3.3.1. Immune Response and Oxidative Stress in Parasitic Flatworms Sources of Exposure to Reactive Oxygen Species

During establishment of the infection, the parasites induce a rapid immune response in the host, although it is nonspecific [125]. In general, this involves the activation of eosinophils, neutrophils, and macrophages, as well as the release of cytokines and the production of antibodies (IgE) [126]. These cells can produce large amounts of ROS and reactive nitrogen species (RNS), capable of directly destroying parasite cells. For example, liver flukes, such as *Opisthorchis viverrini*, induce chronic inflammation of the hepatobiliary system, exposing themselves to large amounts of ROS/RNS released by activated inflammatory cells [127]. Similarly, when an infection by the cestode *Taenia hydatigena* occurs in the peritoneum, an increase in the infiltration of small peritoneal macrophages responsible for a high production of nitric oxide (NO) can be observed, which harms the parasite and

modulates the immune response [128]. However, the presence of immune cells induced by parasitic flatworms may be due to their participation in other processes such as wound repair caused by the migration of *F. hepatica* through the liver parenchyma [129].

The production of ROS/RNS due to the immune response has already been discussed in detail previously [130]. In general, the precursor of all ROS is the superoxide anion radical (O2 •−), which is generated in leukocytes through the integral membrane enzyme NADPH oxidase (NOX), and by transferring an electron from NADPH to O2. O2 •− can undergo a spontaneous dismutation reaction generating hydrogen peroxide (H2O2) and O2. H2O2 can serve as a substrate for the enzyme myeloperoxidase (MPO) to generate the microbicidal compound hypochlorous acid (HClO). In the presence of transition elements such as ferrous (Fe2+) or copper (Cu+) ions, H2O2 can be reduced by the Fenton reaction, which produces the hydroxyl anion (HO−) and the hydroxyl radical (HO•). The HO• radical is highly reactive, so it can subtract electrons from other biomolecules, like proteins, changing their properties and biological activities, with DNA generating mutations and membrane lipids initiating the lipid peroxidation process. This damage can lead to altered metabolism and eventually cell death (Figure 5).

**Figure 5.** The antioxidant system of parasitic flatworms. *Abbreviations:* GSH reduced glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; HO•, hydroxyl radical; O2, molecular oxygen; O2 •− superoxide anion radical; Ph-GPx, glutathione phospholipid peroxidase; Prx, peroxiredoxin; SOD, superoxide dismutase; TGR, thioredoxin-glutathione reductase; Trx-(SH)2, reduced thioredoxin; Trx-(S)2, oxidized thioredoxin.

It is important to clarify that another source of ROS is the metabolism of the parasites themselves, possibly due to their accelerated metabolism and the use of the malate dismutation pathway to obtain energy. It is in this pathway where the mitochondrial complex I continues to work, becoming an important place of ROS generation [131]. In mitochondria isolated from the tegument of *T. crassiceps*, a high production of H2O2 was recorded, unlike that observed in rat liver mitochondria. The high production of H2O2 associated with tegumental aerobic mitochondria has been observed with confocal microscopy in the cysticercus of *T. crassiceps* [94].

An important characteristic of these ROS and RNS is that they are short-lived intermediate products enzymatically synthesized by aerobic organisms and their clearance

is regulated by enzymatic or non-enzymatic antioxidants. In this sense, there is a major co-evolutionary arms race competition between ROS production by the host and ROS scavenger by parasites; both closely related. For example, the production of H2O2 by hemocytes of the snail *Biomphalaria glabrata* when infected with *S. mansoni* sporocysts varies from population to population and in those snails with a high natural resistance against *S. mansoni* a higher production of H2O2 is observed, being preferentially infected by sporocysts with high levels of expression of their antioxidant systems [132].

Functioning and Localization of Enzymatic Antioxidant Systems in Parasitic Flatworms
