(3) Glutathione-S-Transferase and Other Antioxidant Molecules

Glutathione-S-transferase (GST) is a family of highly conserved detoxifying enzymes that participate in the metabolism of many xenobiotics, although, in mammals, it has been found that it can participate in other relevant physiological processes such as the synthesis of leukotrienes, prostaglandins, and steroid hormones, as well as in amino acid catabolism and modulation of signaling processes [168]. GST can conjugate a wide range of substrates of an electrophilic nature, like the glutathione thiolate anion (GSH), to generate glutathionylated compounds, which are less reactive and more soluble and can be eliminated more efficiently by the cell. The enzyme can also detoxify by non-covalently binding to a series of hydrophobic ligands [169]. In the case of parasitic flatworms, it has been proposed that GSTs are essential for survival because they eliminate toxic and xenobiotic compounds derived endogenously or exogenously (generated or administered by the host) [170].

There are three families of GSTs with distinctive structural characteristics and different evolutionary origins: (a) cytosolic GSTs, (b) microsomal GSTs or also called MAPEG (Membrane associated proteins involved in eicosanoid and glutathione metabolism), and (c) kappa-class mitochondrial GSTs [171]. However, cytosolic GSTs have been the most studied, identifying up to seven different classes in mammals: alpha, mu, pi, sigma, theta, zeta, and omega [172]. Parkinson et al. reported the presence of two sigma-type cytosolic GSTs in the larval form of *E. granulosus*; one mu class and one microsomal GST [83]. However, an in-depth analysis of the *C. sinensis* trematode genome suggests the presence of 12 cytosolic GSTs distributed in the mu, sigma, zeta, and omega classes; in addition to mitochondrial GST and microsomal GST [173]. This agrees with findings in cestode genomes, where an important presence of 10 genes for cytosolic GSTs of the mu class have been reported, in addition to two GST genes of the sigma class and one of the MAPEG class [92]. Interestingly, Nguyen et al. reported the presence of a new type of cytosolic sigma GST in the metacestode of *T. solium* (TsMsGST), specifically expressed in the cytosol of the scolex tegument and susceptible to praziquantel (a drug used against neurocysticercosis, and which does not normally interact with sigma GSTs) [170]. Finally, Iriarte et al. analyzed the genomic information available from several flatworm representatives and discovered the potential absence of omega-class GST in cestodes, contrary to observations in other species of *Schistosoma* as the planarian *Schmidtea mediterranea* [174].

Regarding their location and expression in parasitic flatworms, GSTs present complex patterns and depend on both the species and the stage of the life cycle in which they are found. For example, Mei and Loverde reported that, regardless of the parasite stage, when analyzing the transcript levels of different antioxidant enzymes in *S. mansoni*, GST transcripts were 100-times more abundant than GPx transcripts and 10-times more than SOD isoforms. However, when enzymes were localized by immunofluorescence in the adult fluke, GST isoforms were restricted to a reduced subpopulation of parenchymal cells as well as to immature germ cells in both males and females. In contrast, SOD and GPX isoforms were localized in the tegument [137].

Another example is observed in the mRNA expression of the omega class GSTs of *C. sinensis* (CsGSTo 1 and 2). It begins with a growing pattern in juveniles of two to four weeks of age, but there is no expression in the metacercaria form and, in contrast, they are overexpressed in eggs [175]. This was contrasted with immunodetection techniques, locating CsGSTo in the egg, vitelline follicles, seminal receptacles, and testes. Because the expression of CsGSTo remains at high levels, regardless of environmental stimuli, the authors propose that the expression of these GSTs is conditioned by sexual reproduction within the host and that the abundance of CsGSTo in the egg is a preparation for the hostile conditions that the parasite will face when expelled from the definitive host.

Other Detoxifying Proteins That Have Been Reported in Parasitic Flatworms:


from *C. sinensis* (CsMb) showed peroxidase activity and that it may be important for ROS detoxification because of its overexpression after incubation with exogenous H2O2 [185]. This was later corroborated by Kim et al., who showed that incubation of *C. sinensis* flukes under aerobic conditions or in the presence of nitric oxide or nitrite is sufficient to induce the expression of the gene encoding CsMb [72]. Interestingly, overexpression of CsMb was also observed when flukes were co-incubated with human cholangiocytes (bile epithelial cells).


The different antioxidant enzymes do not work in isolation because to successfully face oxidative challenges, all systems must work together and simultaneously to avoid, as much as possible, the generation of highly toxic ROS such as the HO• radical. The latter maintains the peroxidases functioning by regenerating their electron source and repairing the damage caused during oxidative stress. In addition to this, there may be other factors that influence the antioxidant response, such as:

