**3.2. The novel therapeutic strategies mediated by NOx**

through non-heme iron cluster conjugated with a wide range of detoxification mechanisms. Several pathways involving reaction of NO with non-heme iron cluster were described, mainly in *Escherichia coli* [70]. Indeed, NO can lead to a raise of the repression of sulfur assimilation operon (*suf*) exerted by ferric uptake regulator protein (Fur). This reaction is mediated by Fur nitrosylation through NO-derived S-nitrosoglutathione (GSNO). *Suf* products are required for the biogenesis of the [Fe─S] cluster, which, in turn, gives a lure to get rid of NOx collateral damages. The flavohemoglobin (hmpA) is another response against the nitrosative stress.

tions. Interestingly, in the presence of NO, the S-nitrosylation of cysteine residues on MetR and FNR inactivates both the proteins. Thus, the repression exerted by MetR and the fumarate and nitrate reductase regulatory protein (FNR) is raised by *hmpA* allowing its transcription. Furthermore, NO-responsive transcriptional factor (NorR) is directly activated by NO and

sensitive cysteines also take part in the NO-sensing systems. Indeed, NO-activated superoxide regulon (SoxR) protein leads to the transcription increase of superoxide dismutase (*sodA*)

regulon (OxyR) protein is activated by S-nitrosylation. This protein induces gene expression for protective products against nitrosative stress at least by limiting the S-nitrosylation [72]. For example, the expression of catalase KatA, which could buffer free NO, is activated by OxyR [73]. Altogether, these sensing systems lead to the efficient detoxification by generation

Moreover, bacteria also develop repairing system of DNA and [Fe─S] cluster damages related to nitrosative stress. Similar to eukaryotic cells, five DNA repair pathways are available in prokaryotes. Among them, the base excision repair (BER), to repair deaminated base, and the nucleotide excision repair (NER), to remove cross-linking in DNA, are predominant [74]. Concerning the [Fe─S] cluster repair, the iron sulfur cluster S protein (IscS), a cysteine desulfurase, can denytrosylate these protein clusters in the presence of l-cysteine [75]. Moreover, Isc regulator (IscR) protein is able to sense nitrosylated [Fe─S] cluster and thus enhances the

NO could also alter tricarboxylic acid (TCA) metabolic pathway and bacterial respiration through reaction with aconitase and cytochromes of the electron transfer chain, respectively [78]. However, these mechanisms are crucial for the generation of ATP and hence the energetic needs of bacteria [79]. To counteract the inefficiency of this enzyme, some bacteria are able to reprogram their metabolism. For example, *Pseudomonas* spp. uses the citrate lyase, phosphoenolpyruvate carboxylase, and pyruvate phosphate dikinase to convert citrate into

In nonlethal concentrations, NO interplays a signaling role in bacteria through several proteins possessing heme-nitric oxide/oxygen-binding domain and is referred under the name H-NOX proteins. H-NOX exhibits a highly conserved domain among bacteria and also shared in mammals (sGC) [81]. All H-NOX are histidine-ligated protoporphyrin IX hemoprotein able to fix NO on its ferrous iron. The H-NOX-encoding genes are found in operons with diverse bacterial signaling genes [82], whose majority is now divided in two classes: containing (i) histidine

and ONOO<sup>−</sup>

enhances the expression of NorVW protein catalyzing the reduction of NO into N<sup>2</sup>

O) under anaerobic condi-

[71]. Similar to SoxR, peroxide

O. Redox-

This enzyme catalyzes the conversion of NO into nitrous oxide (N2

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

that may react with NO to form NO2

of less toxic compounds or scavenger molecules.

formation and/or repair of [Fe─S] cluster [76, 77].

removing O2

−

pyruvate and ATP [80].

A lot of therapeutic strategies exploiting NOx-mediated pathways has recently emerged, particularly to treat cancer. Here we propose to classify them in three families: (i) NO-donor compounds, materials, or nanoparticles; (ii) modulators of NOS activity; and (iii) last but not least, generators of gaseous NO.

The first family NO donors consist of NO-chelating compounds that can release NO under specific conditions within the organism after administration. As shown below, several NO donors have been developed during the last decade. Their global—direct or not—anticancer effects have been exploited in the case of various carcinoma (**Table 1**).

For further information, we strongly recommend to read the review of Huang et al. [86]. Another extension of the use of NO-donor compounds is their graft on material and nanoparticles. Indeed, prosthetic biomaterials used in medical devices were modified through covalent or noncovalent binding of two NO donors (diazeniumdiolates and S-nitrosothiols).

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


impact on the human health, particularly through methemoglobinemia. This phenomenon occurs when hemoglobin losses its vital function of oxygen carrier following the saturation of its iron heme with NO. Thus, an intermittent high-dose short-duration exposure was tested to determine the dose/duration most effective treatment. An adapted lung mammalian model was also used (macrophages and monocytes and pulmonary epithelial cells) to appreciate the potential effects of this cure on human health [95]. This study reached phase I clinical studies using a promising NO treatment at 160 ppm for 30 min three times daily for two periods of 5 days [96]. However, as reviewed by Petit et al., it is important to keep in mind that "admin-

could also be benefit in the struggle against undesirable microorganism because it seems to impact their metabolism, social behavior, and growth [84]. Moreover, a continuous high-dose

