Microarray analysis of mouse ear tissue exposed to bis-(2-chloroethyl) sulfide

Sulfur mustard (SM) is a potent alkylating agent. We are developing medical countermeasures to reduce the injury caused by SM exposure. Screening in the mouse ear vesicant model has identified three effective compounds: dimercaprol (British anti-lewisite), indomethacin, and octyl homovanillamide (OHV). To identify gene expression changes that correlate with compound efficacy we used oligonucleotide microarrays to compare gene expression profiles in vehicle-exposed skin, SM-exposed skin, and skin pretreated with each compound before SM exposure. Mice were topically exposed on the inner surface of the right ear to SM alone or pretreated for 15 min with one of the compounds and then exposed to SM. Left ears were vehicle-exposed. Ear tissue was harvested 24 hr later for ear weight determination (an endpoint indicating compound efficacy). The exposure groups were: methylene chloride (sulfur mustard vehicle); ethanol (drug vehicle); 0.08 mg sulfur mustard; 6.25 mg dimercaprol 15 min before 0.08 mg sulfur mustard; 1.34 mg indomethacin 15 min before 0.08 mg sulfur mustard; 0.6 mg octylhomovanillamide 15 min before 0.08 mg sulfur mustard; 6.25 mg dimercaprol alone; 1.34 mg indomethacin alone; 0.6 mg octylhomovanillamide alone. RNA was extracted from the tissues and used to generate oligonucleotide microarray probes. Principal component analysis of the gene expression data revealed partitioning of the samples based on drug treatment and SM exposure. Vehicle-exposed mouse ears clustered away from the other treatment groups. SM-exposed mouse ears pretreated with dimercaprol or OHV clustered more closely with vehicle-exposed ears, while SM-exposed mouse ears pretreated with indomethacin clustered more closely with SM-exposed ears. This clustering of the samples is supported by the ear weight data, in which the indomethacin group has ear weights closer to the SM-exposed group, whereas the dimercaprol and OHV groups have ear weights closer to the vehicle-exposed group. Correlation coefficients were calculated for each gene based on the correlation between gene expression level and ear weight. These data provide the basis for understanding what gene expression changes are important in the development of effective SM medical countermeasures. Keywords: ordered Exposure of mouse ears to sulfur mustard alone, sulfur mustard preceded by drug treatment, or vehicle compounds. Naïve controls were also included. Biological replicates of at least n=3 were examined for each exposure condition.

芥子气（sulfur mustard, SM）是一种强效烷化剂。本研究旨在开发可减轻芥子气暴露所致损伤的医学防护措施。通过小鼠耳水疱模型筛选，已确定三种有效化合物：二巯丙醇（dimercaprol, 英国抗路易剂）、吲哚美辛（indomethacin）以及辛基香草酰胺（octyl homovanillamide, OHV）。为筛选与化合物药效相关的基因表达变化，本研究采用寡核苷酸微阵列（oligonucleotide microarray）技术，对比溶剂暴露皮肤、芥子气暴露皮肤以及芥子气暴露前经各化合物预处理的皮肤的基因表达谱。
实验小鼠右侧耳内表面仅暴露于芥子气，或先经其中一种化合物预处理15分钟后再暴露于芥子气；左侧耳均接受溶剂暴露处理。于暴露24小时后收取耳组织，用于测定耳重量（该指标可反映化合物药效）。本次实验设置以下暴露组：二氯甲烷（芥子气溶剂组）、乙醇（药物溶剂组）、0.08 mg芥子气组、0.08 mg芥子气暴露前15分钟给予6.25 mg二巯丙醇组、0.08 mg芥子气暴露前15分钟给予1.34 mg吲哚美辛组、0.08 mg芥子气暴露前15分钟给予0.6 mg辛基香草酰胺组、单独给予6.25 mg二巯丙醇组、单独给予1.34 mg吲哚美辛组以及单独给予0.6 mg辛基香草酰胺组。
从组织中提取RNA，用于制备寡核苷酸微阵列探针。对基因表达数据进行主成分分析（principal component analysis）后发现，样本可根据药物处理方式与芥子气暴露情况进行聚类分组。溶剂暴露组小鼠耳样本与其他处理组样本聚类结果显著分离。经二巯丙醇或辛基香草酰胺预处理的芥子气暴露组小鼠耳样本，与溶剂暴露组聚类结果更为接近；而经吲哚美辛预处理的芥子气暴露组小鼠耳样本，则与单纯芥子气暴露组聚类结果更为接近。
样本的聚类结果与耳重量测定数据一致：吲哚美辛预处理组的耳重量与单纯芥子气暴露组更为接近，而二巯丙醇与辛基香草酰胺预处理组的耳重量则与溶剂暴露组更为接近。基于基因表达水平与耳重量的相关性，为每个基因计算了相关系数。本数据集为解析芥子气有效医学防护措施相关的关键基因表达变化提供了研究基础。
关键词：单纯芥子气暴露小鼠耳样本、药物预处理后芥子气暴露小鼠耳样本、溶剂处理小鼠耳样本；本数据集同时纳入空白对照，每个暴露条件均设置至少3次生物学重复。