**10. Glutathione transferases (GST) and their importance in detoxification**

From an evolutionary point of view, these enzymes emerged over two billion years ago. Based on structural and functional criteria, they can be grouped into four different families: cytoplasmic, microsomal, mitochondrial, and bacterial.

Glutathione transferases are ubiquitous in prokaryotes and eukaryotes, indicating their protective and functional importance. These transferases are a large superfamily of supergene isoenzymes that play important roles in cell detoxification. These enzymes use electrophiles to catalyze the nucleophilic addition of the thiol of reduced glutathione (l-g-glutamyl-l-cysteinyl-glycine) (GSH) to electrophilic centers in organic compounds. The resulting glutathione conjugates are rendered more water-soluble to facilitate their eventual elimination. A wide variety of endogenous (e.g., by-products of reactive oxygen species activity) and exogenous (e.g., polycyclic aromatic hydrocarbons) electrophilic substrates have been identified. In addition, the detoxification functions of these enzymes have been observed not only in one but two mechanisms: passive detoxification and active detoxification. The former, as mentioned by Kostaropoulos et al. [31], refers to a detoxification mechanism characterized by an absence of catalytic function, such as the binding of potentially toxic non-substrate ligands, including porphyrins and lipid peroxides. In fact, GSTs were originally named "ligandins" due to their passive role in detoxification.

Ligandin activity exhibited by GST isoforms was first suggested as a result of the observed affinity for bilirubin, an azo dye carcinogen, and a metabolite of cortisone. The second mechanism was developed by catalytic activity, as described previously **Table 1**.

Glutathione transferases in cestodes were identified several years ago. Initially, these cestode transferase isoforms were associated with the detoxified procedures in several organisms, including *C. elegans* [32, 33]. However, because almost all GSTs


*I50, is a parameter giving the inhibitory concentration causing 50% inhibition; Ki is the inhibition catalytic constant value; %F, is the % of Ts26GST intrinsic fluorescence quenching; ND, not determined. (Exp. Par. 2014;***138***:63-70).*

#### **Table 1.**

*Conditions for inhibition Ts26GST catalytic activity and spectrofluorometric assays.*

have GSH as a nucleophilic substrate, and this is the central redox agent of most aerobic organisms, GST functions encompass other purposes, as reported by Ferguson and Bridge in the *C. elegans* model [34].

### **11. The redox couples in enzyme functions**

As mentioned before, the reduced form of glutathione (GSH) serves as a ubiquitous nucleophile for the conversion of a variety of electrophilic substances under physiological conditions. This is possible when GSH is oxidized to glutathione disulfide (GSSG) by a reaction that involves the transfer of electrons between two species; in other words, when it is affected by the redox reaction.

GSH/GSSG is an example of millions of redox couples that are chemically similar or different, present in cells, organs, tissues, biological fluids, and cell organelles. A considerable number of these redox couples could be linked to each other to form a set of related redox couples, or redox couples that work independently. These reactions are achieved by capturing the energy released via oxidation to build cellular and organismic structures, maintain these structures (some avoid pathogenic action), and provide energy for the processes they support.

The production of a large number of redox couples in aerobic organisms occurs by enzymes and proteins of the glutaredoxin and thioredoxin systems, the former using GSH and the latter thioredoxin (Trx) [35].

### **12. The glutaredoxin and thioredoxin systems**

The glutaredoxin system is composed of glutathione reductase (GR or GSR), glutathione (GSH), and glutaredoxin (Grx), while the thioredoxin system comprises thioredoxin reductase (TrxR) and thioredoxin (Trx). The glutaredoxin and thioredoxin systems are likely to have evolved very early in aerobic organisms. Owing to the cysteine moiety of GSH, the entire system is based on common sulfur biochemistry. Therefore, it requires an electron relay, linking the universal reducing agent NADPH to thiol/disulfide metabolism, and a thiol-containing adapter molecule (GSH, which is considered as a universal adaptor) to transfer electrons to a set of different acceptors, such as flavoproteins, which are widely used as electron relays.

Hence, it is not surprising that the reducing equivalents from NADPH enter the glutathione system either with the help of the FAD-dependent enzyme glutathione reductase (GR) or the thioredoxin reductase/thioredoxin couple (TrxR/Trx).

