It has been described that nitric oxide plays a part in oocyte aging, but it appears to do so by mobilizing more than one signaling pathway. NO can act either as a decelerating factor in oocyte aging [24] or, conversely, as an important cause of unwholesome aging-associated changes, which are caused by high ROS production [73].

Although high levels of NO are related mostly to pathological conditions, e.g., poor oocyte quality, increased protein nitration, and resistance to IVM in women with endometriosis [74], supplementing culture medium with low doses of the NO donor S-nitroso acetyl penicillamine (SNAP) delays manifestation of oocyte aging, *i.e*., decrease of spontaneous cortical granule exocytosis, zona pellucida hardening, and the rate of spindle abnormalities [24]. The significance of NO in sustaining oocyte quality was demonstrated by Goud et al. [75]. Exposure of aged oocytes to l-NAME resulted in a significant disruption of fertilization and apoptosis during early embryonic development.

Different NOS isoforms could play different roles in aging. Lower NO levels produced by eNOS and nNOS could participate in delaying oocyte aging through the activation of sGC, which leads to an increased production of cyclic guanosine monophosphate [76]. However, high NO levels generated by iNOS were associated with higher O2 •− production and promoted oocyte fragmentation and apoptosis [75]. Contrarily, Tripathi and colleagues [73] reported that the generation of NO through iNOS-mediated pathways was associated with the maintenance of meiotic arrest in diplotene-arrested oocytes and the sustained reduction of iNOS expression. Furthermore, they reported that intracellular NO level may induce apoptosis in aged rat oocytes cultured *in vitro*. Similarly, Nevoral et al. [77] described the suppression of apoptosis and lysis after prolonged cultivation of porcine oocytes in media supplemented by the NOS nonspecific inhibitor l-NAME. The decrease of intracellular levels of NO interrupts intracellular signal transduction pathways, especially Ca2+-mediated pathways [75]. Premkumar and Chaube [78] reported that NO increases levels of cytosolic free Ca2+, cGMP, and Wee 1 through an iNOS-mediated pathway. High levels of these signaling molecules trigger parthenogenetic activation of aged oocytes *via* the accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15), a catalytic subunit of MPF. These findings indicate that NO can influence changes associated with oocyte aging in various manners.

#### **11. S-nitrosylation as a posttranslational modification potentially regulating cell cycle**

**10. NO effects on oocyte aging**

162 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

changes, which are caused by high ROS production [73].

apoptosis during early embryonic development.

high NO levels generated by iNOS were associated with higher O2

Mammal oocytes are normally fertilized soon after the completion of meiotic maturation during the MII stage. If ovulated *in vitro,* matured oocytes are not fertilized, they undergo a process called aging, which is characterized by numerous changes. Oocyte aging rapidly decreases their quality and capacity to undergo embryonic development after fertilization. Functional and morphological changes associated with oocyte aging include decreased fertilization rates, polyspermy, parthenogenetic activation, apoptosis, chromosomal abnormalities, cortical granules exocytosis, ooplasmic microtubule dynamics, zona pellucida hardening, decreases in MPF and MAPK activities, epigenetic changes, and abnormal or delayed embryo development [24, 68–71]. Pathological conditions of oocyte aging impose limits for assisted reproduction technologies in animals as well as in humans [72].

It has been described that nitric oxide plays a part in oocyte aging, but it appears to do so by mobilizing more than one signaling pathway. NO can act either as a decelerating factor in oocyte aging [24] or, conversely, as an important cause of unwholesome aging-associated

Although high levels of NO are related mostly to pathological conditions, e.g., poor oocyte quality, increased protein nitration, and resistance to IVM in women with endometriosis [74], supplementing culture medium with low doses of the NO donor S-nitroso acetyl penicillamine (SNAP) delays manifestation of oocyte aging, *i.e*., decrease of spontaneous cortical granule exocytosis, zona pellucida hardening, and the rate of spindle abnormalities [24]. The significance of NO in sustaining oocyte quality was demonstrated by Goud et al. [75]. Exposure of aged oocytes to l-NAME resulted in a significant disruption of fertilization and

Different NOS isoforms could play different roles in aging. Lower NO levels produced by eNOS and nNOS could participate in delaying oocyte aging through the activation of sGC, which leads to an increased production of cyclic guanosine monophosphate [76]. However,

moted oocyte fragmentation and apoptosis [75]. Contrarily, Tripathi and colleagues [73] reported that the generation of NO through iNOS-mediated pathways was associated with the maintenance of meiotic arrest in diplotene-arrested oocytes and the sustained reduction of iNOS expression. Furthermore, they reported that intracellular NO level may induce apoptosis in aged rat oocytes cultured *in vitro*. Similarly, Nevoral et al. [77] described the suppression of apoptosis and lysis after prolonged cultivation of porcine oocytes in media supplemented by the NOS nonspecific inhibitor l-NAME. The decrease of intracellular levels of NO interrupts intracellular signal transduction pathways, especially Ca2+-mediated pathways [75]. Premkumar and Chaube [78] reported that NO increases levels of cytosolic free Ca2+, cGMP, and Wee 1 through an iNOS-mediated pathway. High levels of these signaling molecules trigger parthenogenetic activation of aged oocytes *via* the accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15), a catalytic subunit of MPF. These findings indicate

that NO can influence changes associated with oocyte aging in various manners.

