D'Andrea M R, Alicknavitch M, Nagele R G, Damiano B P
Johnson & Johnson Pharmaceutical Research and Development, Welsh & McKean Roads, Spring House, Pennsylvania, USA.
Biotech Histochem. 2010 Apr 28;85(3):195-204. doi: 10.3109/10520290903547778.
We previously reported a sensitive, quantitative immunohistochemical assay using formalin fixed, paraffin embedded rat testicular tissues to assess the degree of proliferation-related toxicity. An indexing scheme was devised based on the percentage of PCNA-positive cells positioned as a single layer along the basement membrane at the perimeter of similarly staged seminiferous tubules (PCNA index). We observed significant decreases in the PCNA index in testes of rats treated with an experimental compound that has been shown to produce testicular histopathology. This relatively simple assay provided a more quantitative and sensitive assessment of early testicular toxicity. A separate investigation of the rates of apoptosis in adjacent serial sections of affected rat seminiferous tubules showed that the incidence of apoptosis increased as the rate of proliferation of spermatogonial cells in the tubules decreased. Therefore, we developed a simultaneous PCNA immunohistochemical and TUNEL histochemical assay not only to reduce preparation and analysis time but also to allow determination of the relation between effects of various compounds producing testicular toxicity on the two cellular processes within the same tissue section. We show that an experimental compound known to cause testicular toxicity produced a concurrent reduction of proliferation and increase in apoptosis in seminiferous tubules. In dose-response studies, we show that increased apoptosis was apparent at lower doses that did not show a significant decrease in PCNA, thus indicating the greater sensitivity of the TUNEL indexing assay to detect early evidence of toxicity. Detailed analyses show the presence of TUNEL-positive cells in tubules with normal PCNA labeling, which suggests that an effect on apoptosis occurs prior to significant changes in cell proliferation in the meiotic pathway for this particular testicular toxicant. This single assay employing the simultaneous dual labeling of apoptosis and proliferation has potential utility for detecting early testicular toxicity of experimental compounds in preclinical development and shedding light on potential cellular mechanisms for toxicity, which should help identify compounds with reduced testicular toxicity.
我们之前报道了一种灵敏的定量免疫组织化学检测方法，该方法使用福尔马林固定、石蜡包埋的大鼠睾丸组织来评估增殖相关毒性的程度。基于增殖细胞核抗原（PCNA）阳性细胞沿处于相似分期的生精小管周边基底膜呈单层排列的百分比设计了一种索引方案（PCNA指数）。我们观察到，用已显示会导致睾丸组织病理学变化的实验性化合物处理的大鼠睾丸中，PCNA指数显著降低。这种相对简单的检测方法对早期睾丸毒性提供了更定量、更灵敏的评估。对受影响的大鼠生精小管相邻连续切片中的凋亡率进行的另一项研究表明，随着小管中精原细胞增殖率的降低，凋亡发生率增加。因此，我们开发了一种同时进行PCNA免疫组织化学和TUNEL组织化学检测的方法，不仅是为了减少制备和分析时间，还为了能够确定产生睾丸毒性的各种化合物对同一组织切片中这两个细胞过程的影响之间的关系。我们发现，一种已知会导致睾丸毒性的实验性化合物使生精小管中的增殖同时减少，凋亡增加。在剂量反应研究中，我们发现，在未显示PCNA显著降低的较低剂量下，凋亡增加就很明显，这表明TUNEL索引检测法在检测早期毒性证据方面具有更高的灵敏度。详细分析显示，在PCNA标记正常的小管中存在TUNEL阳性细胞，这表明对于这种特定的睾丸毒物，在减数分裂途径中细胞增殖发生显著变化之前，就已对凋亡产生了影响。这种采用凋亡和增殖同时双重标记的单一检测方法在临床前开发中检测实验性化合物的早期睾丸毒性以及揭示潜在的毒性细胞机制方面具有潜在用途，这应该有助于识别睾丸毒性降低的化合物。