Glutaredoxin protein (Grx) was first described in crude enzyme preparations from beef liver by Racker [36] in 1955. Grxs are small (12–18 kDa) GSH-disulfide oxidoreductase members of the thioredoxin family, which includes the cytosolic (Grx1) and mitochondrial (Grx2) isoforms. Oxidized Grxs are reduced by GSH. According to its active site domain, Grxs are classified as dithiols (CPY/FC motif) and monothiols (CGFS motif), wherein monothiols can contain single or multiple monothiol Grx domains. Dithiol Grxs regulate the redox state of various proteins by catalyzing the reversible reduction of oxidized disulfides. For this purpose, Grxs use both cysteine residues from their active sites. In contrast, the monothiol Grxs reduce mixtures of disulfides (glutathionylation) formed between GSH and the thiols of proteins or other small compounds, using the cysteine residues from the active sites in their amino terminals.

#### *Oxygen and Redox Reactions Contribute to the Protection of Free-Living and Parasite Helminths… DOI: http://dx.doi.org/10.5772/intechopen.102542*

With regard to the glutaredoxin genes of *C. elegans*, five have been annotated: glrx-3, glrx-5, glrx-10, glrx-21, and glrx-22 [37]. Except for glrx-5, which is predicted to be a mitochondrial glutaredoxin, the other annotated glutaredoxins are expected to be found in the cytosol. Based on phylogenetic analysis, the *C. elegans* GLRX-3 isoform has been postulated to be an ortholog of the mammalian GLRX3 protein kinase C-interacting cousin of thioredoxin (PICOT), suggesting that it exerts a protective mechanism against DNA-damage-inducing agents, such as some micro pathogens, by acting as an upstream positive regulator of ATR-dependent signaling pathways. On the other hand, a recent report demonstrated that *C. elegans* exhibits changes in the protein S-thiolation patterns (i.e., S-glutathionylation and S-cysteinylation) of targeted cysteine residues. This evidence suggests that glutaredoxins may provide an evolutionarily conserved mechanism for the catalysis of the reversal of S-glutathionylation, preventing the irreversible oxidation of protein thiols in *C. elegans* derived from micro pathogens.

Recently, the human and pig helminth parasite, *Taenia solium*, was cloned, expressed, and characterized for the first time as glutaredoxin (r-TsGrx1) [38]. The full-length DNA of the TsGrx1 gene comprised one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. Conventional enzymes, such as tioredoxin reductase (TrxR) and glutathione reductases (GR or GSR), do not exist in *Theridion solium*. However, the presence of a protein hybrid tioredoxin glutathione reductase (TGR), with tioredoxin and glutathione reductase activity, as described below, makes it possible for the components of Trx and Grx systems in platyhelminth (flatworms) to work as observed in organisms that have independent enzymes for those functions.

Protein S-glutathionylation by glutaredoxins is a widely distributed posttranslational modification of thiol groups with glutathione, which can function as a redoxsensitive switch to mediate redox regulation and signal transduction. Therefore, the presence of Grxs in *C. elegans* and *T. solium* contributes to our understanding of the micro pathogen activation of redox-regulatory processes in these helminths.

GR (also termed GSR, as mentioned before) is a flavoenzyme of the pyridine nucleotide-disulfide oxidoreductase family (EC 1.6.4.2, now 1.8.1.7). This enzyme recycles reduced GSH from its oxidized form GSSG. However, this function was also developed for the thioredoxin system acting as a backup, a trait that is conserved from bacteria to mammals, highlighting its physiological relevance, including protection against toxicity, in both systems.

Glutathione reductase is a GR-isoform from prokaryote and eukaryotes that form stable homodimers of ~110 kDa. From a structural point of view, each subunit is organized into four domains (FAD binding, NADPH binding, central, and interface) and possesses an N-terminal flexible segment of 18 amino acids with a cysteine residue at position 2.

In *C. elegans*, the gsr-1 gene encodes the GSR enzyme, which produces two protein isoforms (GSR-1a and GSR-1b) [39], and its expression of GSR-1 is modulated by the SKN-1 transcription factor.

The GSR-1 gene is vital in *C. elegans* because it supports its embryonic development, and there is no alternative molecule for this purpose. In many organisms, the thioredoxin system exerts GSSG reduction in the absence of GSR. As described previously, this appears not to be the case for *C. elegans*, even though both systems have been shown to cooperate in other processes, such as worm molting.

Therefore, the *C. elegans* thioredoxin and glutaredoxin systems share common functions but also have specific non-overlapping roles in worm physiology. The thioredoxin system was first recognized in the early 1960s as a reductant of methionine sulfoxide and PAPS (3 H-phos- phoadenosine-5 H-phosphosulfate) in yeast and ribonucleotides in *E. coli*.