•− production and pro-

Though cGMP pathway has been reported to be the main road for NO's involvement, evidences have been raised for c-GMP independent pathway, through protein S-nitrosylation (i.e., in porcine oocytes [35]). S-nitrosylation is an established posttranslation modification, whose potential spectra of involvements in cancer cell lines, oocytes, and embryos ranges from cell cycle regulation to embryo implantation [18, 79–81]. The effects of NO may differ among the cellular models considered. For instance, whereas low concentrations of NO donors DETA-NO promote cell proliferation in promyelotic HL-60 cells [82], nitric oxide synthase inhibition drives cell proliferation at blastula stages in *Xenopus* embryos [18]. Such a duality in the effects certainly rely on the diversity of S-nitrosylated proteins.

S-nitrosylation has been reported to modify several regulators of cell cycle progression (**Figure 4**), such as cyclin-dependent kinases (CDKs). CDKs' S-nitrosylation was observed for CDK2, CDK5, and CDK6. While CDK2-nitrosylation enhances its activity independently of any effects on protein levels expression [82], the effect of S-nitrosylation on CDK5 and CDK6 remains elusive. Though G2/M arrests might be observed associated with NO release [83, 84], no S-nitrosylation has been so far reported for Cdk1, the catalytic subunit of MPF. S-nitrosylation of cyclin B was seek in HL-60 cells, but not observed [82]. As well, no S-nitrosylations have yet been reported for polo-like kinases (PLKs), anaphase promoting factor/cyclosome (APC/C), Wee1, and Myt1, which are MPF regulators. Nevertheless, the dual specificity phosphatase Cdc25, which is the main activator of MPF, is clearly impacted by its S-nitrosylation because it annihilates its phosphatase activity [82, 87]. Recently, an

**Figure 4.** Comprehensive scheme of nitrosylation pivotal role in cell cycle, apoptosis, survival, matrix remodelling, and trophoblasts invasiveness.

alternate mechanism than direct S-nitrosylation of Cdc25C for its inhibition was proposed since NOAD, a nitric oxide-releasing derivative of oleanolic acid, induced activation of Chk2, resulting in an increase of the inhibitory phosphorylation of Cdc25C on its residue Serine 216 [84]. However, genotoxicity of nitric oxide might account for the activation of Chk2, the latter being involved in DNA damage response machinery. In the same study, the arrest in G2/M was associated with the upregulation of Cdk inhibitors p21WAF1/CIP1 and P27KIP1, without providing evidence for direct S-nitrosylation of these proteins. p21WAF1/CIP1 downregulation by NO was also reported in *Xenopus* embryos, but through nitric oxide modulation of the RhoA-ROCK pathway [18] (**Figure 1B**). Thus, though there are converging evidences for role of NO in cell cycle regulation [85], the exact mechanisms remain to be fully deciphered.

### **12. S-nitrosylation plays pivotal role in modulating trophoblast motility and survival**

As mentioned above, S-nitrosylation has been also called to play a role in preimplantation embryos and implantation (**Figure 4**). Microenvironnemental presence of NO was reported to contribute to the pathologic effects of endometriose on the development potential of embryos. In this context, NO effects on embryos survival could either rely upon S-nitrosylation, NO/GC/cGMP, or peroxynitrite formation. Lee and collaborators [28] suggested that the apoptotic effects of NO excess on mice embryos could be related to S-nitrosylation, in exclusion to other any mechanisms. The latter effects were closely associated with lipid-rich organelles (mitochondria and endoplasmic reticulum) [28, 86]. On the other hand, trophoblasts might also be protected from apoptosis *via* S-nitrosylation of caspase 3 [87].

Moreover, NO was shown to influence trophoblasts motility [88, 89]: it was further proposed that NO effects trophoblasts migration and invasion, which are critical processes for the successful embryonic development. In human trophoblasts, NO was required for outgrowth since l-NAME prevented this phenomenon in a dose-dependent manner [90]. Nevertheless, one shall keep in mind that high concentrations of NO in the environment have deleterious effects on trophoblasts outgrowth [90]. Effects of NO on trophoblasts motility has been proposed to be mediated by nitrosylation of the matrix metalloprotease MMP9 [79], based on (1) iNOS and MMP9 colocalization in front migration in trophoblast (leading edge with lamellipodium) and (2) observations of MMP9 being S-nitrosylated [91]. The dynamics of iNOS and S-nitrosylated proteins accumulation at the leading edge in trophoblasts led the authors to conclude that iNOS was not likely to be passively piling up [79] (**Figure 5**). In these conditions, iNOS also accumulates in aggregates in the cytoplasm. Taken together, colocalization of Actin, MMP9, and iNOS at the leading edge suggested indeed an active role for S-nitrosylation in cell migration. Invasiveness of cytotrophoblast-derived cell lines induced by adrenomedullin was also associated with an increase in S-nitrosylated protein rate [92]. S-nitrosylation of urokinase plasminogen activator (uPA), whose involvement in extracellular matrix is acknowledged, was reported to increase in these conditions [92]; the mechanisms through which S-nitrosylation increased this enzyme activity remain to be clarified.

