MECHANISMS OF INTESTINAL CARCINOGENESIS OF DISINFECTION

我们研究计划的一个长期目标是确定化学致癌物诱导的结肠肿瘤遗传易感性的基础机制。在之前的研究中，我们已经检测了结肠癌致癌物偶氮甲烷(AOM)，它会在近亲交配的小鼠和大鼠中诱发肿瘤。在接下来的研究中，我们建议评估一种重要的饮用水消毒双产品(DBP)--溴二氯甲烷(BDCM)的致癌潜力。我们将以异常隐窝病灶(ACF)作为早期生物标志物，评价BDCM对Fischer 344/N大鼠和C3B6F1小鼠在饮水暴露后早、中、晚期肠上皮细胞恶性转化的能力。为了评估结肠病变的恶性潜能，含有ACF的组织切片将接受完整的形态计量分析。为了进一步了解肿瘤发生的机制，我们将进一步对组织切片进行免疫组织化学分析，使用本实验室常规使用的一组肿瘤生物标记物，包括以下细胞增殖标记：增殖细胞核抗原、K-ras、细胞周期蛋白D1和CDK4。此外，我们还将评估以下与结肠癌进展相关的基因的状态，包括P53、APC和β-连环素。我们还将使用两种方法测量结肠菌的细胞分化：双花蛇凝集素和氨基己糖苷酶。评估BDCM对结肠上皮细胞死亡率的影响也是很重要的。因此，我们将使用包括TUNEL法在内的商用试剂来检测致癌物质暴露的结肠上皮细胞的凋亡反应。免疫化学分析将通过对候选肿瘤相关基因的突变分析来补充，使用的是从新鲜分离的组织和激光捕获显微镜获取的样本制备的PCR-SSCP分析和直接DNA测序，这是我们实验室常规使用的技术。为了更多地了解这些基因在BDCM诱导的ACF恶性转化中的潜在作用，我们将使用RT-PCR分析它们的mRNA表达。根据我们对BDCM的研究结果，还可能对相关的DBPs进行进一步分析，包括二溴乙酸(DBA)。预计我们对大鼠和小鼠目标上皮细胞群体的比较形态计量学和遗传学分析将为DBPs导致大肠内癌前病变和肿瘤的潜在机制提供重要的新见解。

A long term goal of our research program is to identify mechanisms that underlie genetic susceptibility to colon tumorigenesis induced by chemical carcinogens. In previous studies, we have examined the colon carcinogen, azoxymethane (AOM), which induces tumors in inbred mice and rats. In the following studies, we propose to evaluate the carcinogenic potential of an important drinking water disinfection bi-product (DBP), bromodichloromethane (BDCM). Using aberrant crypt foci (ACF) as an early biomarker, we will evaluate the ability of BDCM to induce malignant transformation within the intestinal epithelium of Fischer 344/N rats and C3B6F1 mice at early, intermediate and late time points after exposure through the drinking water. In order to assess malignant potential of colonic lesions, tissue sections containing ACF will be subjected to a complete morphometric analysis. To gain a further understanding of the mechanisms of tumorigenesis, tissue sections will be further subjected to immunohistochemical analyses using a panel of tumor biomarkers that are routinely used in our laboratory, including the following markers of cell proliferaton: PCNA, K-ras, cyclin D1 and CDK 4. In addition, we will evaluate the status of the following genes are associated with colon tumor progression, including p53, APC and beta-catenin. We will also measure cellular differentiation of colonoctyes using both dolichos biflorus agglutinin and hexosaminidase. It will also be important to assess the effects of BDCM on the rate of cell death within the colonic epithelium. We will therefore assay apoptotic response of carcinogen-exposed colon epithelium using commercially available reagents, including the TUNEL assay. Immunochemical analyses will be complemented by mutational analysis of candidate tumor-related genes, using PCR-SSCP analysis and direct DNA sequencing of cDNA prepared from freshly isolated tissue as well as samples procured by laser capture microscopy, techniques that are routinely used in our laboratory. To learn more about the potential role of these genes in the malignant conversion of BDCM-induced ACF, we will examine their mRNA expression using RT-PCR analysis. Depending on the outcome of our studies of BDCM, further analysis of related DBPs, including dibromacetic (DBA) acid, may also be undertaken. It is anticipated that our comparative morphometric and genetic analyses of target epithelial cell populations in rats and mice will provide important new insights into potential mechanisms by which DBPs induce precancerous lesions and tumors within the large intestine.