MECHANISMS OF INTESTINAL CARCINOGENESIS OF DISINFECTION

我们研究计划的长期目标是确定化学致癌物诱导结肠肿瘤发生的遗传易感性机制。在之前的研究中，我们研究了结肠癌致癌物偶氮氧甲烷（AOM），它在近亲繁殖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副产物溴二氯甲烷（BDCM）的致癌潜力。利用异常隐窝灶（aberrant crypt focal， ACF）作为早期生物标志物，我们将评估BDCM在饮用水暴露后早期、中期和晚期诱导Fischer 344/N大鼠和C3B6F1小鼠肠上皮恶性转化的能力。为了评估结肠病变的恶性潜能，将对含有ACF的组织切片进行完整的形态计量学分析。为了进一步了解肿瘤发生的机制，组织切片将进一步接受免疫组织化学分析，使用我们实验室常规使用的一组肿瘤生物标志物，包括以下细胞增殖标志物：PCNA， K-ras， cyclin D1和cdk4。此外，我们将评估以下与结肠肿瘤进展相关的基因的状态，包括p53、APC和β -catenin。我们也将测量结肠膜细胞的细胞分化使用两种dolichos biflorus凝集素和己糖氨酸酶。评估BDCM对结肠上皮内细胞死亡率的影响也很重要。因此，我们将使用市售试剂（包括TUNEL试验）检测暴露于致癌物质的结肠上皮细胞的凋亡反应。免疫化学分析将辅以候选肿瘤相关基因的突变分析，使用PCR-SSCP分析和对新分离组织制备的cDNA进行直接DNA测序，以及通过激光捕获显微镜获得的样本，这些技术在我们实验室中经常使用。为了进一步了解这些基因在bdcm诱导的ACF恶性转化中的潜在作用，我们将使用RT-PCR分析检测它们的mRNA表达。根据我们对BDCM的研究结果，可能还会对相关dbp进行进一步分析，包括二溴化（DBA）酸。预计我们对大鼠和小鼠靶上皮细胞群的比较形态学和遗传学分析将为DBPs诱导大肠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.