alternate mechanism than direct S-nitrosylation of Cdc25C for its inhibition was proposed since NOAD, a nitric oxide-releasing derivative of oleanolic acid, induced activation of Chk2, resulting in an increase of the inhibitory phosphorylation of Cdc25C on its residue Serine 216 [84]. However, genotoxicity of nitric oxide might account for the activation of Chk2, the latter being involved in DNA damage response machinery. In the same study, the arrest in G2/M was associated with the upregulation of Cdk inhibitors p21WAF1/CIP1 and P27KIP1, without providing evidence for direct S-nitrosylation of these proteins. p21WAF1/CIP1 downregulation by NO was also reported in *Xenopus* embryos, but through nitric oxide modulation of the RhoA-ROCK pathway [18] (**Figure 1B**). Thus, though there are converging evidences for role of NO in cell cycle regulation [85], the exact mechanisms remain to

**12. S-nitrosylation plays pivotal role in modulating trophoblast**

might also be protected from apoptosis *via* S-nitrosylation of caspase 3 [87].

S-nitrosylation increased this enzyme activity remain to be clarified.

As mentioned above, S-nitrosylation has been also called to play a role in preimplantation embryos and implantation (**Figure 4**). Microenvironnemental presence of NO was reported to contribute to the pathologic effects of endometriose on the development potential of embryos. In this context, NO effects on embryos survival could either rely upon S-nitrosylation, NO/GC/cGMP, or peroxynitrite formation. Lee and collaborators [28] suggested that the apoptotic effects of NO excess on mice embryos could be related to S-nitrosylation, in exclusion to other any mechanisms. The latter effects were closely associated with lipid-rich organelles (mitochondria and endoplasmic reticulum) [28, 86]. On the other hand, trophoblasts

Moreover, NO was shown to influence trophoblasts motility [88, 89]: it was further proposed that NO effects trophoblasts migration and invasion, which are critical processes for the successful embryonic development. In human trophoblasts, NO was required for outgrowth since l-NAME prevented this phenomenon in a dose-dependent manner [90]. Nevertheless, one shall keep in mind that high concentrations of NO in the environment have deleterious effects on trophoblasts outgrowth [90]. Effects of NO on trophoblasts motility has been proposed to be mediated by nitrosylation of the matrix metalloprotease MMP9 [79], based on (1) iNOS and MMP9 colocalization in front migration in trophoblast (leading edge with lamellipodium) and (2) observations of MMP9 being S-nitrosylated [91]. The dynamics of iNOS and S-nitrosylated proteins accumulation at the leading edge in trophoblasts led the authors to conclude that iNOS was not likely to be passively piling up [79] (**Figure 5**). In these conditions, iNOS also accumulates in aggregates in the cytoplasm. Taken together, colocalization of Actin, MMP9, and iNOS at the leading edge suggested indeed an active role for S-nitrosylation in cell migration. Invasiveness of cytotrophoblast-derived cell lines induced by adrenomedullin was also associated with an increase in S-nitrosylated protein rate [92]. S-nitrosylation of urokinase plasminogen activator (uPA), whose involvement in extracellular matrix is acknowledged, was reported to increase in these conditions [92]; the mechanisms through which

be fully deciphered.

**motility and survival**

164 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**Figure 5.** Schematic view of iNOS and MMP9 localization in trophoblasts. Adapted from reference [79]. LE, leading edge; TE, trailing edge; N, nucleus; C, cytoplasm; Ag, aggresome (iNOS particle). Actin polymerisation sites were also detected in LE. Noteworthy, Harris and colleagues noted a colocalization of eNOS and actin in trophoblasts.

#### **13. A yin-yang relationship for S-nitrosylation and S-sulfhydration?**

Along with NO, hydrogen sulfide (H2 S) is another gasotransmitter involved in regulating various aspects of cellular life. Many protein sites have been reported to undergo both S-nitrosylation and S-sulfhydration (brought by H2 S), such as Actin [93, 94], GAPDH [95], Parkin [96], PTEN [97], and the p65 subunit of NF-κB [98]. If S-sulfhydration and nitrosylation may occur on the same residue [99], reactive site cysteine, they generally promote different and opposing effects. Indeed, S-nitrosylation typically reduces cysteine thiols reactivity, while S-sulfhydration increases cysteine thiols reactivity, thereby making them more nucleophilic. If one wants to compare S-sulhydration and nitrosylation, it has mainly to outline that (1) proteins are rather S-sulhydrated than S-nitrosylated, and (2) nitrosylation rather inhibits and impairs protein functions. In this regard, the case of tensin (PTEN) provides an example of yinyang relationship for S-nitrosylation and S-sulfhydration (**Figure 6**). Gene suppressor PTEN acts as an inhibitor of the PI-3 kinase/Akt signaling pathway, attenuating cellular growth and survival. S-nitrosylation enabled Akt hyperactivity, and thereby is associated with observed neuroprotective effects [100]. Oxidation of the active site cysteine is acknowledged as a common mechanism for regulating protein tyrosine phosphatases [101]. In addition, a NO-mediated PTEN degradation mechanism has been suggested to be common in neurodegenerative conditions where NO exerts a critical physiopathological role [102]. Finally, S-sulfhydration was

