## 2 The Role of Phytochemical in Oxidative Stress
### Phytochemicals as Pro-Oxidant Agents: Friend or Foe?
Regarding chemical instability, many polyphenols are structurally unstable and can undergo enzymatic or spontaneous oxidation in the presence of metal ions, particularly in cell cultures, to form ROS [29; 70; 71]. For instance, it has been shown that epigallocatechin gallate (EGCG) produces significant amounts of H\({}_{2}\)O\({}_{2}\) in cell cultures [72]. Likewise, the cytotoxicity of green tea and red wine in PC12 rat phaechromocytoma cells, when grown in [PERSON]'s Modified Eagle's Medium (DMEM), can be attributed to H\({}_{2}\)O\({}_{2}\) produced from these drinks, at least partially [73]. The same holds true for phytochemicals, including polyphenols that can become pro-oxidants, even if this has not yet been clearly elucidated. However, transition metal ions are known to be present in cell culture media. An example is DMEM, which contains  
Figure 3: Glutathione (GSH); glutathione \(S\)-transferases (GSTs); glutathione peroxidase (GPX), superoxide dismutase (SOD); catalase (CAT); nuclear factor-erythroid 2-related factor 2 (Nfr2); antioxidant responsive elements (ARE); kelch-like ECH-associated protein-1 (Keap1); nuclear factor-kappaB B (NF-\(\kappa\)B); \(\mathrm{I}\kappa\) kinases (IKK); CREB-binding protein (CBP). inorganic iron, usually Fe(NO\({}_{3}\))\({}_{3}\), which gives it greater pro-oxidant properties. Also, most of the cultured cells are kept in high O\({}_{2}\) conditions (95% air and 5% CO\({}_{2}\)) and low ascorbate, vitamin E and selenium concentrations, which can lead to artefact results [29]. Through in vitro studies, several food polyphenols have been shown to determine cellular GSH depletion, which could contribute to apoptosis activation in tumor cells. For example, [PERSON] and [PERSON] [74] have shown that the flavones, cristine, and apigenin, deplete GSH in cell lines, while hydroxycarbonate and dihydroxyalcone perform this action more effectively in tumor prostate PC-3 cells. These authors found that the enhancement effects of these different flavones are related to mitochondrial dysfunction through the depletion of mitochondrial GSH levels, a decrease in mitochondrial membrane potential, and the increase of cytochrome C release [74]. In this context, isothicyanates are conjugated with GSH by glutathione \(S\)-transferases (GSTs) [75,76]. GPX, which is involved in hydrogen peroxide removal, consumes GSH (Figure 3). On the other hand, it has been suggested that some polyphenols have a pro-oxidant activity, both in vitro and in vivo, which certainly contributes to their antioxidant and anti-cancer effects [29], through inducing antioxidant enzymes, such as SOD, which catalyzes the one-electron dismutation of superoxides into hydrogen peroxide and oxygen, and CAT, allowing two-electron dismutation into oxygen and water (Figure 3). It is well-known that cysteine reduced form (SH) redox status has a primary role in the nuclear factor-erythroid 2-related factor 2 (Nfr2)/ARE pathway (Figure 3). Under physiological conditions, Nfr2 is bound to the kelch-like ECH-associated protein-1 (Keap1) and, thereby, sequestered in the cytoplasm, whereas, when under conditions of oxidative stress, Nfr2 dissociates from Keap1, translocating to the nucleus and inducing antioxidant enzyme transcription. The cysteine residues on Keap1, which are ultrasensitive to electrophiles, are critically important for Nfr2 binding. Antioxidants with catechol and electrophilic moieties induce Nfr2-mediated gene expression in antioxidant enzymes acting as pro-oxidants rather than antioxidants [77]. This effect is well-known for green tea catechins [78], but also other bioactive phytochemicals, like triterpenoids, caffeic acid, and isothicyanates (the breakdown products of glucosinolates), which can induce the Nfr2 pathway, acting on cysteine residues of Keap1 [75,76,79,80,81]. As observed for Nfr2, ROS and some cysteine residues are involved in NF-kB translocation to the nucleus (Figure 3). In particular, cysteine 179 of Ik kinases (IKK) is a target of the S-glutathionylation-induced inactivation [82].