EVALUATION OF CHEMOPREVENTIVE AGENTS BY IN VITRO SCREENI

通过体外筛选评估化学预防药物

• 批准号: 6346788
• 负责人: SHEELA SHARMA
• 金额: $ 38.75万
• 依托单位: ALION SCIENCE AND TECHNOLOGY CORP
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6346788
• 关键词: benzopyrenes; bioassay; cancer prevention; chemoprevention; cytotoxicity; drug screening /evaluation; histology; laboratory rat; neoplastic transformation; respiratory epithelium; retinoate; tissue /cell culture

Primary cultures of RTE cells are exposed to carcinogens on Day 1, then morphologically altered epithelial foci are scored at Day 30. Tracheal epithelial cells are isolated from 8 to 12 weeks old male Fisher 344 rats and plated on collagen coated dishes. Initial cytotoxicity tests with the test chemopreventive agent are used to select appropriate concentration ranges for use in the transformation-inhibition assay. From the initial cytotoxicity test, the highest concentration which permits a CFE of 80% or greater compared to control is determined. This concentration and four half-log lower concentrations are included in the chemopreventive test assays. The attached cells are exposed to: media alone, solvent alone, benzo(a)pyrene alone, benzo(a)pyrene plus retinoic acid (or other positive control), or benzo(a)pyrene plus chemopreventive agent. At Day 2 the benzo(a)pyrene is removed and exposure to the chemopreventive agent, retinoic acid (or other positive control), or solvent continued. At 5-7 days three dishes per group are fixed and stained for cytotoxicity determination. At Day 30 all remaining cultures are fixed, stained, and morphologically altered foci scored. Computerized morphometric analysis is being performed on a subset of the agents at various timepoints. The transformation frequency of the various groups are determined, and the agents prioritized for further development. There are 20 chemopreventive agents to be screened by this assay for chemopreventive activity.

RTE 细胞的原代培养物在第 1 天暴露于致癌物，然后在第 30 天对形态改变的上皮病灶进行评分。从 8 至 12 周龄的雄性 Fisher 344 大鼠中分离气管上皮细胞，并将其铺在胶原蛋白包被的培养皿上。 使用测试化学预防剂进行的初始细胞毒性测试用于选择用于转化抑制测定的适当浓度范围。从最初的细胞毒性测试中，确定了与对照相比允许 CFE 达到 80% 或更高的最高浓度。 该浓度和四个半对数较低浓度包含在化学预防测试分析中。 贴壁细胞暴露于：单独的培养基、单独的溶剂、单独的苯并(a)芘、苯并(a)芘加视黄酸(或其他阳性对照)、或苯并(a)芘加化学预防剂。 第 2 天，去除苯并（a）芘并继续暴露于化学预防剂、视黄酸（或其他阳性对照）或溶剂。 5-7天时，将每组三个培养皿固定并染色以测定细胞毒性。 在第30天，对所有剩余的培养物进行固定、染色，并对形态改变的病灶进行评分。在不同时间点对一部分代理进行计算机形态测量分析。 确定各组的转化频率，并确定代理进一步发展的优先顺序。 通过该测定筛选了 20 种化学预防剂的化学预防活性。