**Figure 6.** PTEN (left: phosphatase domain; right: C2 domain) is either S-nitrosylated (SNO) or S-sulfhydrated (SSH). When S-sulfhydrated, PTEN exerts its activity of phosphatase on PIP3 (phosphatidylinositol—3,4,5-triphosphate), in order to generate PIP2 (phosphatidylinositol-4,5-bisphosphate). In the absence of hydrogen sulfide, low concentrations of NO drive S-nitrosylation of PTEN, leading to its inactivation. In these conditions, Akt activity is maintained. If low concentration of NO drives SNO-PTEN, higher concentrations lead to SNO-Akt and abrogation of survival signal.

reported to maintain the activity of this lipid tyrosine-phosphatase, thereby preventing its S-nitrosylation [97]. PTEN structure reports disparate sites for S-nitrosylation (Cys 83 [100]), S-sulfhydration, and hydrogen peroxide-induced disulfide bond formation (Cys 71 and Cys 124 [103]). One should keep in mind that in this particular example, S-nitrosylation targets a different site than the Cys 124, mandatory in the phosphatase activity of PTEN.

It is tantalizing to hypothesize that sequence of S-nitrosylation and S-sulfhydration could provide a way for fine tuning of signaling pathways and cellular functions regulation. Because protein S-nitrosylation can foster intramolecular disulfide bond formation, a protein S-nitrosylation event might promote the formation of a more enduring S-sulfhydration reaction.

#### **14. Conclusion**

While NO is not necessary for meiotic resumption *per se* in amphibians, NO clearly influences meiotic processes in rodents, porcine, and bovine oocytes. During fertilization, the role of nitric oxide evolved from the hypothesis of being a primary activator to being solely correlated to fertilization, and its role was limited to a particular function such as hardening of the fertilization envelope in sea urchins. Therefore, the physiological relevance of NO has been debated. Nevertheless, NO, together with its effects on spermatozoa (viability, motility, capacitation acrosome reaction, and fusion with oocytes) appeared as a modulator of oocyte aging. The benefits of nitric oxide use in assisted reproduction are now well-considered.

Though NO was not reported to be involved in early developmental processes in ascidians, NO positively affects cell proliferation in early *Xenopus* embryos and impairs cell movement during gastrulation in this model. The involvement of NO during segmentation is emphasized in mammalian models, where NO seemed to be requested for segmentation and blastocysts survival (rodents, porcine, and bovine) and for implantation through blastocysts hatching (rodents and bovine) and trophoblasts motility (humans).

One of the main difficulties when considering the effects of NO remains in the existence of the multiple pathways that can be activated by this gasotransmitter: cGMP-dependent pathway, calcium-related pathways, and reactive oxygen species production. Over the past few years, S-nitrosylation has offered a new angle to decipher NO's actions since S-nitrosylation modulates the activities of many key regulators such as members of the RhoA-ROCK pathway, Cdk2, Cc25, or PTEN. PTEN regulation by nitrosylation offers a new paradigm since sulfhydration and nitrosylation, both provided by gasotransmitters, appeared to play reciprocally in a yin-yang manner.

#### **Acknowledgements**

JFB is affiliated with the Site de Recherche Intégrée en Cancérologie (SIRIC ONCOLILLE). We would like to thank the personnel of the BICeL-Lille1-HB Facility for access to the microscopy systems and technical advices. We are indebted to the Research Federation FRABio for providing the scientific and technical environment to achieving our work [14, 15]. MJ work in Department of Obstetrics and Gynaecology, Center of Assisted Reproduction, University Hospital Brno, and Masaryk University. MJ's work was supported by grant MH CZ—DRO (FNBr, 65269705) and funds from the Faculty of Medicine, Masaryk University Brno, Czech Republic. We thank Brian Kavalir for helpful discussions and comments.

#### **Author details**

reported to maintain the activity of this lipid tyrosine-phosphatase, thereby preventing its S-nitrosylation [97]. PTEN structure reports disparate sites for S-nitrosylation (Cys 83 [100]), S-sulfhydration, and hydrogen peroxide-induced disulfide bond formation (Cys 71 and Cys 124 [103]). One should keep in mind that in this particular example, S-nitrosylation targets

**Figure 6.** PTEN (left: phosphatase domain; right: C2 domain) is either S-nitrosylated (SNO) or S-sulfhydrated (SSH). When S-sulfhydrated, PTEN exerts its activity of phosphatase on PIP3 (phosphatidylinositol—3,4,5-triphosphate), in order to generate PIP2 (phosphatidylinositol-4,5-bisphosphate). In the absence of hydrogen sulfide, low concentrations of NO drive S-nitrosylation of PTEN, leading to its inactivation. In these conditions, Akt activity is maintained. If low concentration of NO drives SNO-PTEN, higher concentrations lead to SNO-Akt and abrogation of survival signal.

