**1. NOx species: the genesis of a major air pollutant**

"Yet, pollution is the largest environmental cause of disease and death in the world today, responsible for an estimated 9 million premature deaths" stated The Lancet Commission in 2017. This phenomenon represents an alarming threat for human health, as a major cause of respiratory and cardiovascular pathologies as well as infertility. Moreover, those atmospheric pollutants have severe impacts on the environment and participate in climatic change,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

acidification, eutrophication, and ecosystem disturbances. Several international and national organizations (e.g. WHO, EEA, and INERIS) aim at reducing the global emission of pollutants, thanks to environmental policies as Kyoto and Gothenburg protocols signed in 1997 and 1999, respectively. After that, an encouraging decrease in air pollutants' levels was measured between 2000 and 2015 [1].

Atmospheric pollutants can be classified into four families: classical, indoor, and organic or inorganic air pollutants. Among the classical ones, which are the principle in amount, sulfur dioxide (SO2 ), particle matter, ozone (O<sup>3</sup> ), and nitrogen oxide (NOx) species are found [2]. The term NOx refers to a wide range of nitrogen-derived compounds, where nitric oxide (NO) and nitrogen dioxide (NO2 ) are predominant [3]. Those compounds can be naturally produced at low level by lightnings [4] and volcanic eruptions [5]. However, NOx is mainly generated by anthropogenic activity (e.g., road transport, energy production, industry, and agriculture) (**Figure 1**) [6].

Even if NO and NO2 represent the main species of NOx, nitrogen exists in several oxidation states in the environment, from N (−III) to N (+V). NO takes a central place in the series of reactive nitrogen species (RNS) [7]. Oxidation and reduction of NO result in the formation of several RNS, including nitrate or ammonium. NO2 is formed by the reaction of NO with O2 and can be dimerized to give N2 O4 . The formed reactive species can, in turn, be involved in a wide range of reactions (**Figure 2**).

Nitrogen is an essential constituent of vital macromolecules (nucleic acids and proteins). This atom also constitutes the dinitrogen (N2 ) gas, usually abbreviated nitrogen. This gas represents 78% of atmospheric air and is continuously recycled in our environment. First, the atmospheric nitrogen is fixed in the soil by prokaryotes to form ammonia (NH<sup>3</sup> ), which could be taken up by plants. NH3 is then converted into ammonium (NH4 + ). Ammonium could be also

> formed by mineralization of organic matter by decomposers (fungi, worms, and prokaryotes). The second part of the nitrogen cycle is the nitrification of ammonium by prokaryotes leading

> **Figure 2.** Redox relationships of NOx with other RNS. Black arrow: oxidation and reduction of NO; purple arrow: a wide range of reactions; roman numbers: oxidation state of the nitrogen atom; color gradient: reduction/oxidation state

> Moreover, nitrogen is also the precursor of NOx. Indeed, NOx formation can be separated into four steps. First, NO is formed in the atmosphere in combination with nitrogen, resulting from the global biogeochemical nitrogen cycle, and oxygen, following combustion (natural or

> Alternatively, nitrogen dioxide can also be directly formed through catalytic ammonia combustion or nitrosyl chloride oxidation. Then, a temperature-dependent equilibrium is estab-

> ing their diverse behavior and reactivity. For example, the solubility of NO is very low

and hydroxyl radicals leads to the generation of ozone (O3

are classified as RNS, their characteristics are very different suggest-

−

O4 −

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

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21

). Finally, nitrate

) to form NO2

) (**Figure 4**).

.

by denitrifying bacteria

). Finally, a photochemical

−

) formation, which is then further oxidized into nitrate (NO3

could be assimilated by plants for their growth or converted into N2

anthropic). Then, this highly reactive compound can be oxidized by oxygen (O2

and its dimeric form, nitrogen tetroxide (N2

to nitrite (NO2

from (−III) to (V).

lishing between NO2

reaction between NO2

Even if NO and NO2

(**Figure 3**).

−

**Figure 1.** NOx sources linked to anthropogenic activities (according to [1]).

The Hidden Face of Nitrogen Oxides Species: From Toxic Effects to Potential Cure? http://dx.doi.org/10.5772/intechopen.75822 21

acidification, eutrophication, and ecosystem disturbances. Several international and national organizations (e.g. WHO, EEA, and INERIS) aim at reducing the global emission of pollutants, thanks to environmental policies as Kyoto and Gothenburg protocols signed in 1997 and 1999, respectively. After that, an encouraging decrease in air pollutants' levels was measured

Atmospheric pollutants can be classified into four families: classical, indoor, and organic or inorganic air pollutants. Among the classical ones, which are the principle in amount, sulfur

