Research Brief In vitro evaluation of the effects of cysticidal drugs in the Taenia crassiceps cysticerci ORF strain using the fluorescent CellTracker CMFDA Hector Trejo-Chávez, David García-Vilchis, Olivia Reynoso-Ducoing, Javier R. Ambrosio * Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Edif. A, 2do. Piso, Ciudad Universitaria, C.P. 04510, Ciudad de México, Mexico article info Article history: Received 25 November 2009 Received in revised form 18 June 2010 Accepted 28 June 2010 Available online 3 July 2010 Keywords: Albendazole sulfoxide Anti-helminthics CMFDA GSH GSSG Praziquantel Taenia crassiceps Vital fluorescent staining abstract Using a murine model of cysticercosis caused by the Taenia crassiceps ORF strain, we developed a fluorescent quantitative evaluation of the action of two well known anti-helminthic drugs: albendazole sulfoxide and praziquantel. The fluorescence emitted by a biotransformed CellTracker Probe known as CellTracker Green CMFDA in the vesicular fluids of cysticerci was estimated, and the results were com- pared with macroscopic observations of the parasites. The pharmacological EC 50 value of each drug and changes in the level of biotransformation of the fluorescent tracker caused by the drugs could be eas- ily calculated. These drug-induced changes in biotransformation could be related to changes in the GSH/ GSSG ratio of parasites. Both the cysticercosis murine model and the CMFDA biotransformation assay could be used as an in vitro screening method to evaluate potential or well known cysticidal drugs. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction An in vitro assay can be a very useful tool for screening anti-par- asitic drugs and may have a variety of applications, including the identification of any potentially interesting new compounds. The success of an in vitro assay depends on a well adapted model and a screening method that does not require visual inspection and is not time-consuming nor expensive (Hemphill et al., 2009). The mur- ine metacestode model Taenia crassiceps ORF strain (Palomares- Alonso et al., 2007, 2009a; Palomares et al., 2004, 2006) has features that would allow it to be used to create a successful in vitro drug- screening assay. It has been considered useful for performing stud- ies applicable to the cysticerci of T. solium (Brehm and Spiliotis, 2008; Willms and Zurabian, 2009) and has been shown to be suit- able for establishing pharmacological parameters of the well known drugs with clinical use, such as albendazole, praziquantel and nita- zoxanide (Jung et al., 2008; Palomares-Alonso et al., 2009a; Palo- mares et al., 2004, 2006) or for the in vitro evaluation of experimental substances with potential cysticidal action (Palo- mares-Alonso et al., 2009a). Despite these features, there is no guar- antee that observed changes in the behavior or the morphology of cysticerci is directly related to its physiological or metabolic state. As a result, further evaluations are necessary. Thus, visual changes in parasites after an in vitro drug treatment are only semi-qualita- tive because they are based on the ability of the examiner and subtle changes in the organisms that must be analyzed at the ultrastruc- tural level (Hemphill et al., 2009; Palomares et al., 2004, 2006). The viability of cells can be evaluated using fluorescent dyes (Haugland, 2005), known as CellTracker Probes, that serve as indi- cators of the metabolic activity of cells or represent the physiolog- ical status of the organism. These fluorescent probes, such as the CellTracker Green 5-chloromethylfluorescein diacetate reagent (CMFDA), have been useful for developing a rapid and objective method for testing fungal susceptibly to drugs (Arunmozhi Balajee et al., 2005), for evaluating the glutathione cellular content in rat thymocytes treated with thimerosal (Ueha-Ishibashi, 2004) and for evaluating the behavior of the parasites during an in vivo ces- tode infection of copepods (Kurtz et al., 2002). There are several properties that account for the effectiveness of this vital fluores- cent probe: in its initial, non-fluorescent state, it can freely diffuse through the cellular plasma membrane; once inside the cell, a detoxification process through the glutathione metabolic pathway, which can only be executed by living cells, results in the conjuga- tion of glutathione to the compound, and the resulting product is an impermeable and brightly fluorescent probe (Haugland, 2005). One aim of this study was to quantitatively evaluate the biotransformation of CMFDA in a fluorescent CellTracker in T. crassiceps cysticerci grown in vitro and to determine, by quanti- tative fluorescent measurements, if this biotransformation can be 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.06.025 * Corresponding author. Fax: +52 5556232381. E-mail address: jrah@servidor.unam.mx (J.R. Ambrosio). Experimental Parasitology 127 (2011) 294–299 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr