**2. Redox homeostasis and ROS generation**

The term "redox" refers to the oxidation-reduction status and is considered a key regulator of several metabolic cellular functions [9] and a fundamental keeper of cellular homeostasis [10].

During redox processes that occur in the cells are generated a variety of reactive oxygen species with functional roles in physiological and pathological conditions dependent on cell's capacity to maintain the ratio between ROS production and ROS disposal in balance. Commonly, the term "redox signaling" is used to express the changes of protein's oxidation status resulted in ROS-mediated events at cellular level [11].

The oxidative stress is characterized by a globally enhancement of intracellular ROS levels appeared from a dysfunction of the mechanisms involved in maintaining redox homeostasis: increased ROS generation or declined capacity of ROS elimination [10]. Mounting evidence suggest that oxidative stress is implicated in various pathologies such as aging, neurodege‐ nerative disorders, development of brain damage, pathogenesis of multiple sclerosis lesions and cancer [10, 12, 13].

ROS are highly reactive molecules, derived from oxygen, ceaselessly generated during oxidative metabolism and exuded into biological systems; outcomes of one or multielec‐ tron reductions of oxygen [7, 14]. The balance between ROS production and ROS dispos‐ al is ensured by the cell's keepers, antioxidant enzymes (superoxide dismutase – SOD, glutathione peroxidase, catalase and thioredoxin reductase) and non-enzymatic scaveng‐ ers (ascorbate, tocopherols, tocotrienols, carotenoids, natural flavonoids, melatonin, gluthatione, thioredoxin) [7, 14].

human body, including: gastrointestinal system, central and peripheral nervous systems, cardiovascular, renal and hematopoietic systems [2]. As regards the toxic mechanism of action of heavy metals, it has been stated that generation of reactive oxygen species represents one of the main mechanisms involved in heavy metals induced-toxicity. It is believed that gener‐ ation of reactive oxygen species is responsible for the hepatotoxicity, neurotoxicity and

Free radicals and reactive oxygen species generated by toxicants were described to hold key roles in lipid peroxidation, DNA damage, oxidation of sulfhydryl groups of proteins, depletion

Reactive oxygen species (ROS) are oxygen-free radicals that contain one or more unpaired electrons, formed during oxidative metabolism and were characterized as exceedingly active compounds which act by inducing oxidative changes of cellular proteins, lipids and polynu‐

Under normal conditions, ROS play essential functions in cellular homeostasis, as signal molecules in several signaling pathways involved in cell differentiation, organogenesis, stress response and wound healing, and as redox regulators [6]. Oxidative stress represents a status characterized by excessive cellular levels of ROS as a result of an imbalance in the redox homeostasis explained by increased production of ROS or declined antioxidant capacity [2, 6, 7]. A considerable number of studies endorse the fact that oxidative stress is linked to a plethora of pathologies including cardiovascular diseases, atherosclerosis, diabetes, chronic inflamma‐

This chapter summarizes an update of available data regarding ROS in physiological and pathophysiological conditions, the roles of ROS in cancer and heavy metals induced toxicity

The term "redox" refers to the oxidation-reduction status and is considered a key regulator of several metabolic cellular functions [9] and a fundamental keeper of cellular homeostasis [10]. During redox processes that occur in the cells are generated a variety of reactive oxygen species with functional roles in physiological and pathological conditions dependent on cell's capacity to maintain the ratio between ROS production and ROS disposal in balance. Commonly, the term "redox signaling" is used to express the changes of protein's oxidation status resulted in

The oxidative stress is characterized by a globally enhancement of intracellular ROS levels appeared from a dysfunction of the mechanisms involved in maintaining redox homeostasis: increased ROS generation or declined capacity of ROS elimination [10]. Mounting evidence suggest that oxidative stress is implicated in various pathologies such as aging, neurodege‐ nerative disorders, development of brain damage, pathogenesis of multiple sclerosis lesions

tory processes, neurodegenerative disorders, and mostly to cancer [6-8].

nephrotoxicity associated to heavy metals [2, 3].

4 Toxicology Studies - Cells, Drugs and Environment

cleotides [5-7].

via ROS generation.

and cancer [10, 12, 13].

of protein, and alteration of calcium homeostasis [2, 4].

**2. Redox homeostasis and ROS generation**

ROS-mediated events at cellular level [11].