It is tantalizing to hypothesize that sequence of S-nitrosylation and S-sulfhydration could provide a way for fine tuning of signaling pathways and cellular functions regulation. Because protein S-nitrosylation can foster intramolecular disulfide bond formation, a protein S-nitrosylation

While NO is not necessary for meiotic resumption *per se* in amphibians, NO clearly influences meiotic processes in rodents, porcine, and bovine oocytes. During fertilization, the role of nitric oxide evolved from the hypothesis of being a primary activator to being solely correlated to fertilization, and its role was limited to a particular function such as hardening of the fertilization envelope in sea urchins. Therefore, the physiological relevance of NO has

a different site than the Cys 124, mandatory in the phosphatase activity of PTEN.

event might promote the formation of a more enduring S-sulfhydration reaction.

**14. Conclusion**

166 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Ješeta Michal1 , Marketa Sedmikova2 , and Jean-François Bodart3 \*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Obstetrics and Gynaecology, Center of Assisted Reproduction, University Hospital Brno and Masaryk University, Brno, Czech Republic

2 Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences in Prague, Prague-Suchdol, Czech Republic

3 University of Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France

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**Miscellaneous Roles of Nitric Oxide Synthase**

## **Role of Endothelial Nitric Oxide Synthase in Breast Cancer**

Tupurani Mohini Aiyengar, Padala Chiranjeevi and Hanumanth Surekha Rani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67493

#### **Abstract**

Breast cancer (BC) is the most common form of carcinoma and a primary cause of morbidity and mortality globally. Oxidative stress represents as an important factor in carcinogenesis and may play a role in initiation and progression of tumors. Oxidative stress–induced NO• damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. It is caused by an unfavorable balance between reactive oxygen species/reactive nitrogen species (ROS/RNS) and antioxidant defenses. ROS/RNS are generated during normal cellular metabolism, as a result of the influence of various environmental factors, as well as during pathological processes. Nitric oxide (NO•) is a ubiquitous, short-lived free radical produced from L-arginine by nitric oxide synthases (NOSs), and isoforms of NOS exist, depending on the site of origin: endothelial (eNOS), neuronal (nNOS), mitochondrial (mtNOS), and inducible (iNOS). eNOS is responsible for the endothelial synthesis of NO• and has shown to modulate cancer-related events such as inflammation, angiogenesis, apoptosis, cell cycle, invasion, and metastasis. Genetic studies also showed that eNOS gene polymorphisms are associated with the development of breast cancer. Therefore, selective targeting of eNOS may prove a potential strategy for prevention and treatment of breast cancer.

 Introduction**

Cancer, a multifaceted disorder, represents one of the most important health problems worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths every

© 2017 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.

year [1]. The transformation from a normal cell into a tumor cell is a multistage process, typically a progression from a precancerous lesion to malignant tumors.

Cancers originate from a single abnormal cell (clonal origin) with an altered DNA sequence (mutation). Successive rounds of mutation and selective expansion of these cells result in the formation of a tumor mass and leads to tumor growth and progression, which eventually breaks through the basal membrane barrier of surrounding tissues and spreads to other parts of the body (metastasis) (**Figure 1**).

 It is estimated as one of the most commonly diagnosed cancers worldwide (11.9%) [2].  The most frequent type of breast cancer is ductal carcinoma *in situ* (DCIS), which affects the cells of the milk ducts. Cancer that starts in lobes or lobules is called lobular carcinoma *in situ* (LCIS); it is the second most common type of breast cancer. While rare breast cancer types include tubular, medullary, metaplastic, mucinous carcinoma, and Paget's disease.

Breast cancer is a clinically heterogeneous disease. Breast cancer cells generally overexpress estrogen receptor (ER)/progesterone receptor (PR), and/or human epidermal growth factor-2 (HER-2) receptor and lead to the tumor formation and progression. Thus, breast cancer can be classified into two subgroups on the basis of receptor status namely: (i) ER/PR positive (luminal A/B) and (ii) triple negative (ER, PR, and HER-2 negative) (basal-like) to know the prognosis and clinical outcome of breast cancer (**Figure 2**). Early breast cancer usually does not cause symptoms. As the cancer grows, symptoms may include: lump in the armpit, change in the size, shape, or feel of the breast or nipples, nipple discharge, etc. Symptoms of advanced breast cancer may include: breast pain or discomfort, bone pain, skin ulcers, and weight loss.

Breast cancer etiology is complex and multifactorial where there is a strong interplay between genetic and environmental factors. The strongest nonmodifiable determinants of breast cancer risk are female gender and age. Other risk factors associated with breast cancer can be grouped into three broad determinants: family history (hereditary) factors, hormonal and reproductive factors, and environmental (including lifestyle) factors (**Figure 3**).