Thioredoxin reductase (EC 1.6.4.5) (TrxR) was originally identified in *E. coli* as part of the ribonucleoside diphosphate reductase system. TrxR catalyzes the reversible electron transfer from NADPH to oxidized thioredoxin (thioredoxin-S2), a 12 kDa protein containing a single oxidation-reduction active disulfide bond. The oxidation of NADPH leads to the formation of the reduced form of thioredoxin (thioredoxin SH2), which has a dithiol. TrxR directly reduces not only Trx from different species but also many non-disulfide substrates, such as selenite lipid hydroperoxides, although not glutathione disulfide (GSSG).

The *C. elegans* genome encodes two thioredoxin reductases: thioredoxin reductase-1 (TRXR-1), which is the sole selenoprotein in *C. elegans*, with a UGA-encoded Sec in the C-terminal active site, and thioredoxin reductase (TRXR-2), a homolog of a UGU-encoded cysteine substitution for Sec. TRXR-1, the cytosolic TrxR, is required for the acidification of the lysosomal compartment in the intestine, whereas TRX-2, the mitochondrial TrxR, is critical for producing stress conditions. Interestingly, the gene expression of both TRX-1 and TRX-2 is induced by heat shock, which results in the production of ROS. Both observations suggest the involvement of these TrxRs in protection against micro pathogens found in the intestine of the worm [40].

Tioredoxin (Trx), the major TrxR substrate, as mentioned previously, is a disulfide reductase with a molecular weight of approximately 12 kDa and has two cysteine residues in its consensus sequence (CGPC motif). When chemically reduced, this allows for the transfer of reducing equivalents to a wide variety of substrates, such as H2O2. Thus, Trxs can, either directly or via 2-Cys peroxidases, catalyze the reduction of hydrogen peroxide (H2O2) to water and lipid hydroperoxides (R∙O∙O∙R) to alcohols in the cell. Trxs can also inhibit and/or activate transcription factors related to immune responses in mammals. For example, the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is inhibited when TRX1 prevents the release of IkB, an inhibitor of NF-κB.

Although the thioredoxin and glutaredoxin systems are vital for aerobic organisms, in platyhelminths (flatworms), both GR and TrxR are missing in their tissues. Instead of these proteins, some platyhelminths have a GR and TrxR molecular link exhibiting the fusion of glutaredoxin (Grx) and thioredoxin reductase (TrxR) domains into a single protein, a selenocysteine-containing enzyme that acts as a thioredoxin glutathione reductase (TGR) [41, 42].

Thus, TGR plays a central role in thiol-disulfide redox reactions by providing electrons to essential detoxification enzymes, such as GR and Prx. GR reduces the tripeptide GSSG to GSH, which acts as the main reducing agent in the catalytic functions displayed by GSTs [43].

Because conventional TrxR and GR are functional in *C. elegans*, no TGR is found in this worm. However, in platyhelminths, TGRs exert an efficient antioxidant defense against lipid peroxidation metabolites. In addition to the essential detoxification function of TGRs in flatworm parasites, as described above, it is necessary to include the TGR activities associated with the Grx domain, such as the deglutathionylase activity of GSH-protein mixed disulfides (protein-S-SG). Protein glutathionylation is the mechanism by which protein-SH groups form mixed disulfides with glutathione to

#### *Oxygen and Redox Reactions Contribute to the Protection of Free-Living and Parasite Helminths… DOI: http://dx.doi.org/10.5772/intechopen.102542*

avoid protein-SH group oxidation. In addition, TGRs play essential roles in redox cell signaling and sensing. Cell signaling transduction is the mechanism by which external stimuli are transferred to their inner compartments, resulting in the activation or inhibition of genes; cell sensing is the oxidative modification of protein cysteines with consequent events, such as changes in its activities and interactions with other biomacromolecules, such as native immunity receptors.

*Haemonchus contortus* has been used as an excellent model to gain insights into the oxidative defenses of hosts against nematodes [44]. *H. contortus* is a blood-sucking nematode parasite of the abomasum of small ruminants that causes a disease known as haemonchosis, in which the abomasal epithelium and highly toxic heme molecules are released [45]. Free heme catalyzes the formation of cytotoxic lipid peroxides via lipid peroxidation using hydrogen peroxide (H2O2) as Fenton reaction.