ROS can be generated by multiple endogenous and exogenous sources. The main endogenous source of ROS is mitochondria, which produces reactive species as by-products of normal cell metabolism during the electron leakage that passes in the conversion of molecular oxygen process. Hereupon it might be added the activity of some enzymes, like: membrane-associated NADPH oxidases, cytochrome p450s, P-450-dependent monooxygenases, lipoxygenase, cyclooxygenase and xanthine oxidase [6, 13]. At mitochondrial level responsible for the formation of ROS are complexes I (NADH dehydrogenase (ubiquinone)) and II (succinate dehydrogenase), which produce ROS on the inner side of mitochondrial matrix, and complex III (ubiquinol-cytochrome c reductase) that delivers the generated species (superoxide radical) into the intermembrane space or mitochondrial matrix [14].

Other endogenous sources of ROS are the microsomes and peroxisomes (generate especially H2O2) and it was, also, demonstrated that immune cells' (neutrophils and macrophages) mechanism of action against invading microorganisms involves ROS [10]. The biosynthesis of prostaglandins, prostacyclins and thromboxane A2 from arachidonic acid, process catalyzed by cyclooxygenases, it is also a source of ROS [13, 15].

As exogenous sources of ROS, there were indicated the following agents: atmospheric pollutants, tobacco smoke, irradiation (UV irradiation, x-ray, gamma-ray), chemicals, iron salts, heavy metals and chemicals [2, 10, 14].

The group of reactive oxygen species comprises two different kinds of species:


Free radicals (also known as pro-oxidants) can be recognized by some specific features, like: high instability and reactivity, the presence of unpaired electrons in the outmost orbital of their atoms, the need to acquire equilibrium by bonding with electrons of neighboring atoms, what leads to chain reactions and the ability to react with different cellular molecules [16, 17].

Some of the main ROS species will be presented in Table 1, outlining the generation process and their specific characteristics.



**Table 1.** Description of the main ROS species.

**ROS name Generation Characteristics**

mitochondria formed during aerobic metabolism; there are two sites of mitochondrial ROS production, complex I (NADH – ubiquinone oxidoreductase) and complex III (ubiquinol - cytochrome c oxidoreductase)

*-* is described as the first free radical obtained during mitochondrial electron transfer chain process and it is easily converted to hydrogen peroxide via a dismutation reaction catalyzed

*-* it's a short-lived molecule and presents only

*-* it cannot be considered a candidate molecule for signal transduction in the cell since the ability of this radical to cross the mitochondrial

is the precursor of most ROS and a mediator in

*-* presents a two-electron reduction state [2] *-* is a mitochondrial ROS, a electrophobic molecule able to pass through membranes

*-* mitochondrial concentrations of hydrogen peroxide are 100 times greater than that of

*-* it can be regarded as a fundamental ROS in

*-* its conversion into oxygen and water is mediated by catalases and gluthatione

*-* is a strong oxidant, precursor of hydroxy radical (⋅OH) via Haber Weiss reaction [18]. *-* due to its lipophilic character lightly crosses mitochondrial and plasmatic membranes reaching into the cytosol and extracellular environment where asserts its effects [14]. *-* has the capacity to generate highly reactive hydroxyl radicals via reactions with metals (iron

oxidative chain reactions [19]

by superoxide dismutase (SOD) [6]

outer membrane is rather low [14] *-* at mitochondrial level it is involved in the generation of peroxynitrite (ONOO2 .-), a noxious oxidant that induces DNA damage, disruption of mitochondrial integrity, and irreversible modification of proteins [14] *-* releases Fe2+ from iron-sulfur proteins and

one reduction equivalent

ferritin [2]

H2O2 [2, 11].

superoxide anion [11].

carcinogenesis [18].

peroxidases [18].

and copper) [20, 27].

*- enzymatically:* it is produced is by NADPH oxidase (Nox) expressed in the phagocytes' cell membrane, by cytochrome P450-dependent oxygenases in the endoplasmic reticulum of the liver, lung and small intestine and by the xanthine oxidase (XO) located in

*- non-enzymatically*: the generation process consists in the transfer of a single electron to oxygen by coenzymes in reduced form, flavins or iron sulfur clusters or xenobiotics that suffered a reduction reaction [19, 23].



the cytosol [14, 16, 18, 19, 21, 22]..

*-* by direct reduction of O2 [2]

mitochondrial matrix) [11, 16, 24].

⋅−→ H2O2+ O2

2 O2

*- at mitochondrial level:* as a side-product of

[10, 18, 19] O2+ e- <sup>→</sup>O2 . [20]

6 Toxicology Studies - Cells, Drugs and Environment

**O2.-, superoxide**

**H2O2, hydrogen peroxide**

**anion**
