JoVE Journal > Environment
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Traduction automatique
Please note that all translations are automatically generated. Click here for the English version.
Environnement

斑马鱼细胞系的细胞毒性测定

Published: January 06, 2023
doi:
10.3791/64860
, , , , , , , , ,
1Graduate Program in Genetics, Department of Genetics,Federal University of Paraná (UFPR), 2Safety of Product Department,Grupo Boticário, 3Department of Genetics,Federal University of Paraná (UFPR)

Summary

该协议介绍了常用的细胞毒性测定（阿拉马尔蓝 [AB]、CFDA-AM、中性红和 MTT 测定），适用于评估斑马鱼胚胎 （ZEM2S） 和肝脏 （ZFL） 细胞系的细胞毒性 96 孔板。

Abstract

鱼细胞系越来越多地用于生态毒性研究，细胞毒性测定已被提出作为预测鱼类急性毒性的方法。因此，该协议提出了修改的细胞毒性测定，以评估斑马鱼（Danio rerio）胚胎（ZEM2S）和肝脏（ZFL）细胞系中的细胞活力，该板在96孔板中。评估的细胞毒性终点是线粒体完整性（Alamar Blue [AB] 和 MTT 测定）、通过酯酶活性 的 膜完整性（CFDA-AM 测定）和溶酶体膜完整性（中性红 [NR] 测定）。在96孔板中暴露测试物质后，进行细胞毒性测定;在这里，AB和CFDA-AM同时进行，然后在同一板上进行NR，而MTT测定在单独的板上进行。这些测定的读数通过AB和CFDA-AM的荧光以及MTT和NR的吸光度获取。用这些鱼细胞系进行的细胞毒性测定可用于研究化学物质对鱼的急性毒性。

Introduction

需要对化学物质进行人体健康和环境安全性测试。监管机构和/或立法（例如，REACH、OECD、US EPA）在安全性评估中越来越多地考虑分子和细胞生物标志物来预测对生物体的影响1，2，因为它们可以先于体内不良结果（例如，内分泌干扰、免疫反应、急性毒性、光毒性）3，4，5，6，7.在这种情况下，细胞毒性已被作为预测鱼类急性毒性的测量方法5，8;然而，它可以在生态毒性研究中有许多其他应用，例如定义化学物质的亚细胞毒性浓度，以研究它们对鱼类最多样化的影响（例如，内分泌干扰效应）。

在细胞培养系统（体外 系统）中，化学物质的细胞毒性可以通过终点类型的不同方法确定。例如，一种细胞毒性方法可以基于与细胞死亡过程中观察到的特定形态相关的终点，而另一种方法可以通过测量细胞死亡、活力和功能、形态、能量代谢以及细胞附着和增殖来确定细胞毒性。化学物质可以通过不同的机制影响细胞活力，因此覆盖不同细胞活力终点的细胞毒性评估对于预测化学效应是必要的9。

MTT和Alamar Blue（AB）是根据细胞代谢活性确定对细胞活力影响的测定。MTT测定评估线粒体酶琥珀酸脱氢酶10的活性。淡黄色的3-[4，5-二甲基噻唑-2基]-2，5-二苯基四唑溴化物（MTT）还原为甲臜蓝仅发生在活细胞中，其光密度与活细胞的数量成正比10。AB测定是一种灵敏的氧化还原指示剂，由线粒体酶介导，线粒体酶在活细胞将刃天青还原为试卤灵时发出荧光并改变颜色11;然而，胞质和微粒体酶也有助于减少AB和MTT12。这些酶可能包括几种还原酶，例如醇和醛氧化还原酶、NAD（P）H：醌氧化还原酶、黄素还原酶、NADH脱氢酶和细胞色素11。

中性红（NR）测定是基于将该染料掺入活细胞的溶酶体中的细胞活力测定13。NR的摄取取决于细胞维持pH梯度的能力。溶酶体内的质子梯度保持低于细胞质的pH值。在正常的生理pH值下，NR呈现的净电荷约为零，这使其能够穿透细胞膜。因此，染料带电并保留在溶酶体内。因此，保留NR的量越大，活细胞的数量就越多14。破坏细胞表面或溶酶体膜的化学物质会损害这种染料的摄取。

