Kilemade Michael, Lyons-Alcantara Maria, Rose Tina, Fitzgerald Richard, Mothersill Carmel
Environmental and Radiation Science Centre, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.
Aquat Toxicol. 2002 Oct 2;60(1-2):43-59. doi: 10.1016/s0166-445x(01)00269-7.
Little or no work has been carried out on primary cell cultures in terms of cellular proliferation and toxicity studies. Cell proliferation represents one of the most relevant cellular functions. Anti-PCNA antibodies have aroused considerable interest recently as potential immunocytochemical markers of proliferation for use in toxicity studies. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to rainbow trout (Oncorhynchus mykiss (R.)) primary cultured epidermal cells exposed in vivo i.e. whole animal exposures and in vitro for the study of the ecotoxicological potential of the aromatic amine, 2,4-dichloroaniline (2,4-DCA), a member of a little studied and widespread class of aquatic pollutants. There are many approaches to assess the proliferative activity of cells. Immunocytochemical methods offer a high sensitivity and specificity. The immunohistochemical avidin-biotin complex (ABC) method was used for the detection and quantification of PCNA, one of the best-known endogenous proliferation markers, applying the mammalian monoclonal antibody PC-10 to formalin-fixed primary cultures of rainbow trout skin. Here we describe our experience with the immunocytochemical detection and quantification of this proliferation marker. Results indicate that the antibody cross reacts with the corresponding rainbow trout epitope and that the alterations in PCNA labelling in the in vivo and in vitro exposed cultures followed similar patterns. This paper presents data on the validation of rainbow trout primary epidermal culture as an in vitro ecotoxicity model with epidermal proliferation as an endpoint. It can be concluded that cellular proliferation could be used as an indicator of the aquatic toxicity potential of xenobiotics. Correlations between cellular proliferation responses in primary cultures derived from in vivo exposed rainbow trout and primary cultures exposed in vitro were assessed. A dose-response was evidenced in both approaches, however the in vivo exposures appeared to be approximately two orders of magnitude more sensitive than the in vitro exposures. Responses in vitro occurred between 200 and 1000 micro M while in vivo responses were between 2 and 10 micro M. The good qualitative correspondence between the in vitro and in vivo results indicates that studies using trout epidermal cells allow the identification of xenobiotic effects in fish skin. However, further work is required before quantitative predictions i.e. effective concentrations in vivo, can be made from in vitro studies. This study suggests that the in vitro exposed rainbow trout primary cultured cell model with proliferation as an endpoint can be used as an alternative testing procedure to the whole animal assay.
就细胞增殖和毒性研究而言，针对原代细胞培养开展的工作很少或几乎没有。细胞增殖是最相关的细胞功能之一。抗增殖细胞核抗原（PCNA）抗体作为毒性研究中潜在的增殖免疫细胞化学标志物，最近引起了相当大的关注。在本研究中，主要针对哺乳动物组织开发的PCNA方法，被应用于虹鳟（Oncorhynchus mykiss (R.)）原代培养的表皮细胞，这些细胞在体内（即对整个动物进行暴露）和体外暴露，以研究芳香胺2,4-二氯苯胺（2,4-DCA）的生态毒理学潜力，2,4-DCA是一类研究较少但广泛存在的水生污染物中的一种。有许多方法可用于评估细胞的增殖活性。免疫细胞化学方法具有高灵敏度和特异性。免疫组织化学抗生物素蛋白-生物素复合物（ABC）方法用于检测和定量PCNA（最著名的内源性增殖标志物之一），将哺乳动物单克隆抗体PC-10应用于福尔马林固定的虹鳟皮肤原代培养物。在此，我们描述了我们在免疫细胞化学检测和定量这种增殖标志物方面的经验。结果表明，该抗体与相应的虹鳟表位发生交叉反应，并且在体内和体外暴露培养物中PCNA标记的变化遵循相似的模式。本文提供了以表皮增殖为终点的虹鳟原代表皮培养作为体外生态毒性模型的验证数据。可以得出结论，细胞增殖可作为异生物素水生毒性潜力的指标。评估了体内暴露的虹鳟原代培养物与体外暴露的原代培养物中细胞增殖反应之间的相关性。两种方法均证明了剂量反应关系，然而，体内暴露似乎比体外暴露敏感约两个数量级。体外反应发生在200至1000微摩尔之间，而体内反应发生在2至10微摩尔之间。体外和体内结果之间良好的定性对应表明，使用虹鳟表皮细胞进行的研究能够识别鱼类皮肤中的异生物素效应。然而，在能够从体外研究进行定量预测（即体内有效浓度）之前，还需要进一步的工作。本研究表明，以增殖为终点的体外暴露虹鳟原代培养细胞模型可作为全动物试验的替代测试程序。
