Wheeldon E B, Williams S M, Soames A R, James N H, Roberts R A
Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, United Kingdom.
Toxicol Pathol. 1995 May-Jun;23(3):410-5. doi: 10.1177/019262339502300317.
Nongenotoxic hepatocarcinogens have been proposed to inhibit hepatocyte apoptosis, preventing the removal of DNA-damaged cells. Conclusive proof of this hypothesis has been hindered by the lack of a marker or stain for apoptosis suitable for high throughput counting. A method is described for the detection of apoptotic bodies (AB) in paraffin sections of rat liver using an in situ end labeling (ISEL) technique that detects DNA damage. Results of this AB quantitation were compared with routine hematoxylin-eosin (H&E)-stained sections. The number of apoptotic bodies was enhanced by the withdrawal of the rodent nongenotoxic hepatocarcinogens and hepatomitogens nafenopin (naf) or cyproterone acetate (CPA) after 7 days of daily dosing. In rat livers 24-96 hr after cessation of dosing with naf, and in control livers, an AB index of approximately 0.1% of hepatocyte nuclei was seen when stained by H&E or ISEL. However, livers examined 48 hr after cessation of 7 days of daily dosing with CPA had an AB index of approximately 1% hepatocyte nuclei when stained with H&E, but only approximately 0.4% hepatocyte nuclei using the ISEL technique. Thus, CPA withdrawal from the hyperplastic liver generated a wave of apoptosis in contrast to naf withdrawal where little was seen up to 96 hr after withdrawal. The differing kinetics of CPA and naf clearance may explain this discrepancy. Less apoptosis was detected by the ISEL method following CPA withdrawal; this could arise if the stage of apoptosis labeled by ISEL is shorter than the morphologically recognizable stages (using H&E). The ISEL method for the evaluation of AB indices is useful in parallel with H&E, although more validation is required before it can be used routinely for the quantitation of AB in tissue sections.
非遗传毒性致癌物被认为可抑制肝细胞凋亡，从而阻止DNA损伤细胞的清除。由于缺乏适用于高通量计数的凋亡标记物或染色方法，这一假设的确凿证据一直受到阻碍。本文描述了一种使用检测DNA损伤的原位末端标记（ISEL）技术，检测大鼠肝脏石蜡切片中凋亡小体（AB）的方法。将该AB定量结果与常规苏木精-伊红（H&E）染色切片进行了比较。在每天给药7天后，撤除啮齿动物非遗传毒性致癌物和肝有丝分裂原萘酚平（naf）或醋酸环丙孕酮（CPA）后，凋亡小体数量增加。在停用naf后24 - 96小时的大鼠肝脏以及对照肝脏中，用H&E或ISEL染色时，可见AB指数约为肝细胞核的0.1%。然而，在每天给药7天停药48小时后检查的肝脏中，用H&E染色时CPA组的AB指数约为肝细胞核的1%，而使用ISEL技术时仅约为0.4%肝细胞核。因此，与naf停药后96小时内几乎未见凋亡不同，从增生性肝脏中撤除CPA会引发一波凋亡。CPA和naf清除动力学的差异可能解释了这种差异。在撤除CPA后，ISEL方法检测到的凋亡较少；如果ISEL标记的凋亡阶段比形态学上可识别的阶段（使用H&E）短，就可能出现这种情况。尽管在将ISEL方法常规用于组织切片中AB的定量之前还需要更多验证，但与H&E平行使用ISEL方法评估AB指数是有用的。
