Mazon Aurélia de Fátima, Nolan Declan Thomas, Lock Robert A C, Wendelaar Bonga Sjoerd E, Fernandes Marisa Narciso
Department of Physiological Sciences, Federal University of São Carlos, Rod. Washington Luiz, km 235, 13565-905 São Carlos, SP, Brazil.
Toxicology. 2007 Jan 25;230(1):53-63. doi: 10.1016/j.tox.2006.10.027. Epub 2006 Nov 12.
This study evaluated the efficacy of fish opercular external (skin) and inner (opercular membrane) epithelium as an in vitro model for toxic and other substances studies. The rainbow trout (Oncorhynchus mykiss) operculum was cultured in 12-well dishes containing sterile Leibovitz 15 (L-15) supplemented with glutamine medium during 24h at 9 degrees C, and the effect of copper, a toxic agent, and/or cortisol, an endogenous agent, on the epithelial cells was analyzed using light microscopy techniques. The opercula were submitted to four treatments: (i) control (Cont), L-15 medium only, (ii) 0.28 microM cortisol (Cort), (iii) 100 microM CuSO(4) (Cu), and (iv) 0.28 microM cortisol+100 microM CuSO(4) (Cort-Cu). The tissue condition after 24h incubation was analyzed by staining the mucous cells for neutral and acid mucosubstances. Cellular necrosis was evaluated by measuring the lactate dehydrogenase (LDH) leakage at 12 and 24h incubation. Cellular proliferation, apoptosis, metallothionein (MT) and glucocorticoid receptor (GR) expression were evaluated by immunohistochemistry. The LDH leakage was higher and the proliferating cell nuclear antigen (PCNA) positive-stained cells were lower in Cu treatment in both, epidermis and opercular membrane. Apoptotic cells in the opercular membrane were higher in the Cort and Cort-Cu treatments while, in the epidermis, they were higher in Cu and Cort-Cu treatments. GR-positive stained cells decreased significantly in all treatments in both epithelia and the MT-positive cells increased in the Cu and Cort-Cu treated groups. Copper showed to be a potent toxic stressor killing the cells via necrosis, decreasing the number of PCNA-positive cells and inducing MT synthesis while cortisol did not affect the MT synthesis, although it might stimulate apoptosis. The results are evidence that the opercular epithelia serve as a suitable model for studying in vitro effects of toxic agents, as well as endogenous factors on the cellular responses without interference of the physiological state of fish being useful to predict in vivo toxicity.
本研究评估了鱼类鳃盖外部（皮肤）和内部（鳃盖膜）上皮作为有毒物质及其他物质研究的体外模型的有效性。虹鳟（Oncorhynchus mykiss）鳃盖在含有补充了谷氨酰胺的无菌Leibovitz 15（L-15）培养基的12孔培养板中于9摄氏度培养24小时，使用光学显微镜技术分析有毒剂铜和/或内源性物质皮质醇对上皮细胞的影响。鳃盖接受四种处理：（i）对照（Cont），仅L-15培养基；（ii）0.28微摩尔皮质醇（Cort）；（iii）100微摩尔硫酸铜（Cu）；（iv）0.28微摩尔皮质醇 + 100微摩尔硫酸铜（Cort-Cu）。孵育24小时后，通过对黏液细胞进行中性和酸性黏液物质染色来分析组织状况。通过测量孵育12小时和24小时时乳酸脱氢酶（LDH）泄漏来评估细胞坏死。通过免疫组织化学评估细胞增殖、凋亡、金属硫蛋白（MT）和糖皮质激素受体（GR）表达。在表皮和鳃盖膜中，Cu处理组的LDH泄漏较高，增殖细胞核抗原（PCNA）阳性染色细胞较低。在Cort和Cort-Cu处理组中，鳃盖膜中的凋亡细胞较高，而在表皮中，Cu和Cort-Cu处理组中的凋亡细胞较高。在两种上皮中，所有处理组中GR阳性染色细胞均显著减少，Cu和Cort-Cu处理组中MT阳性细胞增加。铜显示为一种强效的毒性应激源，通过坏死杀死细胞，减少PCNA阳性细胞数量并诱导MT合成，而皮质醇虽可能刺激凋亡，但不影响MT合成。结果证明鳃盖上皮可作为研究有毒剂以及内源性因素对细胞反应的体外效应的合适模型，且不受鱼类生理状态干扰，有助于预测体内毒性。