**Figure 1.** Carcinogenesis, a multistep process.

Role of Endothelial Nitric Oxide Synthase in Breast Cancer http://dx.doi.org/10.5772/67493 181

**Figure 2.** Classification of breast cancer.

year [1]. The transformation from a normal cell into a tumor cell is a multistage process, typi-

Cancers originate from a single abnormal cell (clonal origin) with an altered DNA sequence (mutation). Successive rounds of mutation and selective expansion of these cells result in the formation of a tumor mass and leads to tumor growth and progression, which eventually breaks through the basal membrane barrier of surrounding tissues and spreads to other parts

Cancers may be classified by their primary site of origin such as brain cancer, oral cancer, lung cancer, prostate cancer, liver cancer, renal cell carcinoma (kidney cancer), breast cancer, etc. Breast cancer is defined as a malignant growth that begins in the epithelium of the breast. It is estimated as one of the most commonly diagnosed cancers worldwide (11.9%) [2].  The most frequent type of breast cancer is ductal carcinoma *in situ* (DCIS), which affects the cells of the milk ducts. Cancer that starts in lobes or lobules is called lobular carcinoma *in situ* (LCIS); it is the second most common type of breast cancer. While rare breast cancer types include tubular, medullary, metaplastic, mucinous carcinoma, and Paget's disease. Breast cancer is a clinically heterogeneous disease. Breast cancer cells generally overexpress estrogen receptor (ER)/progesterone receptor (PR), and/or human epidermal growth factor-2 (HER-2) receptor and lead to the tumor formation and progression. Thus, breast cancer can be classified into two subgroups on the basis of receptor status namely: (i) ER/PR positive (luminal A/B) and (ii) triple negative (ER, PR, and HER-2 negative) (basal-like) to know the prognosis and clinical outcome of breast cancer (**Figure 2**). Early breast cancer usually does not cause symptoms. As the cancer grows, symptoms may include: lump in the armpit, change in the size, shape, or feel of the breast or nipples, nipple discharge, etc. Symptoms of advanced breast cancer may include: breast pain or discomfort, bone pain, skin ulcers, and weight loss. Breast cancer etiology is complex and multifactorial where there is a strong interplay between genetic and environmental factors. The strongest nonmodifiable determinants of breast cancer risk are female gender and age. Other risk factors associated with breast cancer can be grouped into three broad determinants: family history (hereditary) factors, hormonal and reproduc-

cally a progression from a precancerous lesion to malignant tumors.

tive factors, and environmental (including lifestyle) factors (**Figure 3**).

of the body (metastasis) (**Figure 1**).

180 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**Figure 1.** Carcinogenesis, a multistep process.

**Figure 3.** Breast cancer etiology.

*Family history (hereditary) factors* are associated primarily with early-onset of breast cancer. Previous genetic analyses of breast cancer–prone families have identified the BRCA1 and BRCA2 genes (breast cancer type 1 and 2 susceptibility protein). Women with mutations in either BRCA1 or BRCA2 (over 250 mutations have been identified) are at a significantly elevated lifetime risk (55–85% compared with 12% for the general population) for developing breast or ovarian cancer [4].

*Reproductive and hormonal factors* may increase the time and/level of steroid hormone exposure, consequently stimulating cell growth, and have been associated with breast cancer susceptibility. Major reproductive factors such as early menarche, late menopause, later age at first full-term pregnancy, and nulliparity are known to be associated with a higher risk of breast cancer [5].

*Environmental (including lifestyle) factors* such as the use of exogenous estrogens, radiation exposure, alcohol consumption, and socioeconomic status are also well-known risk factors for the disease [6]. Physical inactivity is a major risk factor for several types of cancer [7– 9]. Exposure to ionizing radiation such as X-rays at a young age, alcohol consumption, and smoking habits have consistently been shown to increase breast cancer risk.

Radiations are classified into two major types namely: ionizing radiation and nonionizing radiation. The ionizing radiation transmits energy via X-rays and alpha particles disrupt chemical bonds, which results in chemically reactive free radical formation, a phenomenon known as ionization. These ionizing radiations may directly pass through the cell and may cause DNA damage. Such damage if unrepaired can result in nonlethal DNA modifications or cell death. The nonlethal DNA modifications thus eventually may cause malignant transformations. Ionizing radiation is, therefore, a known mutagen and an established breast carcinogen. Nonionizing radiation (e.g., microwaves and extremely low-frequency electric and magnetic fields (ELF-EMF)) does not have enough energy to break chemical bonds and produce ionization [10].

Several epidemiological studies have shown that alcohol consumption has been associated with breast cancer susceptibility. The alcohol metabolism occurs via multiple stages which may increase the risk of carcinogenesis. Alcohol metabolized into an acetaldehyde and other products subsequently damages DNA by inducing DNA modifications. Apart from this, acetaldehyde alone may also cause breast tumorigenesis by interfering with DNA repair mechanisms. Free radicals generated in the second stage of alcohol metabolism are thought to cause DNA damage, strand breakage, and base alterations and have been implicated for their role in alcohol-associated carcinogenesis [11].

There are over 60 carcinogens in cigarette smoke, which have been evaluated by the International Agency for Research on Cancer [12]. Cigarette smoke is rich in carcinogens such as nitrosamines and polycyclic aromatic hydrocarbons and induces oxidative damage. The gas phase of freshly generated cigarette smoke has large amounts of nitric oxide and other unstable oxidants. The presence of such free radicals and oxidants can lead to oxidative DNA damage [13]. The environmental risk factors that alter the levels of free radicals (reactive species) generated in the body are known to react with DNA to cause mutations in critical genes and consequently promote carcinogenesis.