The term NOx refers to a wide range of nitrogen-derived compounds, where nitric oxide

produced at low level by lightnings [4] and volcanic eruptions [5]. However, NOx is mainly generated by anthropogenic activity (e.g., road transport, energy production, industry, and

states in the environment, from N (−III) to N (+V). NO takes a central place in the series of reactive nitrogen species (RNS) [7]. Oxidation and reduction of NO result in the formation of

Nitrogen is an essential constituent of vital macromolecules (nucleic acids and proteins). This

sents 78% of atmospheric air and is continuously recycled in our environment. First, the atmo-

is then converted into ammonium (NH4

), and nitrogen oxide (NOx) species are found [2].

is formed by the reaction of NO with O2

), which could be

). Ammonium could be also

) are predominant [3]. Those compounds can be naturally

. The formed reactive species can, in turn, be involved in a

) gas, usually abbreviated nitrogen. This gas repre-

+

represent the main species of NOx, nitrogen exists in several oxidation

between 2000 and 2015 [1].

(NO) and nitrogen dioxide (NO2

and can be dimerized to give N2

taken up by plants. NH3

wide range of reactions (**Figure 2**).

atom also constitutes the dinitrogen (N2

agriculture) (**Figure 1**) [6].

Even if NO and NO2

), particle matter, ozone (O<sup>3</sup>

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

several RNS, including nitrate or ammonium. NO2

**Figure 1.** NOx sources linked to anthropogenic activities (according to [1]).

O4

spheric nitrogen is fixed in the soil by prokaryotes to form ammonia (NH<sup>3</sup>

dioxide (SO2

**Figure 2.** Redox relationships of NOx with other RNS. Black arrow: oxidation and reduction of NO; purple arrow: a wide range of reactions; roman numbers: oxidation state of the nitrogen atom; color gradient: reduction/oxidation state from (−III) to (V).

formed by mineralization of organic matter by decomposers (fungi, worms, and prokaryotes). The second part of the nitrogen cycle is the nitrification of ammonium by prokaryotes leading to nitrite (NO2 − ) formation, which is then further oxidized into nitrate (NO3 − ). Finally, nitrate could be assimilated by plants for their growth or converted into N2 by denitrifying bacteria (**Figure 3**).

Moreover, nitrogen is also the precursor of NOx. Indeed, NOx formation can be separated into four steps. First, NO is formed in the atmosphere in combination with nitrogen, resulting from the global biogeochemical nitrogen cycle, and oxygen, following combustion (natural or anthropic). Then, this highly reactive compound can be oxidized by oxygen (O2 ) to form NO2 . Alternatively, nitrogen dioxide can also be directly formed through catalytic ammonia combustion or nitrosyl chloride oxidation. Then, a temperature-dependent equilibrium is establishing between NO2 and its dimeric form, nitrogen tetroxide (N2 O4 − ). Finally, a photochemical reaction between NO2 and hydroxyl radicals leads to the generation of ozone (O3 − ) (**Figure 4**).

Even if NO and NO2 are classified as RNS, their characteristics are very different suggesting their diverse behavior and reactivity. For example, the solubility of NO is very low

**Figure 3.** Nitrogen cycle at the interfaces among air, soil, and ecosystem. (1) Fixation of gaseous nitrogen by prokaryotes leading to ammonia (NH3 ) and ammonium (NH4 + ); (2) nitrification of NH<sup>4</sup> + by prokaryotes resulting in nitrite formation (NO2 − ) subsequently oxidized into nitrate (NO3 − ); (3) denitrifying prokaryotes catalyzing the conversion of nitrate into gaseous N2 .

(5.7 × 102 g/L), and NO<sup>2</sup> is highly reactive in water resulting in the formation of HNO2 and HNO3 [8]. Moreover, the half-life time of those compounds highly differs because NO<sup>2</sup> half-life is around 35 h, whereas that of NO is almost impossible to determine because of its high reactivity [9]. The permeability of both compounds is also totally unrelated: NO has a lipophilic behavior with consequently a high-membrane permeability. This can be illustrated by the high-diffusion ability of NO toward the membrane. Conversely, the permeability coefficient of NO<sup>2</sup> is estimated about 5 cm s−1 suggesting a lower diffusion power toward biological membranes [10]. In spite of these differences, the penetration of NO and NO2 inside cells and their high reactivity are at the origin of their pathogenic potential on human health, particularly when physiological elimination thresholds are exceeded. Since skin, respiratory tract, and lungs are the first barrier toward those gases, these organs are evidently the principle targets of these compounds and their derived species [11–13]. However, their diffusion deeper in the organism can lead afterward to other severe effects, for instance, on the cardiovascular and immune systems [12, 14]. Human health effects range from reversible (nausea, breathing difficulties, and asthma symptoms) to irreversible (cardiovascular defects, emphysema, and immunopathologies), including cancer induction in the worst cases (**Figure 5**) [15].

