**4. Biochimical and morphological features to define cell death in parasite trichomonas**

#### **4.1 Caspases pathway**

Caspases are essential proteins involved in cellular death that exist in cytosol of most cells in its inactive form as a polypeptide. They are, activated by cleavage, and apoptosis is considered a consequence of their activation cascade [39].

In some cases caspases can induce cellular death and in others they seem to be irrelevant in decision between death and life. In both situations, caspases participate in morphology of apoptosis [39]. According to Mariante et al. [4], caspases dependent cell death in trichomonads can occur through different known mechanisms, either by death receptors pathway, or through unknown signaling pathways, like the release of hydrogenosomes molecules, which may have analogous functions of mitochondrial proteins.

Although it is stablished that the apoptosis regulated by caspases are an important form of PCD, in many instances, PCD is caspase independent and nonapoptotic. Necrosis-like might or might not require caspases to activate cell death, while paraptosis and autophagic/vacuolar cell death traditionally do not call for the participation of caspases [10, 40]. Despite, some studies in mammalian cells indicate that caspases can regulate both apoptotic and nonapoptotic cell death, as autophagy [41].

In eukaryotic organisms, it is known that the caspases possess a fundamental role during the process of cellular death, especially on apoptosis, being the primary site of interaction with Bcl-2 proteins family. However, despite of the lack of mitochondria in trichomonads, Mariante et al. [42] confirm the participation of proteases of the Caspase-3 family in *T. foetus* cellular death, after treatment with H2O2. They showed that hydrogen peroxide may have degenerative effects. Studies suggest that progressive destabilization of the membranes of intracellular organelles, which would be caused for short-term exposure to low concentrations of H2O2 may induce lysosomal rupture indirectly by activation of phospholipases [43]. PDT treatment also leads to the release of lysosomes enzymes in *T. foetus* citoplasm, which by the increase of reactive oxygen species (ROS) activate procaspases that in turn active caspases inducing the occurrence of the cell death process [44].

#### **4.2 Nuclear abnormalities**

Trichomonads treated with different concentrations of H2O2 showed severe nuclear changes like unusual DNA condensation. Peripheral heterochromatin masses and nuclear DNA fragmentation can be observed in the nucleus of some cells, probably due to the activation of different endonucleases. One the other hand, in mammals the same treatment lead a different nuclear organization [42]. Nuclear changes was observed in *T. foetus* after treatment with H2O2 and griseofulvine, which may be related to the presence of activated molecules through caspases pathway, and/or, molecules released from hydrogenosomes [5]. After treatment of *T. foetus* with AlPcS4, cellular disorder such as nucleus fragmentation was observed in several cells [6]. *T. foetus* PDT treatment associated with the same photosensitizer (AlPcS4) showed a "ladder pattern" of DNA fragments of *T. foetus* in electrophoresis assay which can be a hallmark of apoptosis. Comet Assay testes confirmed DNA fragmentation in this condition as longer tails was observed (unpublished data). However, taxol and nocodazole induced the formation of multinucleated cells, abnormal distribution of the nuclear contents and, sometimes, nuclear fragmentation [22]. In recent studies utilizing tetracycline, it was observed DNA fragmentation in *T. vaginalis* [25].

*Cell Death after Photodynamic Therapy Treatment in Unicellular Protozoan Parasite… DOI: http://dx.doi.org/10.5772/intechopen.94140*

## **4.3 Morphology cell changes**

*T. foetus* has a simple life cycle that consists of only a trophozoitic form, which is characterized by a pear-shaped (PS) body, three anterior flagella and one recurrent flagellum [24, 45]. The trophozoite undergoes profound morphological alterations and takes on an endoflagellar form (EFF), also known as pseudocyst, under unfavorable environmental conditions, such as abrupt changes in temperature or in the presence of drugs, e.g. colchicine [22]. In *T. foetus* treated with AlPcS4, the internalized flagella and fragmented axostyle-pelta complex was seen, whereas changes in the elongated shape were not observed after 24 h. Although, after 48 h of treatment was observed changes in the shape of parasites. The same changes were observed in *T. foetus* after treatment with PDT [6]. However, after treatment with H2O2, the parasites exhibit a change in their morphology, changing from elongated shape to spherical one [42]. After treatment with taxol, nocodazole or colchicine, a large number of cellular changes and drastic effects were observed such as the loss of the cells original shape, the flagella were gradually internalized and the cells assumed the pseudocyst form. During treatment with taxol cell size was increased, and giant, multinucleate and abnormal cells were observed, as well as the presence of zoids and membrane blebbing formation [22]. In *T. vaginalis* tetracycline-treatment the cell exhibited a round shape, a not disrupted plasma membrane, and a rough cell surface [25]. However, the parasites treated with mepoxomicin and bortezomib showed the appearance of wrinkled or rounded cells with externalized flagella, membrane blebbing, cell lysis, intense cytosolic and nuclear vacuolization, cytoplasmic disintegration and abnormal Golgi reduction [24]. The flagella were internalized after treatment of *T. foetus* with thiabendazole, and about 70% of the parasites were observed as giant form. Furthermore, after treatment with mebendazole, more than 90% of these parasites presented drastically altered morphologies [23]. The EFF occurs in living cells able to undertake nuclear division to form multinucleated cells and able to provoke damage to host cell. It is a reversible form of parasite and can have the ecto-phosphatase activities responsible to signaling this process [45, 46].