#### **2. Free radicals**

*Family history (hereditary) factors* are associated primarily with early-onset of breast cancer. Previous genetic analyses of breast cancer–prone families have identified the BRCA1 and BRCA2 genes (breast cancer type 1 and 2 susceptibility protein). Women with mutations in either BRCA1 or BRCA2 (over 250 mutations have been identified) are at a significantly elevated lifetime risk (55–85% compared with 12% for the general population) for developing

*Reproductive and hormonal factors* may increase the time and/level of steroid hormone exposure, consequently stimulating cell growth, and have been associated with breast cancer susceptibility. Major reproductive factors such as early menarche, late menopause, later age at first full-term pregnancy, and nulliparity are known to be associated with a higher risk of

*Environmental (including lifestyle) factors* such as the use of exogenous estrogens, radiation exposure, alcohol consumption, and socioeconomic status are also well-known risk factors for the disease [6]. Physical inactivity is a major risk factor for several types of cancer [7– 9]. Exposure to ionizing radiation such as X-rays at a young age, alcohol consumption, and

Radiations are classified into two major types namely: ionizing radiation and nonionizing radiation. The ionizing radiation transmits energy via X-rays and alpha particles disrupt chemical bonds, which results in chemically reactive free radical formation, a phenomenon known as ionization. These ionizing radiations may directly pass through the cell and may cause DNA damage. Such damage if unrepaired can result in nonlethal DNA modifications or cell death. The nonlethal DNA modifications thus eventually may cause malignant transformations. Ionizing radiation is, therefore, a known mutagen and an established breast carcinogen. Nonionizing radiation (e.g., microwaves and extremely low-frequency electric and magnetic fields (ELF-EMF)) does not have enough energy to break chemical bonds and produce ionization [10].

Several epidemiological studies have shown that alcohol consumption has been associated with breast cancer susceptibility. The alcohol metabolism occurs via multiple stages which may increase the risk of carcinogenesis. Alcohol metabolized into an acetaldehyde and other products subsequently damages DNA by inducing DNA modifications. Apart from this, acetaldehyde alone may also cause breast tumorigenesis by interfering with DNA repair mechanisms. Free radicals generated in the second stage of alcohol metabolism are thought to cause DNA damage, strand breakage, and base alterations and have been implicated for their role

There are over 60 carcinogens in cigarette smoke, which have been evaluated by the International Agency for Research on Cancer [12]. Cigarette smoke is rich in carcinogens such as nitrosamines and polycyclic aromatic hydrocarbons and induces oxidative damage. The gas phase of freshly generated cigarette smoke has large amounts of nitric oxide and other unstable oxidants. The presence of such free radicals and oxidants can lead to oxidative DNA damage [13]. The environmental risk factors that alter the levels of free radicals (reactive species) generated in the body are known to react with DNA to cause mutations in critical genes

smoking habits have consistently been shown to increase breast cancer risk.

breast or ovarian cancer [4].

182 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

in alcohol-associated carcinogenesis [11].

and consequently promote carcinogenesis.

breast cancer [5].

Free radicals are molecules with high instability and reactivity due to the presence of an odd number of electrons in the outermost orbit of their atoms; their action derives from their attempts to attain "balance" by binding with electrons of neighboring atoms, giving rise to chain reactions (**Figure 4**) [14].

Free radical can be classified as reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS include the superoxide anion (O2 − ), hydroxyl radical (OH− ), hydrogen peroxide (H2 O2 ), while RNS include nitric oxide (NO**•**− ) and peroxynitrite (ONOO− ) the radical nitrogen dioxide (NO2 − ), and nitrite (NO2 − ) [15].

Free radicals are key players in the initiation and progression of tumor cells and enhance their metastatic potential. They are now considered a hallmark of cancer.

**Figure 4.** Formation and elimination of ROS.

#### **3. Oxidative stress**

The cell generates free radicals and also degrades, which is strictly necessary to avoid the damage derived from free radicals. However, various intrinsic and extrinsic circumstances and the biochemical activity of the cell can make it lose control over the formation and management of free radicals and results in "oxidative stress." It results from a disturbance in balance between the formation of ROS/RNS and the defense provided by cell antioxidants. This imbalance may cause damage related to various human diseases (**Figure 5**) [16].

During endogenous metabolic reactions, aerobic cells produce ROS such as superoxide anion (O2 − ), hydrogen peroxide (H2 O2 ), hydroxyl radical (OH•), and organic peroxides as normal products of the biological reduction of molecular oxygen [17]. Under hypoxic conditions, the mitochondrial respiratory chain also produces nitric oxide (NO•− ), which can generate other reactive nitrogen species (RNS) [18].

