**2.1. Exogenous NOx: a hostile intruder inside the eukaryotic cells**

Much work was already performed on NOx targets at the cell scale. NO and NO2 , thanks to their chemistry properties mentioned earlier, are highly diffusible through living membranes and exhibit a strong reactivity. It is easy to imagine the potential of such compounds. Since NO is highly reactive in lipophilic media, it can react inside the lipid bilayer, first protection of cells against the environment. Indeed, polyunsaturated fatty acids (PUFAs) are susceptible to NO in the presence of O2 and ONOO<sup>−</sup> [17]. The interaction of these toxic compounds with PUFA double bounds leads to the formation of nitrated FA. This process is considered as a protective strategy because it redirects O2 − and ONOO<sup>−</sup> -mediated cytotoxic reactions to other oxidative pathways. Post-translational modification of several proteins is also observed in the presence of NOx and RNS. For example, tyrosine nitration on phenol residue mediated by peroxynitrite derived from OH and NO2 leads to 3-nitrotyrosine formation [18]. This molecule represents a useful biomarker of nitrosative stress in various pathologies such as atherosclerosis [19]. Protein thiol residues are highly susceptible to NO and ONOO− leading to the formation of nitrosothiol (RS-NO) and nitrothiol (RS-ONO), respectively [20]. This reversible modification modulates the activity of several proteins similar to phosphorylation. For example, S-nitrosylated cysteine thiol residue can be denitrosylated by S-nitrosoglutathione reductase or thioredoxin systems [21]. NO and other RNS can also nitrosylate [Fe─S] cluster of transition metal centers, which are essential for protein function. This modification can activate a wide range of enzymes such as the soluble guanylate cyclase (sGC) mentioned below [22]. On the contrary, this reaction can also alter the protein function, for example, in the case of hemoglobin [23]. NO and other higher RNS products (ONOO<sup>−</sup> or NO2 ) are able to generate nitration, nitrosation, or deamination reactions on DNA bases leading to mutagenesis [24]. This phenomenon is enhanced by the inhibition of DNA repair triggered by NO [25]. More recently, a role of NO in epigenetic modification was suggested. To be more precise, this simple molecule seems to modulate histone acetylation and methylation through direct and indirect modulations of histone acetyltransferases and deacetylases, lysine demethylases, histone methyltransferases activity, thus modifying the expression of several genes [26]. These pleiotropic activities of NO and derived compounds highlight here the necessity of fine regulation pathways to prevent the development of several diseases.

### **2.2. Endogenous NO: an essential mediator of cellular signalization**

**2. The double face of NOx in the eukaryotic world**

24 Emerging Pollutants - Some Strategies for the Quality Preservation of Our Environment

according to [1]. ppm, parts per million; ND, not determined.

forming each part of our body: the eukaryotic cells themselves.

to NO in the presence of O2

according to [9]; (C) NO<sup>2</sup>

protective strategy because it redirects O2

by peroxynitrite derived from OH and NO2

**2.1. Exogenous NOx: a hostile intruder inside the eukaryotic cells**

and ONOO<sup>−</sup>

To better understand why NOx impact on so many organs, it is essential to focus on the units

**Figure 6.** Key features concerning NOx. (A) Lewis structure of NOx and conversion factors; (B) NO threshold (ppm)

threshold (ppm) according to [9]; (D) and (E) NO and NO<sup>2</sup>

their chemistry properties mentioned earlier, are highly diffusible through living membranes and exhibit a strong reactivity. It is easy to imagine the potential of such compounds. Since NO is highly reactive in lipophilic media, it can react inside the lipid bilayer, first protection of cells against the environment. Indeed, polyunsaturated fatty acids (PUFAs) are susceptible

