**2. Redox molecules as metabolic regulators**

It was thought that reactive oxygen species (ROS) were damaging molecules that were associated with the main pathological consequences of oxidative stress. However, recently, numerous reports have demonstrated that ROS and reactive nitrogen species (RNS) also play important roles in signaling pathways. In this way, the nowadays perspective of prooxidant reactions in the cellular milieu is that they form part of the physiological response to internal and environmental regulatory factors [8]. Indeed, the liver being one of the major metabolic organs, the signaling consequences of ROS and RNS are very relevant in the hepatic tissue.

It is well known that mitochondrial activity is the major source for ROS production, with con‐ sequences in the oxidative phosphorylation coupling affecting the Δ*Ψ* (mitochondrial mem‐ brane potential) as well as several mitochondrial metabolic networks. Eventually, important processes such as cell proliferation and apoptosis can be triggered by the mitochondrial pro‐ oxidant condition [9].

During the electron transfer through the mitochondrial respiratory complexes, O2 is eventually reduced to water by receiving two electrons, along the creation of a [H+ ] gradient that makes possible the formation of ATP. However, some electrons "leak" without completing the path‐ way until the cytochrome oxidase complex is achieved. In this case, one electron is received by an oxygen molecule forming the anion superoxide (O2 **−** ). O2 **−** is produced in the interphase between sites I and II, as well in site III of the electron transfer chain. Within the mitochon‐ dria, O2 **−** is transformed to hydrogen peroxide (H2 O2 ) by mitochondrial superoxide dismutase (SOD2); when it reaches the cytosolic compartment, the O2 **−** is turned into H2 O2 by SOD1. In cytosol, the mitochondrial O2 **−** and H2 O2 are incorporated to the ROS pool generated mainly by the activity of the family of NADPH oxidases to potentially act as the signaling molecules [8].

H2 O2 is itself a metabolic regulator, but at high levels can act as a deleterious factor. H2 O2 can be converted into H2 O by several antioxidant enzymes: H2 O2 can be reduced by peroxiredoxins or glutathione peroxidases, which couple reduction of H2 O2 with oxidation of glutathione (GSH). Oxidized peroxiredoxins can be reduced by thioredoxins. Subsequently, oxidized thioredoxins become reduced by thioredoxin reductase in a NADPH‐dependent manner. Oxidized gluta‐ thione disulfide (GSSG) is reduced by glutathione reductase again in the presence of NADPH.

H2 O2 acts as a signaling factor by oxidizing thiol groups (‐SH HS‐) into disulfide bridges (‐S‐S‐) in cysteine residues of key regulatory proteins (enzymes and receptors). Several years ago, some reports postulated that H2 O2 showed insulin‐like properties in hepatocytes. This observation was eventually confirmed in the yeast *Saccharomyses cerevisiae* and supported the notion, fully accepted nowadays, that H2 O2 could be acting as a signaling molecule in many cellular systems [8]. **Figure 2** shows the important role that mitochondria play in the prooxi‐ dant reactions of many cellular functions.

#### **2.1. Cellular proliferation**

reactions are preponderant in mitochondria and peroxisomes whereas reductive reactions are more common in cytosol), (2) available coenzymes (oxidized nicotinamide adenine dinucleo‐

dinucleotide phosphate (NADPH) for anabolism), (3) adenine nucleotides pool (low energy charge, AMP‐activated protein kinase (AMPK) activation for catabolism and high energy

It was thought that reactive oxygen species (ROS) were damaging molecules that were associated with the main pathological consequences of oxidative stress. However, recently, numerous reports have demonstrated that ROS and reactive nitrogen species (RNS) also play important roles in signaling pathways. In this way, the nowadays perspective of prooxidant reactions in the cellular milieu is that they form part of the physiological response to internal and environmental regulatory factors [8]. Indeed, the liver being one of the major metabolic organs, the signaling consequences of ROS and RNS are very relevant in the hepatic tissue. It is well known that mitochondrial activity is the major source for ROS production, with con‐ sequences in the oxidative phosphorylation coupling affecting the Δ*Ψ* (mitochondrial mem‐ brane potential) as well as several mitochondrial metabolic networks. Eventually, important processes such as cell proliferation and apoptosis can be triggered by the mitochondrial pro‐

During the electron transfer through the mitochondrial respiratory complexes, O2

possible the formation of ATP. However, some electrons "leak" without completing the path‐ way until the cytochrome oxidase complex is achieved. In this case, one electron is received

between sites I and II, as well in site III of the electron transfer chain. Within the mitochon‐

the activity of the family of NADPH oxidases to potentially act as the signaling molecules [8].

