Selden J R, Dolbeare F, Clair J H, Miller J E, McGettigan K, DiJohn J A, Dysart G R, DeLuca J G
Department of Safety Assessment, Merck Research Laboratories, West Point, PA 19486.
Mutat Res. 1994 Sep;315(2):147-67. doi: 10.1016/0921-8777(94)90015-9.
An in vitro flow cytometric (FCM) DNA repair assay has been developed and validated by comparison to conventional autoradiography (ARG). Both assays measure unscheduled DNA synthesis (UDS). Cultures of hepatocytes from young male Sprague-Dawley rats were exposed to a battery of 26 chemicals plus bromodeoxyuridine (BrdUrd) or 3H-thymidine (3H-dT) for 18-20 h before harvest. Selection of test chemicals was based upon both their genotoxicity classifications and carcinogenicity bioassay results in male rats. DNA repair in chemically treated cultures was detected flow cytometrically by measuring the uptake of BrdUrd in non-replicating (G1, G2, mitotic and 4C) cells. Intracellular levels of incorporated BrdUrd were visualized by immunochemical labeling with fluorescein isothiocyanate (FITC), and total cellular DNA content was simultaneously estimated by counterstaining samples with the nucleic acid intercalator, propidium iodide (PI). Information was obtained from 10(4) cells/sample. Since repairing cells incorporate significantly less BrdUrd per unit of time than replicating cells, low intensity BrdUrd-FITC fluorescent signals from repairing cells are readily discriminated from high intensity signals from replicating cells when displayed on linear univariate histograms. Further distinction between repairing and replicating cells was achieved by displaying the DNA contents of all cells on linear bivariate histograms. Thus, repairing cells were resolved without subjecting these cultures to agents which suppress replicative synthesis (e.g., hydroxyurea). Results from these concurrent FCM and ARG investigations include the following: (1) conclusions (DNA repair positive or negative) were in agreement, with one exception, cinnamyl anthranilate, for which cytotoxic doses produced a positive FCM response, but lack of intact hepatocytes in parallel ARG preparations prevented analysis; (2) similar sensitivities for most of the positive chemicals were reported; (3) a high correlation (85%) exists between the reported genotoxicity classification and these DNA repair results in the absence of overt cytotoxicity; (4) a poor correlation exists between these DNA repair results and hepatocarcinogenesis (only 4/11 liver carcinogens tested positive) or overall carcinogenesis in the male rat (only 9/21 carcinogens tested positive). This FCM assay provides a rapid, sensitive, safe and reliable means of identifying agents which induce DNA repair in mammalian cells.
一种体外流式细胞术（FCM）DNA修复检测方法已经开发出来，并通过与传统放射自显影术（ARG）比较进行了验证。两种检测方法都测量了非预定DNA合成（UDS）。在收获前，将来自年轻雄性Sprague-Dawley大鼠的肝细胞培养物暴露于一组26种化学物质以及溴脱氧尿苷（BrdUrd）或3H-胸腺嘧啶核苷（3H-dT）中18至20小时。测试化学物质的选择基于它们的遗传毒性分类以及在雄性大鼠中的致癌性生物测定结果。通过测量非复制（G1、G2、有丝分裂和4C）细胞中BrdUrd的摄取，以流式细胞术检测化学处理培养物中的DNA修复。通过用异硫氰酸荧光素（FITC）进行免疫化学标记来可视化掺入的BrdUrd的细胞内水平，并通过用核酸嵌入剂碘化丙啶（PI）对样品进行复染来同时估计总细胞DNA含量。从每个样品的10^4个细胞中获取信息。由于修复细胞每单位时间掺入的BrdUrd比复制细胞少得多，因此当在线性单变量直方图上显示时，修复细胞的低强度BrdUrd-FITC荧光信号很容易与复制细胞的高强度信号区分开来。通过在线性双变量直方图上显示所有细胞的DNA含量，进一步区分了修复细胞和复制细胞。因此，无需对这些培养物使用抑制复制合成的试剂（例如羟基脲）就能分辨出修复细胞。这些同时进行的FCM和ARG研究的结果如下：（1）结论（DNA修复阳性或阴性）基本一致，但有一个例外，即邻氨基苯甲酸肉桂酯，其细胞毒性剂量产生了阳性FCM反应，但平行ARG制剂中缺乏完整的肝细胞，无法进行分析；（2）报告了大多数阳性化学物质具有相似的敏感性；（3）在没有明显细胞毒性的情况下，报告的遗传毒性分类与这些DNA修复结果之间存在高度相关性（85%）；（4）这些DNA修复结果与雄性大鼠肝癌发生（仅11种肝致癌物中的4种检测为阳性）或总体致癌作用（仅21种致癌物中的9种检测为阳性）之间存在较差的相关性。这种FCM检测方法为鉴定在哺乳动物细胞中诱导DNA修复的试剂提供了一种快速、灵敏、安全和可靠的方法。