PUFA double bounds leads to the formation of nitrated FA. This process is considered as a

oxidative pathways. Post-translational modification of several proteins is also observed in the presence of NOx and RNS. For example, tyrosine nitration on phenol residue mediated

ecule represents a useful biomarker of nitrosative stress in various pathologies such as ath-

the formation of nitrosothiol (RS-NO) and nitrothiol (RS-ONO), respectively [20]. This reversible modification modulates the activity of several proteins similar to phosphorylation. For example, S-nitrosylated cysteine thiol residue can be denitrosylated by S-nitrosoglutathione reductase or thioredoxin systems [21]. NO and other RNS can also nitrosylate [Fe─S] cluster of transition metal centers, which are essential for protein function. This modification can activate a wide range of enzymes such as the soluble guanylate cyclase (sGC) mentioned below

and ONOO<sup>−</sup>

[17]. The interaction of these toxic compounds with

leads to 3-nitrotyrosine formation [18]. This mol-


, thanks to

guideline values, respectively,

leading to

Much work was already performed on NOx targets at the cell scale. NO and NO2

−

erosclerosis [19]. Protein thiol residues are highly susceptible to NO and ONOO−

Interestingly, NO can also be produced by several living organisms: plants, animals, and bacteria, thanks to a specific enzyme called nitric oxide synthase (NOS) [27, 28]. The large distribution of this enzyme through the different reigns emphasizes the importance of NO synthesis. Evolutionary studies highlight the necessity for the first living organisms during primitive era to eradicate toxic O3 present in the paleoatmosphere as a survival strategy. Indeed, the liberation of gaseous NO in extracellular environment could have subsequently neutralized O3 , thus limiting harmful oxidative reactions [29]. In eukaryotic cells, three isoforms of NOS have been described [30]. The neuronal nNOS (NOS-1) and the endothelial eNOS (NOS-3) are constitutively expressed but are only activated through calcium-dependent mechanisms. The third one is the inducible iNOS (NOS-2) expressed in macrophages following infection by pathogens, virus, or tumors. Contrary to NOS-1 and 3, NOS-2 is constitutively functional. Interestingly, these NOSs have high structural similarities with an oxygenase and a reductase domain (**Figure 7**) [27].

To be fully functional, NOS requires to associate with homodimers, and this form of NOS is crucial for the generation of NO [32]. When conditions are favorable (high level of l-arginine

**Figure 7.** The structural similarities of NOS (adapted from [31]). BH4 , tetrahydrobiopterin; l-arg, l-arginine-binding site; Heme Fe, iron protoporphyrin IX; CaM, calmodulin-binding site; FMN, flavin mononucleotide-binding site; FAD, flavin adenine dinucleotide-binding site; NADPH, nicotinamide adenine dinucleotide phosphate-binding site; \*, palmitoylation and myristoylation of eNOS oxygenase domain.

and cofactors like BH4 ), this enzyme catalyzes the conversion of l-arginine into l-citrulline using O2 and leading to NO formation with a ratio of 1:1 [33]. However, if these parameters are not available or if NOS remains monomeric, the enzyme only produces superoxide (O2 − ) [32]. Even if NOS isoforms catalyze the same reaction, their distribution is largely related to their respective functions.

#### *2.2.1. The endothelial eNOS and vasodilatation*

Endothelial nitric oxide synthase (eNOS) can be activated through calcium-dependent and independent pathways. On the one hand, through activation of specific receptors, such as acetylcholine muscarinic receptors, bradykinin receptors, and H1 histamine receptors, distributed in the endothelial cell membrane, agonists can trigger an increase in intracellular concentration of calcium (Ca2+) through the well-known polyphophoinosides pathway. Indeed, activation of those receptors stimulates membrane-associated PLCϒ and PI3K activation. This results into inositol trisphosphate (IP3) formation, which induces the release of intracellular calcium stock from endoplasmic reticulum [34]. Then, Ca2+ binds to calmodulin (CaM), which could later fix on the calmodulin-binding domain of eNOS controlling its enzymatic activity. On the other hand, in response to hormones or growth factors acting on their corresponding receptors, phosphate kinase A (PKA) or B (AKT) pathway can be induced mediating a phosphorylation cascade. The post-translational phosphorylation of eNOS on three specific sites (Ser617 and Ser1179 for AKt, Ser635 and Ser1177 for PKA) enhances the activity of the enzyme [35]. Moreover, the lipidation of eNOS (palmitoylation and myristoylation) could also enhance its activity [36]. This other post-translational modification promotes eNOS association with cell membrane and is stabilized by interaction with membrane chaperone proteins (caveolin-1 and HSP70/90). Both mechanisms are essential for linking upstream signal transduction pathway to eNOS activity in cells [37]. eNOS could then produce a large amount of NO, which latter diffuses freely inside smooth muscles. Herein, NO activates soluble guanylate cyclase (sGC) by reaction with the heme of the enzyme leading to the increase of cyclic guanosine monophosphate level (cGMP). cGMP subsequently activates phosphate kinase G (PKG) favoring a cytosolic Ca2+ reuptake into sarcoplasmic reticulum [38]. The decrease of intracellular level of Ca2+ leads to the relaxation of smooth muscle. Moreover, NO can inhibit caspases 3 and 8 and thus apoptosis through protein nitrosylation. NO released by endothelium is also reducing platelet aggregation and platelet or leukocyte adhesion (**Figure 8**) [39, 40].

