**5.4 In vitro assessment of the reproductive fitness of adult worms**

The effects of natural or synthetic products on the reproductive fitness of *S. mansoni* have been previously reported in several studies (Braguine et al., 2009; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Mohamed et al., 2005; Moraes et al., 2011; Sanderson et al., 2002). To evaluate drug effects on schistosome during *in vitro* screening drug assays, cultures are continually monitored to assess the sexual fitness of worms treated with sublethal concentrations of drug. In this case, the following parameters are assessed: (1) changes in the pairing, an indicator of the mating process; (2) egg production, an indicator of egg output per worm; and (3) egg development.

In the experiments, adult worm pairs (male and female coupled) are incubated in a 24-well culture plate, as previously described here, and parasites are monitored on daily basis for 5 days using an inverted microscope and a stereomicroscope (Figure 2). Therefore, it is important that, after collection by the perfusion technique, the parasites are carefully washed to prevent the separation of the worm pairs.

Schistosome egg output *in vitro* is usually determined by counting the number of eggs. Egg development can be analysed quantitatively and scored as developed or undeveloped on the basis of the presence or absence of the miracidium (de Melo et al., 2011; Magalhães et al., 2009, 2010). This is a simple and recommended method because conventional light microscopy is able to distinguish morphologic differences in eggs. However, the characterisation of the viability of immature eggs is very difficult. Alternatively, the analysis of egg viability, distinguishing live immature eggs from dead immature ones, can be performed using a fluorescent label, as described by Sarvel et al. (2006). In this assay, the eggs obtained in culture are stained with the Hoescht 33258 probe and observed with fluorescent microscopy. The authors evaluated fluorescent labels and vital dyes, aiming at differentiating live and dead eggs, and showed the only the fluorescent Hoechst 33258 can be considered a useful tool to differentiate between dead and live eggs.

### **5.5 Cytotoxicity assays**

348 Current Topics in Tropical Medicine

Fig. 3. Dorsal region of a *Schistosoma mansoni* adult male worm, on which the effect of antischistosomal compounds on the tegument is evaluated quantitatively. The parasite was fixed in FAA solution, and fluorescent images were obtained using a confocal microscope. A: General view of the anterior helminth region showing, in red, the location where tubercles were counted. Bar = 500 µm. B: View of an area of 20,000 µm2, calculated with the

Zeiss LSM Image Browser software, showing the tubercles. This image is a higher

sucker; Vs: ventral sucker; Tu: tubercles

magnification of the dorsal region of the *S. mansoni* adult worm marked in red in panel A. X and Y: three-dimensional images obtained from laser scanning confocal microscopy. Os: oral Finding a new compound capable of killing a parasite is not difficult. However, it is difficult to find a substance that can kill the parasite without affecting the host. Therefore, early *in vitro* studies of new compounds must include comparative cytotoxicity data from human or animal cells in tissue culture to establish that the compound has selective antischistosomal activity and may be a realistic prospect for future clinical use in humans. In our operating procedures for antischistosomal drug screening, mammalian cells are exposed to concentrations of at least two times higher than what is needed to elicit a schistosomicidal effect. Thus, the *in vitro* schistosomicidal activity of compounds cannot be associated with cytotoxic effects.

General toxicity tests can be conducted in many cell types (e.g., fibroblasts and epithelial and hepatoma cells). Peripheral blood mononuclear cells and erythrocytes are widely used in *in vitro* studies to detect cytotoxicity or cell viability following exposure to antischistosomal compounds. Vero mammalian cells (African green monkey kidney broblasts) are also commonly used to examine whether natural or synthetic antiparasitic compounds are tolerated by mammalian cells (da Silva Filho et al., 2009; Moraes et al., 2011; Parreira et al., 2010).

The crystal violet staining method and the neutral red and MTT assays are the most common methodologies used to detect cytotoxicity or cell viability following exposure to toxic substances. In our *in vitro* cytotoxicity assays with cultured cells, the crystal violet

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 351

Discovering untapped natural sources of new anthelmintic compounds remains a major challenge and a source of novelty in the era of combinatorial chemistry and genomics. To find new anthelmintics, all sources of natural, synthetic and semi-synthetic lead compounds must be investigated. *In vitro* bioassays using parasitic worms have played a central role in the early pre-clinical stages of most research on potential natural anthelmintics. The identification of the antiplasmodial and antischistosomal activity of the sesquiterpene lactone artemisinin has stimulated interest in natural products, and soon, promising leads will be identified with new chemical types and active agents against schistosomiasis. Therefore, bioprospecting programmes related to the isolation of bioactive compounds must be rewarded, and the screening *in vitro* of chemical constituents belonging to different