CFDA-AM测定是基于保留5-羧基荧光素二乙酸乙酰氧基甲酯（CFDA-AM）的荧光细胞活力测定15。5-CFDA-AM是一种酯酶底物，被转化为羧基荧光素，羧基荧光素是一种极性且不可被活细胞膜渗透的荧光物质15;因此，它保留在完整细胞膜的内侧，表明活细胞。

最近，三种细胞毒性测定（CFDA-AM、NR 和 AB 测定）在经过验证的 ISO（国际标准化组织）指南 （ISO 21115：2019）16 和 OECD（经济合作与发展组织）测试方法 （OECD TG 249） 中合并，以使用 RTgill-W1 细胞系（虹鳟鱼 [Oncorhynchus mykiss] 鳃的永久性细胞系）在 24 孔板中评估鱼类急性毒性17.虽然有一种现有的基于细胞的方法来预测鱼类的急性毒性，但已经投入了努力开发与其他鱼类物种类似的方法，并提高该方法的通量。一些例子包括用特定毒性途径18，19的报告基因转染的ZFL细胞系的开发，RTgill-W1细胞系20中的光毒性测试，以及使用ZFL和ZF4细胞系（来自1天龄胚胎的斑马鱼成纤维细胞）通过几种细胞毒性测定来评估毒性21。

Danio rerio（斑马鱼）是水生毒性研究中使用的主要鱼类之一;因此，使用斑马鱼细胞系进行基于细胞的鱼类毒性测试方法可能非常有用。ZFL细胞系是一种斑马鱼上皮肝细胞系，具有肝实质细胞的主要特征，可代谢异生素7，22，23，24，25。同时，ZEM2S细胞系是源自胚泡阶段的胚胎斑马鱼成纤维细胞系，可用于研究对鱼类26，27的发育影响。因此，该协议描述了四种细胞毒性测定（MTT，AB，NR和CFDA-AM测定），并在96孔板中使用ZFL和ZEM2S细胞系进行修饰。

Protocol

注意：有关本协议中使用的材料列表，请参阅 材料 表，有关本协议中使用的溶液和介质的组成，请参阅 表1 。 1. 制备ZFL和ZEM2S细胞 从具有80%汇合度的ZFL或ZEM2S细胞的T75烧瓶开始，在28°C的相应完全培养基中培养，不含CO2。 从烧瓶中取出培养基，并通过加入 10 mL 1x 磷酸盐缓冲盐水 （PBS） （0.01 M） 洗涤细胞。向培?…

Representative Results

图3显示了AB，CFDA-AM，NR和MTT测定的板。对于AB测定（图3A），空白孔和没有或减少活细胞数量的孔显示蓝色和低荧光，而具有大量活细胞的孔呈粉红色并呈现高荧光值，因为刃天青（AB）转化为试卤灵（粉红色物质）被活细胞。对于CFDA-AM测定，板上孔的颜色没有明显差异;然而，由于CFDA-AM的保留并随后转化为羧基荧光素（荧光物质），含有活细胞的孔中?…

Discussion

细胞毒性测定广泛用于体外毒性评估，本协议文章介绍了四种常用的细胞毒性测定，修改后可在斑马鱼细胞系中进行（即，96孔板的细胞密度，MTT测定中的孵育时间，化学暴露条件下的FBS耗竭和SC的最大可接受浓度）。由于这些测定通过不同的细胞活力终点（代谢功能、溶酶体膜完整性和细胞膜完整性）量化细胞毒性，因此它们的组合提供了斑马鱼细胞系中化学细胞毒性的准确评估。该协议…

Divulgations

The authors have nothing to disclose.

Acknowledgements

为了纪念这部作品的合著者Márcio Lorencini博士，他是化妆品领域的优秀研究人员，致力于促进巴西的化妆品研究。作者感谢生理学系（UFPR）的多用户实验室提供设备，并感谢高等教育人员改进协调（CAPES，巴西）（财务代码001）和Grupo Boticario的财政支持。

Materials

5-CFDA, AM (5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester) Invitrogen C1345
Cell culture plate, 96 well plate Sarstedt 83.3924 Surface: Standard, flat base
DMEM Gibco 12800-017 Powder, high glucose, pyruvate
Ham's F-12 Nutrient Mix, powder Gibco 21700026 Powder
HEPES (1 M) Gibco 15630080
Leibovitz's L-15 Medium Gibco 41300021 Powder
Neutral red  Sigma-Aldrich N4638 Powder, BioReagent, suitable for cell culture
Orbital shaker  Warmnest KLD-350-BI 22 mm rotation diameter
Dulbeccos PBS (10X) with calcium and magnesium Invitrogen 14080055
Penicillin-Streptomycin (10,000 U/mL) Gibco 15140122
Resazurin sodium salt  Sigma-Aldrich R7017 Powder, BioReagent, suitable for cell culture
RPMI 1640 Medium Gibco 31800-014 Powder
SFB – Fetal Bovine Serum, qualified, USDA-approved regions Gibco 12657-029
Sodium bicarbonate Sigma-Aldrich S5761 Powder,  bioreagent for molecular biology
Thiazolyl Blue Tetrazolium Bromide  98% Sigma-Aldrich M2128
Trypan blue stain (0.4%) Gibco 15250-061
Trypsin-EDTA (0.5%), no phenol red Gibco 15400054
ZEM2S cell line ATCC CRL-2147 This cell line was kindly donated by Professor Dr. Michael J.
Carvan (University of Wisconsin, Milwaukee, USA)
ZFL cell line BCRJ 256
DOWNLOAD MATERIALS LIST

References

  1. ECHA. Non-Animal Approaches-Current Status of Regulatory Applicability Under the REACH, CLP and Biocidal Products Regulations. ECHA. , (2017).
  2. Alternative Methods Accepted by US Agencies. National Toxicology Program, and US Department of Health and Human Services Available from: https://ntp.niehs.nih.gov/whatwestudy/niceatm/accept-methods/index.html (2022)
  3. Schirmer, K. Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology. 224 (3), 163-183 (2006).
  4. Scholz, S., et al. Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians-screening for estrogen, androgen and thyroid hormone disruption. Critical Reviews in Toxicology. 43 (1), 45-72 (2013).
  5. Tanneberger, K., et al. Predicting fish acute toxicity using a fish gill cell line-based toxicity assay. Environmental Science & Technology. 47 (2), 1110-1119 (2013).
  6. Roesler, R., Lorencini, M., Pastore, G. Brazilian cerrado antioxidant sources: cytotoxicity and phototoxicity in vitro. Food Science and Technology. 30, 814-821 (2010).
  7. Ruyra, A., et al. Zebrafish liver (ZFL) cells are able to mount an anti-viral response after stimulation with Poly (I:C). Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 182, 55-63 (2015).
  8. Natsch, A., Laue, H., Haupt, T., von Niederhäusen, V., Sanders, G. Accurate prediction of acute fish toxicity of fragrance chemicals with the RTgill-W1 cell assay. Environmental Toxicology and Chemistry. 37 (3), 931-941 (2018).
  9. Freshney, R. I. Cytotoxicity. Culture of Animal Cells: A Manual of Basic Technique. , (2005).
  10. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 65 (1-2), 55-63 (1983).
  11. O’Brien, J., Wilson, I., Orton, T., Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry. 267 (17), 5421-5426 (2000).
  12. Gonzalez, R. J., Tarloff, J. B. Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity. Toxicology In Vitro. 15 (3), 257-259 (2001).
  13. Borenfreund, E., Puerner, J. A. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology Letters. 24 (2-3), 119-124 (1985).
  14. Repetto, G., del Peso, A., Zurita, J. L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols. 3 (7), 1125-1131 (2008).
  15. Kamiloglu, S., Sari, G., Ozdal, T., Capanoglue, E. Guidelines for cell viability assays. Food Frontiers. 1 (3), 332-349 (2020).
  16. Water Quality-Determination of Acute Toxicity of Water Samples and Chemicals to a Fish Gill Cell Line (RTgill-W1) (ISO 21115:2019). International Organization for Standardization Available from: https://www.iso.org/standar/69933.html (2019)
  17. Organisation for Economic Co-operation and Development. . Test Guideline No. 249: Fish Cell Line Acute Toxicity-The RTgill-W1 Cell Line Assay. OECD Guidelines for the Testing of Chemicals, Section 2. Effects on Biotic Systems. , (2021).
  18. Lungu-Mitea, S., Lundqvist, J. Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines. Archives of Toxicology. 94 (8), 2769-2784 (2020).
  19. Lungu-Mitea, S., Han, Y., Lundqvist, J. Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes. Cell Biology and Toxicology. , 1-23 (2021).
  20. Schirmer, K., Chan, A. G., Greenberg, B. M., Dixon, D. G., Bols, N. C. Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture. Toxicology In Vitro. 11 (1-2), 107-119 (1997).
  21. Lungu-Mitea, S., et al. Modeling bioavailable concentrations in zebrafish cell lines and embryos increases the correlation of toxicity potencies across test systems. Environmental Science & Technology. 55 (1), 447-457 (2021).
  22. Cavalcante, D. G. S. M., et al. Cytotoxic, biochemical and genotoxic effects of biodiesel produced by different routes on ZFL cell line. Toxicology In Vitro. 28 (6), 1117-1125 (2014).
  23. Meng, Q., Yeung, K., Chan, K. M. Toxic effects of octocrylene on zebrafish larvae and liver cell line (ZFL). Aquatic Toxicology. 236, 105843 (2021).
  24. Kwok, M. L., Chan, K. M. Oxidative stress and apoptotic effects of copper and cadmium in the zebrafish liver cell line ZFL. Toxicology Reports. 7, 822-835 (2020).
  25. Yang, J., Chan, K. M. Evaluation of the toxic effects of brominated compounds (BDE-47, 99, 209, TBBPA) and bisphenol A (BPA) using a zebrafish liver cell line, ZFL. Aquatic Toxicology. 159, 138-147 (2015).
  26. Bradford, C. S., Sun, L., Collodi, P., Barnes, D. W. Cell cultures from zebrafish embryos and adult tissues. Journal of Tissue Culture Methods. 16 (2), 99-107 (1994).
  27. He, S., et al. Genetic and transcriptome characterization of model zebrafish cell lines. Zebrafish. 3 (4), 441-453 (2006).
  28. Mansoury, M., Hamed, M., Karmustaji, R., Al Hannan, F., Safrany, S. T. The edge effect: A global problem. The trouble with culturing cells in 96-well plates. Biochemistry and Biophysics Reports. 26, 100987 (2021).
  29. Funk, D., Schrenk, H. -. H., Frei, E. Serum albumin leads to false-positive results in the XTT and the MTT assay. BioTechniques. 43 (2), 178 (2007).
  30. Dayeh, V. R., Bols, N. C., Tanneberger, K., Schirmer, K., Lee, L. E. J. The use of fish-derived cell lines for investigation of environmental contaminants: An update following OECD’s fish toxicity testing framework no. 171. Current Protocols in Toxicology. 1, (2013).
  31. Stepanenko, A. A., Dmitrenko, V. V. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 574 (2), 193-203 (2015).