#### **Figure 3.**

*Kinetic evidences of TR and GR activity from HcTrxR3. (A) Reduction of ebselen by NADPH catalyzed by HcTrxR3 produced ebselen diselenide and ebselen selenol. To 1 ml solutions containing 50 mM Tris-Cl, 1 mM EDTA, pH 7.5, 100 mM NADPH, and 0.1 mM ebselen, 2 μg (*▪*) or 4 μg (*•*) HcTrxR3, was added, and A340 was measured against a blank without ebselen (*∆*). Ebselen reduction was shown when absorbance decreased followed by ebselen selenol formation in the highest enzyme concentration. (B) Effect of NADP<sup>+</sup> on the glutathione reductase activities of HcTrxR3. IC50 plots were obtained; an enzyme aliquot (about 2 μg) was pre-incubated at 25°C in the presence of 100 μM NADPH and different concentrations of NADP<sup>+</sup> . To start the reaction GSSG at a final concentration of 0.2 mM was added. (C) Show a competitive type inhibition where the 1/v versus 1/[NADPH<sup>+</sup> ] plot of initial velocities HcTrxR3 activity in absence (*◆*) and the presence of 0.1 mM (*•*) and 0.5 mM (*▪*) of NADP<sup>+</sup> with various concentrations of NADPH<sup>+</sup> (0.01–10 μM). Inset shows secondary plot of the slope values derived from the primary 1/v versus 1/[NADPH<sup>+</sup> ] plot versus NADP<sup>+</sup> concentration for the determination of Ki [47].*

The range of antioxidant enzyme systems available to *H. contortus* for the detoxification of H2O2 has been investigated using molecular biology tools. As a result, full-length sequences were obtained for a 2-Cys peroxiredoxin (Prx), a catalase, and a selenium-independent glutathione peroxidase (GPx), indicating that *H. contortus* expresses several antioxidant systems with the potential to detoxify peroxide, most of them within the host's immune response. Other studies identified additional three thioredoxin reductases (TrxRs) (HcTrxR-1, HcTrxR-2, and HcTrxR-3), two mitochondrial thioredoxins (HcTrx-1, HcTrx-5), and one cytosolic (Trx-3) thioredoxin (Trx), increasing the possible mechanisms of *H. contortus* detoxification. All the abovementioned detoxification enzymes and proteins, except catalase, work closely with the two major detoxification and redox systems in animal cells: thioredoxin (Trx) and glutathione (GSH) [46].

Interestingly, for the first time in the study of the TGR system [47], HcTrxR3 was found to catalyze the direct reduction of GSSG, the specific substrate for GR, in the same catalytic range as that of any GR. Its affinity for GSSG, measured as Km value, was higher than that of the 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) substrate for TrxR, demonstrating its preference for the GSSG substrate. Until now, no TrxR has been identified that is able to directly reduce GSSG.

This GR activity from HcTrxR3 is important not only because the enzyme is a TrxR, but also because information on the presence of GR in the *H. contortus* tissues is lacking thus far **Figure 3**.

### **13. The models of detoxified and innate immunity in pioneer earthworms**

In addition to being essential for soil fertility, earthworms are also an excellent model for the study of the protection mechanisms used by helminths against micro pathogens [48], as in *C. elegans*.

Earthworms are terrestrial invertebrates belonging to the order Oligochaeta, class Chaetopoda, and phylum Annelidae. They range in size from a fraction of a centimeter to exceptional individuals of Megascolides australis, which can measure up to 2.75 m in length and 3 cm in diameter. Approximately 1800 species are distributed all over the world.

Earthworms became a model for comparative immunology in the early 1960s with the publication of results from transplantation experiments that proved the existence of self/non-self-recognition in earthworms. This initiated extensive studies on the immune mechanisms of earthworms, which evolved to prevent invasion by pathogens. In recent decades, important cellular and humoral pathways have been discovered, and numerous biologically active compounds have been characterized and cloned [49].

For example, earthworm coelomocytes (macrophage-like cells) are part of the cellular immune response and are both morphologically and functionally analogous to vertebrate phagocytes. Coelomocyte subpopulations (named as hyaline-, granular amoebocytes, and eleocytes) possess distinct functions, such as phagocytosis, encapsulation, and cellular cytotoxicity.

Additionally, phagocytic defense by the earthworm *Eisenia foetida* against certain pathogenic bacteria has been found to be aided by bacteriostatic or bactericidal substances, which may also play an opsonic role, as in vertebrates. Therefore, given the molecular tools of earthworm coelomocytes, there is a possibility that these organisms also use a functional NOX/DUOX system to eliminate invading micro pathogens via ROS production, as in *C. elegans*.

*Oxygen and Redox Reactions Contribute to the Protection of Free-Living and Parasite Helminths… DOI: http://dx.doi.org/10.5772/intechopen.102542*