The literature regarding antischistosomal compounds contains a large number of natural products screened for their schistosomicidal properties. However, only a few of these may be promising drug leads in the development of a therapeutic reserve for schistosomiasis. Therefore, it is important to continue to identify new drugs and to explore alternative strategies to improve screening efficacy. Most of the extracts or natural compounds were only evaluated with *in vitro* studies; it is expected that they will be evaluated using *in vivo* experimental models. Further, it must be mentioned that the results of *in vitro* assays with many drugs do not correspond to what is observed *in vivo*; however, *in vitro* screening could identify novel anthelmintics that could eventually translate into practical applications. Thus, while *in vitro* tests are recommended initially, the assessment of therapeutic activity using *in* 

The analysis of the *S. mansoni* genome and transcriptome offers great possibilities for identifying possible new drug targets and will facilitate further exploration of differences between host and parasite metabolic pathways. In addition to the isolation and structural determination of new drugs from natural products and information from the originating plant, the integration of the pharmacological properties of natural products with the functional genomic and proteomic studies in schistosome and *in vitro* screening methods with improved automatic high-content screening will be important tools to identify possible new drugs in the future and shed light on the approaches of helminth chemotherapy. Attempting new combinations of natural or synthetic drugs will be also important in

I thank Dr. Eliana Nakano and Mr. Alexsander S. Souza (Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brazil) for assistance with confocal microscopy. I also thank Ms. Edinéia C. Moraes for drawing the lifecycle diagram and Ms. Aline A. L.

Abdul-Ghani, R.A.; Loutfy, N. & Hassan, A. (2009). Myrrh and trematodoses in Egypt: an

Abdulla, M.H.; Ruelas, D.S.; Wolff, B.; Snedecor, J.; Lim, K.C.; Xu, F.; Renslo, A.R.; Williams,

overview of safety, efficacy and effectiveness profiles. *Parasitology International*,

J.; Mckerrow, J.H. & Caffrey, CR. (2009). Drug discovery for schistosomiasis: hit

classes must be evaluated on the blood fluke *S. mansoni.*

discovering alternative drugs to replace the use of praziquantel.

*vivo* models should be performed.

**7. Acknowledgments** 

**8. References** 

Carvalho for comments and suggestions.

Vol.58, pp. 210-214, ISSN 1383-5769

staining method is routinely used because it is rapid and inexpensive. This method measures the effects of compounds on cell growth through the colorimetric evaluation of fixed cells stained with crystal violet. Briefly, cells maintained in culture medium are seeded into 96-well culture microplates in the presence of different concentrations of extracts, fractions or isolated compounds. After different timepoints of incubation (e.g., 2, 24, 48, 72, and 96 h), the supernatants are removed and the remaining live cells are assessed by xing and staining them with crystal violet (0.2% in 20% methanol). Viable cells attach to the bottom of the well plate, and the absorbance is measured by reading each well at 595 nm in a microplate reader (Moraes et al., 2011).

Neutral red is another cell viability assay often used to determine cytotoxicity following exposure to toxic substances (Borenfreund & Puerner, 1985). It has been used as an indicator of cytotoxicity in cultures of primary hepatocytes and other cell lines (Fautz et al., 1991; (Fotakis & Timbrell, 2006; Morgan et al., 1991). Living cells take up the neutral red, which is concentrated within the lysosomes of cells.

The MTT assay is also used to measure cell viability. MTT is a water-soluble tetrazolium salt, which is converted to an insoluble purple formazan upon the cleavage of the tetrazolium ring by succinate dehydrogenase within the mitochondria. The formazan product is impermeable to the cell membranes, and therefore, it accumulates in healthy cells. The MTT assay has been tested for its validity in various cell lines (Fotakis & Timbrell, 2006; Mossmann, 1983).

Alternatively, the lactate dehydrogenase (LDH) leakage assay and protein assay are also used to detect cytotoxicity, despite the fact that they have low sensitivity when compared to the methods already described. The LDH leakage assay is based on the measurement of LDH activity in the extracellular medium. The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage (Decker & Lohmann-Matthes, 1988; Fotakis & Timbrell, 2006). The protein assay is an indirect measurement of cell viability because it measures the protein content of viable cells. Despite the existence of several protocols to establish total protein concentration (e.g., biuret, bicinchoninic acid, Lowry and Bradford protocols), the two most commonly used methods for protein quantification are the Lowry and Bradford assays (Bradford et al., 1976; Lowry et al., 1951).

Finally, tritiated thymidine-based methods, which act through the incorporation of tritium into the DNA of cells, have also been used currently to detect cytotoxicity, especially in immune cells (Pechhold et al., 2002).
