。此外，本研究证实cmQTL效应具有可重复性，并展示了如何利用小鼠遗传学结合系统毒理学方法筛选候选基因，为实验验证提供支持。

综上，本研究表明cmQTLs能够反映急性砷暴露过程中已知的关键分子事件，且该分析框架可推广应用于其他化学物与细胞培养模型。

总体实验设计：为评估暴露于MMAIII的遗传多样性小鼠成纤维细胞的转录变化，我们基于小鼠10号染色体82.9 Mbp（GRCm38参考基因组）区域的单倍型，从DO小鼠中筛选得到16株成纤维细胞系。我们对这16株成纤维细胞系分别在未暴露及0.75 μM MMAIII暴露的培养条件下进行RNA测序，获取基因表达数据。除基于整体表达水平与MMAIII暴露后表达差异，在cmQTL区间内筛选候选基因外，我们还利用这些数据开展了差异表达分析与基因集富集分析。