Hendawi M, Sauvé S, Ashour M, Brousseau P, Fournier Michel
INRS-Institut Armand Frappier, Université du Québec, Pointe-Claire, QC, Canada.
Ecotoxicol Environ Saf. 2004 Sep;59(1):17-22. doi: 10.1016/j.ecoenv.2003.07.015.
There is mounting evidence that earthworms could be used as a sentinel species for soil ecotoxicity evaluation. In this aspect, phagocytosis by coelomocytes was shown to be a sensitive biomarker of exposure to xenobiotics. In this paper, we introduce a simple method for ultrasound extrusion of earthworm coelomocytes that generates a high cell yield, does not interfere with phagocytic competence, and requires a minimum of manipulations. Coelomocytes were extruded from the earthworm Eisenia fetida using this new ultrasound method and compared with ethanol and electrical extrusion. The ultrasonic extrusion showed the highest cell recovery with 3.17 +/ -0.8 x 10(6) cells per earthworm compared with 2.22 +/- 0.8 x 10(6) cells per earthworm for electrical extrusion and 1.57 +/- 0.07 x 10(6) cells per earthworm for ethanol extrusion. No significant differences in the cell viability were observed using propidium iodide and flow cytometry with viability for extrusion with ethanol of 63.8 +/- 12.7%, electrical 76.8 +/- 7.5%, and ultrasound 68.2 +/- 7.8%. To compare the potential effect of extrusion on cell quality, the cells extruded using the three methods were subjected to an 18-h in vitro exposure to methylmercury chloride (MeHgCl; CH3HgCl) with concentrations ranging from 10(-9) to 10(-4)M. The half-maximal effective concentration (EC50) for inhibition of phagocytosis occurred between 10(-7) and 10(-6)M. We found no significant differences among the extrusion methods for the phagocytic potential of the coelomocytes. This method does not harm the worms and can certainly improve collection of coelomocytes from earthworms and therefore contribute to the development of bioassays using invertebrates.
越来越多的证据表明,蚯蚓可作为土壤生态毒性评估的指示物种。在这方面,体腔细胞的吞噬作用被证明是接触异生物素的敏感生物标志物。在本文中,我们介绍了一种简单的蚯蚓体腔细胞超声挤压方法,该方法细胞产量高,不干扰吞噬能力,且操作最少。使用这种新的超声方法从赤子爱胜蚓中挤出体腔细胞,并与乙醇挤压法和电挤压法进行比较。超声挤压法的细胞回收率最高,每条蚯蚓为3.17±0.8×10⁶个细胞,而电挤压法为每条蚯蚓2.22±0.8×10⁶个细胞,乙醇挤压法为每条蚯蚓1.57±0.07×10⁶个细胞。使用碘化丙啶和流式细胞术观察细胞活力,未发现显著差异,乙醇挤压法活力为63.8±12.7%,电挤压法为76.8±7.5%,超声挤压法为68.2±7.8%。为了比较挤压对细胞质量的潜在影响,使用这三种方法挤出的细胞在体外暴露于浓度范围为10⁻⁹至10⁻⁴M的氯化甲基汞(MeHgCl;CH₃HgCl)18小时。吞噬作用抑制的半数有效浓度(EC₅₀)在10⁻⁷至10⁻⁶M之间。我们发现,对于体腔细胞的吞噬潜力,挤压方法之间没有显著差异。该方法不会伤害蚯蚓,肯定可以改善从蚯蚓中收集体腔细胞的情况,因此有助于开发使用无脊椎动物的生物测定法。