Thanks to epidemiological and clinical studies, thresholds of NO and NO2

**Figure 5.** Representation of NOx targets and associated pathologies [11–15].

limit value (**Figure 6E**).

**Figure 4.** Atmospheric genesis of NOx (adapted from [3]).

totally different effects as discussed later.

spheric NO2

structure and conversion factors are represented **Figure 6A**, have been determined for different human health effects (**Figure 6B** and **C**, respectively). However, no guideline values are available for NO (**Figure 6D**) due to its complete and rapid reaction [16]. Thus, in order to limit health issues, international organizations, such as WHO, set up guidelines for atmo-

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23

NOx is then highly toxic compounds and has a myriad of deleterious impacts on the human physiology when the bearable thresholds are exceeded. However, NOx is also naturally produced by cellular processes in a wide range of living organisms. In this case, NOx can have

, whose chemical

The Hidden Face of Nitrogen Oxides Species: From Toxic Effects to Potential Cure? http://dx.doi.org/10.5772/intechopen.75822 23

**Figure 4.** Atmospheric genesis of NOx (adapted from [3]).

(5.7 × 102 g/L), and NO<sup>2</sup>

leading to ammonia (NH3

meability coefficient of NO<sup>2</sup>

tion in the worst cases (**Figure 5**) [15].

HNO3

(NO2 −

gaseous N2 .

NO2

is highly reactive in water resulting in the formation of HNO2

+

); (3) denitrifying prokaryotes catalyzing the conversion of nitrate into

is estimated about 5 cm s−1 suggesting a lower diffusion power

 [8]. Moreover, the half-life time of those compounds highly differs because NO<sup>2</sup> half-life is around 35 h, whereas that of NO is almost impossible to determine because of its high reactivity [9]. The permeability of both compounds is also totally unrelated: NO has a lipophilic behavior with consequently a high-membrane permeability. This can be illustrated by the high-diffusion ability of NO toward the membrane. Conversely, the per-

**Figure 3.** Nitrogen cycle at the interfaces among air, soil, and ecosystem. (1) Fixation of gaseous nitrogen by prokaryotes

); (2) nitrification of NH<sup>4</sup>

+

−

) and ammonium (NH4

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

) subsequently oxidized into nitrate (NO3

toward biological membranes [10]. In spite of these differences, the penetration of NO and

 inside cells and their high reactivity are at the origin of their pathogenic potential on human health, particularly when physiological elimination thresholds are exceeded. Since skin, respiratory tract, and lungs are the first barrier toward those gases, these organs are evidently the principle targets of these compounds and their derived species [11–13]. However, their diffusion deeper in the organism can lead afterward to other severe effects, for instance, on the cardiovascular and immune systems [12, 14]. Human health effects range from reversible (nausea, breathing difficulties, and asthma symptoms) to irreversible (cardiovascular defects, emphysema, and immunopathologies), including cancer induc-

and

by prokaryotes resulting in nitrite formation

**Figure 5.** Representation of NOx targets and associated pathologies [11–15].

Thanks to epidemiological and clinical studies, thresholds of NO and NO2 , whose chemical structure and conversion factors are represented **Figure 6A**, have been determined for different human health effects (**Figure 6B** and **C**, respectively). However, no guideline values are available for NO (**Figure 6D**) due to its complete and rapid reaction [16]. Thus, in order to limit health issues, international organizations, such as WHO, set up guidelines for atmospheric NO2 limit value (**Figure 6E**).

NOx is then highly toxic compounds and has a myriad of deleterious impacts on the human physiology when the bearable thresholds are exceeded. However, NOx is also naturally produced by cellular processes in a wide range of living organisms. In this case, NOx can have totally different effects as discussed later.

[22]. On the contrary, this reaction can also alter the protein function, for example, in the case

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 regu-

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

tion of gaseous NO in extracellular environment could have subsequently neutralized O3

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

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; \*,

present in the paleoatmosphere as a survival strategy. Indeed, the libera-

or NO2

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

) are able to generate

25

, thus

, tetrahydrobiopterin; l-arg, l-arginine-binding

of hemoglobin [23]. NO and other higher RNS products (ONOO<sup>−</sup>

lation pathways to prevent the development of several diseases.

**Figure 7.** The structural similarities of NOS (adapted from [31]). BH4

palmitoylation and myristoylation of eNOS oxygenase domain.

to eradicate toxic O3

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

**Figure 6.** Key features concerning NOx. (A) Lewis structure of NOx and conversion factors; (B) NO threshold (ppm) according to [9]; (C) NO<sup>2</sup> threshold (ppm) according to [9]; (D) and (E) NO and NO<sup>2</sup> guideline values, respectively, according to [1]. ppm, parts per million; ND, not determined.
