Effects of Phenylethyl Isothiocyanate on N-Nitrosomethylbenzylamine-induced Cytotoxicity in Rat Esophagus. Rattus norvegicus

To date, there is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA) induced esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short–term NMBA-treatment and modulated by co-treatment with phenethylisothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for three weeks. During the third week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg b.w.). Rats were sacrificed 24 h after the last treatment, esophagi excised and processed for histological grading, microarray and real-time PCR analysis. Our histopathological data showed that treatment of rats with PEITC had protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2261 genes that were differentially expressed in the NMBA-treated vs. control esophagi and 1936 genes in the NMBA+PEITC- vs. NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1323 genes in NMBA-treated esophagus that were modulated by PEITC to near-normal levels of expression. The measured changes in the expression levels of 10 selected genes were validated using real-time PCR. Principle components analysis (PCA) was applied to all twelve microarrays in the study, which suggested that in terms of global gene expression, PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or co-treated with NMBA were more similar to controls than they were to NMBA treatment alone. Keywords: Toxicogenomics,reference design Overall design: For each treatment we used a pooling strategy to enable the inclusion of samples from numerous animals. Three replicate microarrays were completed for each of the four treatments (control, PEITC, NMBA and NMBA+PEITC) for a total of twelve microarrays. Each microarray was hybridized using a pooled RNA sample, and each pool was created from equal amounts of total RNA from two or three independent RNA samples, representing individual animals. Each of these samples was obtained from a different animal, and no sample was included in more than one pool. In total, samples from nine animals per group were pooled for control, NMBA, PEITC and NMBA+PEITC microarrays. To facilitate comparisons of gene expression across all treatments, we utilized a reference design in which each microarray was co-hybridized with a common reference sample labeled with Cy3, and RNA from the treatment pool was labeled with Cy5. Stratagene’s Universal Rat reference RNA (Stratagene, La Jolla, CA) was used as the common reference in all microarrays.

迄今为止，关于N-亚硝基甲基苄胺（N-nitrosomethylbenzylamine, NMBA）诱导食管肿瘤发生过程中的早期分子事件，以及化学预防剂对这些事件的调控作用，相关研究资料仍较为匮乏。本研究中，我们对大鼠食管组织中因短期NMBA处理而出现差异表达、且可通过苯乙基异硫氰酸酯（phenethylisothiocyanate, PEITC）联合处理进行调控的基因进行了筛选鉴定。将大鼠分为两组，分别饲喂AIN-76A基础饲料及添加PEITC的AIN-76A饲料，持续三周。在饲喂处理的第三周，对大鼠予以3次皮下注射（s.c.）NMBA（剂量为0.5 mg/kg体质量）。末次处理24小时后处死大鼠，摘取食管组织，分别进行组织学分级、基因芯片分析及实时荧光定量PCR（real-time PCR）检测。组织病理学结果显示，PEITC处理可对NMBA诱导的大鼠食管癌前病变起到保护作用。我们筛选得到2261个在NMBA处理组与对照组食管组织中存在差异表达的基因，以及1936个在NMBA+PEITC联合处理组与单独NMBA处理组食管组织中存在差异表达的基因。对这两组差异基因取交集后，共鉴定得到1323个在单独NMBA处理组食管组织中、可被PEITC调控至接近正常表达水平的基因。我们选取10个差异基因，通过实时荧光定量PCR验证了其表达量的变化趋势。本研究对全部12张芯片数据进行了主成分分析（Principle Components Analysis, PCA），结果显示，从全局基因表达谱来看，PEITC处理可对NMBA诱导的基因表达产生全基因组水平的调控作用。单独经PEITC处理或联合NMBA处理的动物样本，其基因表达谱与对照组的相似度高于与单独NMBA处理组的相似度。关键词：毒理基因组学（Toxicogenomics），参考设计（reference design）实验整体设计：本研究针对各处理组采用混合样本策略，以纳入多只动物的样本。四个处理组（对照组、PEITC组、NMBA组及NMBA+PEITC组）各设置3次生物学重复芯片实验，总计完成12张芯片。每张芯片均采用混合RNA样本进行杂交，每个混合样本由2~3份独立的动物个体总RNA等量混合制备而成，每份独立RNA样本均来自不同的实验动物，且每份样本仅可加入一个混合池。对照组、NMBA组、PEITC组及NMBA+PEITC组的芯片实验，分别使用了9只动物的样本混合制备的混合RNA。为便于比较各处理组间的基因表达差异，本研究采用参考设计方案：每张芯片均与经Cy3荧光标记的通用参考样本共杂交，而处理组混合RNA则采用Cy5荧光标记。本研究所有芯片实验均采用Stratagene公司（美国加利福尼亚州拉霍亚市）的通用大鼠参考RNA作为通用对照样本。