**4. ROS in pathophysiological conditions**

Pan and coworkers stated that ROS are involved in the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and act as inducers of oxidative damage

**Figure 1.** ROS cellular functions in physiological conditions (the picture was obtained by using Servier Medical Art

Most of the free radicals generated by various cellular metabolic systems originate from oxygen. The percent of molecular oxygen that is converted into superoxide and hydroxyl radicals at mitochondrial level is 5%. Mounting evidence indicates that the free radicals resulted have major function in the normal metabolism of cells: they are used in the synthesis of prostaglandins, cholesterol and steroidal hormones. Furthermore, the biosynthesis of

The effects and functions of ROS are distinct and dependent of their concentration, for example: low concentrations of mitochondrial ROS are associated with metabolic adapta‐ tion in hypoxic conditions; moderate concentrations are involved in the regulation of inflammatory response and high levels stimulate apoptosis/autophagy pathways responsi‐

collagen demands the participation of hydroxyl free radicals [16].

templates).

ble of inducing cell death [11].

to macromolecules (nucleic acids, proteins, and lipids) (see Figure 1) [29].

8 Toxicology Studies - Cells, Drugs and Environment

The phenomenon characterized by an impaired balance between ROS generation and ROS elimination is known as oxidative stress. The oxidative damage was frequently associated with different pathologies including: diabetes type II, neurodegenerative disease, atherosclerosis, multiple sclerosis, chronic inflammatory disease, aging and carcinogenesis (see Figure 2). The ability of free radicals to chemically react with most of cell components it was considered a risk, especially for large molecules (nucleic acids, proteins, polymerized carbohydrates polysaccharides), and lipids, since these components are targets for the oxygenated free radicals [16,20].

The free radical induced-toxicity at cellular level is expressed as lipid and protein peroxidation and damaged nucleic acids (see Figure 2) [20]. The hydroxyl radical is the most reactive free radical molecule capable to cause severe cell damage and to other intracellular structures due to its ability to induce covalent cross-linking of a variety of biological molecules [20].

Superoxide anion is a reactive oxygen molecule that plays important roles in the body because is considered the precursor of the other free radicals that determine cell injury. The toxic mechanism of action of this free radical involves the disassembly of iron-sulphur ([Fe–S]) clusters in proteins via the inactivation of iron regulatory protein-1 (IRP-1), leading to clearing of iron and damage of –SH residues [20,34].

The noxious effects of ROS might be exerted at: DNA level, also known as DNA oxidation what leads to mutations and possibly cancer [20,33]; at protein level causing enzyme inhibition, denaturation and protein degradation, and at lipid level leading to lipid peroxidation [20]. ROS-induced DNA peroxidation inhibits gene transcription and determines gene mutations. The main toxic products resulted from ROS-induced DNA damage are 8-hydroxyadenine (8- OH-Ade), 8-hydroxyguanine (8-OH-Gua), 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, Tg) [19].

The lipid peroxidation process is a chemical chain reaction that consists of three stages: initiation, propagation and termination [10,20]. During the initiation stage occur the following processes: a ROS (usually a hydroxyl radical due to its high reactivity) reacts with a peroxide-free lipid system in order to remove a hydrogen (H) atom from a methyl‐ ene group (-CH2-), the result being the generation of free radicals such as conjugated dienes and peroxyl radical [10,20].

**Figure 2.** ROS cellular functions in pathophysiological conditions (the picture was obtained by using Servier Medical Art templates).

The propagation stage is characterized by the attack of the peroxyl radical resulted during the initiation stage to other lipid molecules (fatty acids) via an autocatalytic chain reaction catalyzed by metals such as iron or copper and it results a lipid hydroperoxide (or peroxide) [10,20]. Polyunsaturated fatty acids are considered easy targets for lipid peroxidation due to the unsaturated chemical structure. The ROS-induced peroxidation end products (α, β unsaturated reactive aldehydes, such as malondialdehyde - MDA, 4-hydroxy-2-nonenal - HNE, acrolein and isoprostanes) have also deleterious effects on tissues (see Figure 3) [10,20,35].

The injury caused by ROS to membrane phospholipids can affect in a cascade manner the membrane integrity, followed by altered membrane integrity, suppression of enzymes and membrane receptors, an increased tissue permeability, an impaired cellular function and cell death [10,20,29].

The end-products of lipid peroxidation possess a high reactivity and can easily interact with proteins, phospholipids and nucleic acids via different reactions resulting stable products (adduct between proteins and lipid peroxides or glycation products) with potential roles in the pathogenesis of several maladies [20, 36]. The stable products mediate cell death by aggregation of bulky protein complexes which inhibit the activity of 26S and 20S proteosome and determine accumulation of injured proteins (see Figure 3) [10].

**Figure 3.** ROS-induced lipid peroxidation (the picture was obtained by using Servier Medical Art templates).

The propagation stage is characterized by the attack of the peroxyl radical resulted during the initiation stage to other lipid molecules (fatty acids) via an autocatalytic chain reaction catalyzed by metals such as iron or copper and it results a lipid hydroperoxide (or peroxide) [10,20]. Polyunsaturated fatty acids are considered easy targets for lipid peroxidation due to the unsaturated chemical structure. The ROS-induced peroxidation end products (α, β unsaturated reactive aldehydes, such as malondialdehyde - MDA, 4-hydroxy-2-nonenal - HNE, acrolein and isoprostanes) have also deleterious effects on tissues (see Figure 3)

**Figure 2.** ROS cellular functions in pathophysiological conditions (the picture was obtained by using Servier Medical

The injury caused by ROS to membrane phospholipids can affect in a cascade manner the membrane integrity, followed by altered membrane integrity, suppression of enzymes and membrane receptors, an increased tissue permeability, an impaired cellular function and cell

The end-products of lipid peroxidation possess a high reactivity and can easily interact with proteins, phospholipids and nucleic acids via different reactions resulting stable products (adduct between proteins and lipid peroxides or glycation products) with potential roles in the pathogenesis of several maladies [20, 36]. The stable products mediate cell death by

[10,20,35].

Art templates).

10 Toxicology Studies - Cells, Drugs and Environment

death [10,20,29].

Recent studies demonstrated that ROS is involved in the mitochondrial energetically dysfunction associated to ethanol-induced gastric mucosa damage [29,37]. It was stated that impairment of mitochondria and/or up-regulation of NADPH-oxidase complex are linked to various cancers and play major function in establishing the anticancer therapeutic strategies [14].

In the last years, the subject regarding the roles of ROS in cancer gained a lot of interest. The cellular injury initiated by ROS is considered an important step in cancer development. It seems like the signal transduction messenger function of ROS is associated with cancer initiation by activation of different signaling pathways like MAPK, PI3K and NF-κB [7].

The mechanism of action of ROS in ageing is still a matter of debate. There are studies that sustain the fact that elevated ROS levels along with mitochondrial dysfunction and metabolic alterations are features of cellular senescence. Other studies have proposed the hypothesis that ROS is cause and consequence of NF-κB pathway activation during senescence [38].