Oxidized peroxiredoxins can be reduced by thioredoxins. Subsequently, oxidized thioredoxins become reduced by thioredoxin reductase in a NADPH‐dependent manner. Oxidized gluta‐ thione disulfide (GSSG) is reduced by glutathione reductase again in the presence of NADPH.

 acts as a signaling factor by oxidizing thiol groups (‐SH HS‐) into disulfide bridges (‐S‐S‐) in cysteine residues of key regulatory proteins (enzymes and receptors). Several years

is itself a metabolic regulator, but at high levels can act as a deleterious factor. H2

O2

**−** ). O2 **−**

**−**

O2

O2

reduced to water by receiving two electrons, along the creation of a [H+

O2

O2

O by several antioxidant enzymes: H2

by an oxygen molecule forming the anion superoxide (O2

is transformed to hydrogen peroxide (H2

(SOD2); when it reaches the cytosolic compartment, the O2

**−** and H2

glutathione peroxidases, which couple reduction of H2

dized nicotinamide adenine dinucleotide phosphate (NADP+

charge, reduced AMPK activity for anabolism) [7].

**2. Redox molecules as metabolic regulators**

), reduced nicotinamide adenine dinucleotide (NADH) for catabolism and oxi‐

), reduced nicotinamide adenine

is eventually

by SOD1. In

O2

can be

] gradient that makes

is produced in the interphase

O2

) by mitochondrial superoxide dismutase

can be reduced by peroxiredoxins or

with oxidation of glutathione (GSH).

is turned into H2

are incorporated to the ROS pool generated mainly by

showed insulin‐like properties in hepatocytes. This

tide (NAD+

158 Redox - Principles and Advanced Applications

oxidant condition [9].

cytosol, the mitochondrial O2

ago, some reports postulated that H2

converted into H2

dria, O2 **−**

H2 O2

H2 O2 Another system of ROS modulation well characterized in the liver is the control of growth fac‐ tors by the redox regulation of cysteine residues in tyrosine phosphatases. H2 O2 downregulates cyclin D1 and cyclin E to inhibit proliferation and upregulates Bcl‐2‐associated X protein (BAX) to induce apoptosis in hepatocytes and MCF‐7 cells (cell line from cancerous mammary gland) [10].

Another example is the modulation of redox‐sensitive cysteine residues in the epidermal growth factor (EGF) receptor, which is activated by the action of H2 O2 .

Other protein factors such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF) have been identified as redox regulators of this process.

In response to hypoxic conditions, the hypoxia‐inducible transcription factors (HIFs) are upregulated by ROS, especially the ones from mitochondrial source. Paradoxically, in liver

**Figure 2.** Mitochondrial handling of reactive oxygen species (ROS). Pro‐oxidant reactions (synthesis of O2 − and H2 O2 ) are upregulated by a variety of physiological or pathological events. Mitochondrial activation promotes responses such as cell proliferation and activation, as well as cancerous processes.

and vascular smooth muscle cells, in hypoxic condition there is an increase in mitochondrial ROS production from complex III; however, the exact mechanism is not well understood.

In liver and muscle cells, angiotensin II signaling also promotes higher production of mito‐ chondrial ROS, which are necessary to activate downstream responses such as mitogen‐acti‐ vated protein kinases (MAPK) [11].

#### **2.2. Immune system**

When an organism is infected primordial T‐cells rapidly proliferate, and differentiate into effector T‐cells, and mitochondria contribute to this activation through the production of ROS. This has been analyzed adding antioxidants to mice after a viral infection and showing that they exhibited a depressed immune system. Data suggest that ROS play an important role in T‐cell activation, proliferation and adaptive immune function [12].

#### **2.3. Cancer**

A fundamental characteristic of cancerous cells is their uncontrolled proliferation. It has been observed that they generate high levels of ROS, especially H2 O2 ; in addition, elevated levels of antioxidants have been detected, maybe to implement a protective cellular response. The final equilibrium allows a high rate of ROS synthesis concomitant with a transformed functional phenotype.

ROS are responsible of oncogenes activation and/or loss of tumor suppressors enhancing mitogenic signaling.

Mutations in mitochondrial DNA result in ROS increase that has been associated to a great variety of human cancers. One example is the mutation in the gene of NADH dehydrogenase in the mitochondrial complex I, which promotes an elevation of ROS production. The pro‐ oxidant stimulus leads to the proliferation of several human and mouse cell lines, as well as tumor formation in rodents. Interestingly, this condition could be rescued by the reconstitu‐ tion of the wild‐type enzymatic activity [13].