  32. Ulukaya, E., Colakogullari, M., Wood, E. J. Interference by anti-cancer chemotherapeutic agents in the MTT-tumor chemosensitivity assay. Chemotherapy. 50 (1), 43-50 (2004).
  33. Sebaugh, J. L. Guidelines for accurate EC50/IC50 estimation. Pharmaceutical Statistics. 10 (2), 128-134 (2011).
  34. Weimer, M., et al. The impact of data transformations on concentration-response modeling. Toxicology Letters. 213 (2), 292-298 (2012).
  35. Green, J. W., Holbech, T. A., Henrik, Chapter 4: Analysis of Continuous Data (Regression). Statistical Analysis of Ecotoxicity Studies. , (2018).
  36. Proença, S., et al. Effective exposure of chemicals in in vitro cell systems: A review of chemical distribution models. Toxicology In Vitro. 73, 105133 (2021).
  37. Guidony, N. S., et al. ABC proteins activity and cytotoxicity in zebrafish hepatocytes exposed to triclosan. Environmental Pollution. 271, 116368 (2021).
  38. da Silva, N. D. G., et al. Interference of goethite in the effects of glyphosate and Roundup® on ZFL cell line. Toxicology In Vitro. 65, 104755 (2020).
  39. Yang, Y., et al. Temperature is a key factor influencing the invasion and proliferation of Toxoplasma gondii in fish cells. Experimental Parasitology. 217, 107966 (2020).
  40. Lopes, F. M., Sandrini, J. Z., Souza, M. M. Toxicity induced by glyphosate and glyphosate-based herbicides in the zebrafish hepatocyte cell line (ZF-L). Ecotoxicology and Environmental Safety. 162, 201-207 (2018).
  41. Lachner, D., Oliveira, L. F., Martinez, C. B. R. Effects of the water soluble fraction of gasoline on ZFL cell line: Cytotoxicity, genotoxicity and oxidative stress. Toxicology In Vitro. 30, 225-230 (2015).
  42. Morozesk, M., et al. Effects of multiwalled carbon nanotubes co-exposure with cadmium on zebrafish cell line: Metal uptake and accumulation, oxidative stress, genotoxicity and cell cycle. Ecotoxicology and Environmental Safety. 202, 110892 (2020).
  43. Dognani, G., et al. Nanofibrous membranes for low-concentration Cr VI adsorption: kinetic, thermodynamic and the influence on ZFL cells viability. Materials Research. , 24 (2021).
  44. ZEM2S (ATCC®CRL-2147™). American Type Culture Collection Available from: https://www.atcc.org/products/crl-2147 (2023)
  45. Chen, Y., et al. Acute toxicity of the cationic surfactant C12-benzalkonium in different bioassays: how test design affects bioavailability and effect concentrations. Environmental Toxicology and Chemistry. 33 (3), 606-615 (2014).
  46. Pomponio, G., et al. In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments. Toxicology In Vitro. 30, 36-51 (2015).
  47. Mori, M., Wakabayashi, M. Cytotoxicity evaluation of chemicals using cultured fish cells. Water Science and Technology. 42 (7-8), 277-282 (2000).
  48. Caminada, D., Escher, C., Fent, K. Cytotoxicity of pharmaceuticals found in aquatic systems: comparison of PLHC-1 and RTG-2 fish cell lines. Aquatic Toxicology. 79 (2), 114-123 (2006).
  49. Giltrap, M., et al. In vitro screening of organotin compounds and sediment extracts for cytotoxicity to fish cells. Environmental Toxicology and Chemistry. 30 (1), 154-161 (2011).
  50. Hollert, H., Duerr, M., Erdinger, L., Braunbeck, T. Cytotoxicity of settling particulate matter and sediments of the Neckar River (Germany) during a winter flood. Environmental Toxicology and Chemistry. 19 (3), 528-534 (2000).
  51. Pannetier, P., et al. Toxicity assessment of pollutants sorbed on environmental sample microplastics collected on beaches: Part I-adverse effects on fish cell line. Environmental Pollution. 248, 1088-1097 (2019).
  52. Ternjej, I., Srček, V. G., Mihaljević, Z., Kopjar, N. Cytotoxic and genotoxic effects of water and sediment samples from gypsum mining area in channel catfish ovary (CCO) cells. Ecotoxicology and Environmental Safety. 98, 119-127 (2013).
  53. Hamid, R., Rotshteyn, Y., Rabadi, L., Parikh, R., Bullock, P. Comparison of alamar blue and MTT assays for high throughput screening. Toxicology In Vitro. 18 (5), 703-710 (2004).
  54. Vistica, D. T., et al. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Recherche en cancérologie. 51 (10), 2515-2520 (1991).
  55. Knauer, K., Lampert, C., Gonzalez-Valero, J. Comparison of in vitro and in vivo acute fish toxicity in relation to toxicant mode of action. Chemosphere. 68 (8), 1435-1441 (2007).
  56. Stadnicka-Michalak, J., Tanneberger, K., Schirmer, K., Ashauer, R. Measured and modeled toxicokinetics in cultured fish cells and application to in vitro-in vivo toxicity extrapolation. PLoS One. 9 (3), 92303 (2014).

Tags

Keywords: Cytotoxicity AssayZebrafish Cell LinesZEM2SZFL96-well PlateCell ViabilityAcute ToxicityEcotoxicityAlternative Fish AssaysHepatocytesEmbryonic CellsCell SuspensionMulti-channel PipetteTest ChemicalsTriton X-100Exposure Media
check_url/fr/64860?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
Rodrigues de Souza, I., Wilke Sivek, T., Vaz de Oliveira, J. B., Di Pietro Micali Canavez, A., de Albuquerque Vita, N., Cigaran Schuck, D., Rodrigues de Souza, I., Cestari, M. M., Lorencini, M., Leme, D. M. Cytotoxicity Assays with Zebrafish Cell Lines. J. Vis. Exp. (191), e64860, doi:10.3791/64860 (2023).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image