Althaus F R, Lawrence S D, Sattler G L, Longfellow D G, Pitot H C
Cancer Res. 1982 Aug;42(8):3010-5.
Technical modifications of the quantitative determination of unscheduled DNA synthesis in cultured hepatocytes are described which allow for the rapid identification of potentially carcinogenic chemicals on a large-scale screening basis. The test is based on the biochemical quantification of [methyl-3H]thymidine incorporation into DNA in the presence of hydroxyurea following isolation of nuclei from hepatocytes treated with the agent under study. This procedure ("nuclei procedure") eliminates most of the background radioactivity which otherwise obscures the stimulation of DNA repair synthesis by agents that induce a relatively weak response. By combining the nuclei procedure with a double-labeling technique, test results can be obtained within a few hr after exposure of hepatocytes to the test agents. A test series involving 41 agents confirmed the reliability of the nuclei procedure for the assay of DNA repair synthesis. In addition, chemicals which had yielded conflicting results previously in the autoradiographic hepatocyte DNA repair test, such as 4-acetylaminofluorene, or which had passed unrecognized in previous in vitro tests, such as the potent liver carcinogen methapyrilene hydrochloride, scored clearly positive in our test protocol.
本文描述了对培养肝细胞中DNA非预定合成定量测定的技术改进，这些改进使得在大规模筛选的基础上能够快速鉴定潜在致癌化学物质。该测试基于在用研究试剂处理过的肝细胞分离出细胞核后，在羟基脲存在的情况下，对[甲基-³H]胸腺嘧啶核苷掺入DNA进行生化定量。这个程序（“细胞核程序”）消除了大部分背景放射性，否则这些背景放射性会掩盖诱导相对较弱反应的试剂对DNA修复合成的刺激作用。通过将细胞核程序与双标记技术相结合，在肝细胞暴露于测试试剂后几小时内就能获得测试结果。一个涉及41种试剂的测试系列证实了细胞核程序用于测定DNA修复合成的可靠性。此外，以前在放射自显影肝细胞DNA修复测试中产生相互矛盾结果的化学物质，如4-乙酰氨基芴，或在以前的体外测试中未被识别的化学物质，如强效肝致癌物盐酸美吡拉敏，在我们的测试方案中都明确显示为阳性。