In summary, this review of the literature shows that NOx is ubiquitous and essential in our lives. We are exposed to these compounds through environmental contaminations, but they also result from physiological processes, infections, diseases, or even drugs. NO and derivatives are implicated in a large diversity of vital functions including brain functions; motricity; cardiac, vascular, and pulmonary functions; and immunity. For these reasons, NOx is key molecules of major biochemical interest. As all active substances, NOx can exert both beneficial and adverse effects with regard to the exposition dose. The physiological adaptation to NOx results from a fine-tuned equilibrium where detoxification plays crucial roles. Myriad of finely regulated processes has emerged in response to nitrosative stress. Our knowledge still remains incomplete, and future studies are necessary to finely precise the synergy of each NOx implemented in nitrosative stress, especially when they are presented in gaseous phase that remains less explored. However, in spite of all previously described mechanisms to counteract the nitrosative stress, abnormal NOx thresholds could trigger a wide range of pathologies and even death. These antagonists, positive and negative effects, of NOx are actually intriguing. So, many researches are now focusing on NOx producing pathways to find the most effective treatments and drugs. More investigations are still needed to better understand the real potential of NOx as antitumor, antibacterial agents, and their safe clinical use.

We thank Dr. Annabelle Merieau, Djouhar Souak, and Kévin Guérin for their constructive comments. LMSM is supported by grants from Evreux Portes de Normandie, Région Normandie, French research ministry, and European Union (FEDER). S. Depayras is a recipient of a PhD

exposure seems to lead in membrane alteration through an increased permeability [98].

," thanks to the

exposure

35

[97]. They also reported that the higher NO doses

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

. In contrast, a direct gaseous NO2

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

istration of inhaled NO is associated with and unavoidable codelivery of NO2

spontaneous high reactivity of NO with O2

NO2

**4. Conclusion**

**Acknowledgements**

grant from Région Normandie.

are used, the faster is the formation rate of NO2

**Table 1.** The different classes of NO donor (adapted from [86]).

Several clinical trials have shown their usefulness in the context of preventing thrombus formation [87]. NO-donating nanoparticle systems represent a new promising tumoricidal agents, thanks to their unique properties: (i) strengthening NO-donor stability, (ii) loading a large amount of NO, and (iii) possibility to trigger NO releasing [88].

The modulation of NOS activity is another interesting strategy. In some pathologies inducing exacerbating inflammation (sepsis), a localized "nitrosative burst" occurs, and inhibition of NOS function is a crucial element. Thus, several l-arginine precursor analogs (N<sup>ω</sup>-substituted l-arginine) were developed and tested. However, since l-arginine is the common point between all NOS isoforms, these analogs represent nonselective drugs. Moreover, such therapy did not reach clinical studies on human stage despite its effects on canine vascular tone [89]. Thus, investigation on a more selective inhibition of iNOS, crucial for inflammation, was conducted through transgenic animals. Unfortunately, iNOS knockout mice exhibit an increase in mortality following polymicrobial sepsis [90]. More recently, researcher focused on increasing NO delivery within cancers. Thus, thanks to technical progress and scientific knowledge updating, iNOS-based suicide gene therapy was investigated through viral vector use. This treatment exhibits promising tumoricidal effects and appears interesting for its specific and localized iNOS expression on animal models [91].

The third strategy is the direct gaseous delivery of NO in the treatment of various infection diseases related to antibiotic resistant bacteria. Ghaffari et al. provided a considerable work on this topic. First, they developed an ingenious delivery system usefulness for the monitoring of gNO effects on microorganisms (bacteria and fungi) and eukaryotic cells [92]. Then, they reported an effective concentration of gNO up to 200 ppm in continuous 4 h delivery exhibiting bacteriostatic effects on a representative group of microorganisms (*Staphylococcus aureus*, *Pseudomonas aeruginosa*, *E. coli*, *Candida albicans*, and *Group B Streptococcus*). Moreover, no toxic effects were observed on representative mammalian cell lines (dermal fibroblasts) of wound-healing pathology such as leg ulcer or burn injuries [93]. Thus, these promising results placed gNO as a potential topical antibiotic agent [94]. Later, they also investigated the potential use of inhaled gNO in the treatment of pathogenic infection in the case of cystic fibrosis. Indeed, this pathology is a threatening pulmonary disease where microbial infection could be lethal. However, a continuous exposure of gNO at such concentration can lead to severe impact on the human health, particularly through methemoglobinemia. This phenomenon occurs when hemoglobin losses its vital function of oxygen carrier following the saturation of its iron heme with NO. Thus, an intermittent high-dose short-duration exposure was tested to determine the dose/duration most effective treatment. An adapted lung mammalian model was also used (macrophages and monocytes and pulmonary epithelial cells) to appreciate the potential effects of this cure on human health [95]. This study reached phase I clinical studies using a promising NO treatment at 160 ppm for 30 min three times daily for two periods of 5 days [96]. However, as reviewed by Petit et al., it is important to keep in mind that "administration of inhaled NO is associated with and unavoidable codelivery of NO2 ," thanks to the spontaneous high reactivity of NO with O2 [97]. They also reported that the higher NO doses are used, the faster is the formation rate of NO2 . In contrast, a direct gaseous NO2 exposure could also be benefit in the struggle against undesirable microorganism because it seems to impact their metabolism, social behavior, and growth [84]. Moreover, a continuous high-dose NO2 exposure seems to lead in membrane alteration through an increased permeability [98].