*2.2.2. The neuronal nNOS: a crucial element in neurotransmission*

caveolin-1; NOSIP, NOS-interacting protein; NOSTRIN, NOS traffic inducer.

*2.2.2.1. nNOSα in the central nervous system*

muscle where NO controls muscle contractility and local blood flow.

nNOS, also called NOS-1, was the first isoform discovered in the neuronal tissue. It should be distinguished between two subfamilies: nNOSα and nNOSμ, thanks to their locations and functions. Indeed, nNOSα is found in central nervous system and plays a major role in the neurotransmission at neuronal synapses, whereas nNOSμ is found in skeletal and cardiac

**Figure 8.** Physiology of vasodilatation: implication of eNOS and NO (adapted from [41]). eNOS, endothelial nitric oxide synthase; ach, acetylcholine; BK, bradykinin; H, histamine; R, receptor; CaM, calmodulin; PLCϒ, phospholipase C ϒ; PI3K, phosphoinositide 3-kinase; PKA, phosphate kinase A; AKT, phosphate kinase B (PKB); sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate; PKG, phosphate kinase G; A, protein complex of HSP70, HSP90, and

The Hidden Face of Nitrogen Oxides Species: From Toxic Effects to Potential Cure?

http://dx.doi.org/10.5772/intechopen.75822

27

Glutamate is the major excitatory neurotransmitter in the brain. When deliver into the synapse, this molecule is activating a specific receptor (NMDAR) located inside the postsynaptic membrane. Activated NMDAR thus allows the entry of calcium. Ca2+ are able to activate calmodulin by physical interaction [44]. Afterward calmodulin binds to nNOSα, which, in turn, is activated. Interestingly, nNOSα can indirectly interact with the NMDAR through its fixation on an adapter protein PSD95 by their PDZ interaction domain [45]. This allows the high speed of NO synthesis in response to NMDAR activation. Thereafter, NO can diffuse back to the presynaptic area leading to an increase glutamate release in the synaptic cleft and thus neurotransmission. CAPON, a chaperon protein of DexRas, was found altering the level

Another source of NO inside endothelial cells was also described. Indeed, nitrite and nitrate reservoirs could be converted into NO by several enzymes such as cytochrome P450, hemoglobin, myoglobin, and others, under specific conditions [42]. However, several pathways could abolish NO production. Indeed, NO can be eliminated in combination with reactive oxygen species (ROS). eNOS could be inactivated by several phosphatases. The removal of eNOS membrane sequestration by interaction between NOS-interacting protein (NOSIP) and NOS-traffic inducer (NOSTRIN) complex favors eNOS recycling [36]. Since NO plays a central role in this phenomenon, any abnormality altering its production leads to pathogenesis and vascular disorders such as atherosclerosis and hypertension [43].

The Hidden Face of Nitrogen Oxides Species: From Toxic Effects to Potential Cure? http://dx.doi.org/10.5772/intechopen.75822 27

**Figure 8.** Physiology of vasodilatation: implication of eNOS and NO (adapted from [41]). eNOS, endothelial nitric oxide synthase; ach, acetylcholine; BK, bradykinin; H, histamine; R, receptor; CaM, calmodulin; PLCϒ, phospholipase C ϒ; PI3K, phosphoinositide 3-kinase; PKA, phosphate kinase A; AKT, phosphate kinase B (PKB); sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate; PKG, phosphate kinase G; A, protein complex of HSP70, HSP90, and caveolin-1; NOSIP, NOS-interacting protein; NOSTRIN, NOS traffic inducer.