**Figure 5.** Oxidative stress.

#### **4. Antioxidants**

Cells have natural defense systems against ROS that consist of antioxidant enzymes, vitamins, etc., some of these antioxidants are produced inside the human body, mostly falling into the enzymatic category, as they are predominantly protein in nature. These proteins include the superoxide dismutase (SOD) enzymes (which have differential subcellular localization and superoxide dismutation to H2 O2 ), glutathione peroxidase (GPx), and catalase (both of which clear peroxide), thioredoxins (Trxs; reduce oxidized proteins), and glutathione synthetase (GSS; synthesizes glutathione [GSH], an important antioxidant), among others. Vitamins are mostly obtained from nutritional sources and include ascorbic acid (vitamin A), tocopherol. Therefore, a fine balance exists between the levels of ROS and antioxidants within the cell [19]. Increased ROS/RNS can result in a greater number of mutations, oxidation of critical proteins, and other alterations, finally culminating in cell death. Identification of potentially modifiable factors that affect oxidative stress in breast cancer patients is an increasingly important task.

#### **5. Nitric oxide**

Nitric oxide (NO•) is a ubiquitous, short-lived, water soluble and endogenously produced gas that exerts a wide range of biological effects. It is a pleiotropic regulator, critical to numerous biological processes, including vasodilation, neurotransmission, and macrophage-mediated immunity.

The general function of NO• protects against the effects of free radicals but at excessive concentrations, NO• or its derivatives may lead to DNA damage and impair the tumor suppressor function of p53, which may cause cancer development [20].

#### **5.1. Intracellular mechanisms of NO•−**

When NO• is synthesized, it has a half-life of only a few seconds. Its bioavailability is reduced due to the high affinity binding of superoxide anion (high reactivity in both molecules is due to the unpaired electrons). NO• has an ability to tightly bind to the heme moiety of both hemoglobin (Hb) and guanylyl cyclase (GC) and found mostly in the vascular smooth muscle cells and other cells. Thus, NO• when produced in endothelium is quickly diffused into the blood circulation, binds to hemoglobin in blood and forms nitrates. NO• may also activate guanylyl cyclase (GC), an enzyme that catalyzes the dephosphorylation of guanosine triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP) and serves as a second messenger for many important cellular functions, particularly for signaling smooth muscle relaxation [21].

#### **5.2. NO•− biological functions**

**4. Antioxidants**

**Figure 5.** Oxidative stress.

184 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**5. Nitric oxide**

ated immunity.

superoxide dismutation to H2

O2

Cells have natural defense systems against ROS that consist of antioxidant enzymes, vitamins, etc., some of these antioxidants are produced inside the human body, mostly falling into the enzymatic category, as they are predominantly protein in nature. These proteins include the superoxide dismutase (SOD) enzymes (which have differential subcellular localization and

clear peroxide), thioredoxins (Trxs; reduce oxidized proteins), and glutathione synthetase (GSS; synthesizes glutathione [GSH], an important antioxidant), among others. Vitamins are mostly obtained from nutritional sources and include ascorbic acid (vitamin A), tocopherol. Therefore, a fine balance exists between the levels of ROS and antioxidants within the cell [19]. Increased ROS/RNS can result in a greater number of mutations, oxidation of critical proteins, and other alterations, finally culminating in cell death. Identification of potentially modifiable factors that affect oxidative stress in breast cancer patients is an increasingly important task.

Nitric oxide (NO•) is a ubiquitous, short-lived, water soluble and endogenously produced gas that exerts a wide range of biological effects. It is a pleiotropic regulator, critical to numerous biological processes, including vasodilation, neurotransmission, and macrophage-medi-

), glutathione peroxidase (GPx), and catalase (both of which

Nitric oxide (NO•− ) is known to play important functional roles in a variety of physiological systems [22].

While, NO also inhibits the contractility of the smooth muscle wall of the uterus. During child birth, as the moment of birth approaches the production of NO decreases.

**5.** NO•− affects secretion from several endocrine glands—NO regulates the release of Gonadotropin-releasing hormone (GnRH) from the hypothalamus and adrenaline from the adrenal medulla.

In mammals, under normoxic conditions, NO• is generated endogenously "on demand" when the guanidino nitrogen of L-arginine undergoes a five-electron oxidation to yield the gaseous free radical, nitric oxide, and citrulline in equimolar concentrations (**Figure 6**) [23].

The constitutive forms of NOS are endothelial NOS (eNOS; type III) and inducible NOS (iNOS; type II). Cofactors for NOS include oxygen, NADPH, tetrahydrobiopterin, and flavin adenine nucleotides.

The inducible (calcium-independent) isoform (iNOS) produces much larger amounts of NO and is only expressed during inflammation. Whereas iNOS can produce injurious amounts of RNS (check), eNOS and nNOS produce beneficial amounts under physiological conditions. The constitutive (calcium-dependent) isoform and endothelial NOS (eNOS) produce small amounts of NO, which act as a vasodilator. The third form, neural NOS (nNOS; type I) serves as a transmitter in the brain and in different nerves of the peripheral nervous system to produce vasodilation. mtNOS constitutively expressed and membrane-bound nNOS isoform alpha, precluding a novel alternative spliced product. The mitochondrial production of nitric oxide is catalyzed nitric-oxide synthase (mtNOS). This enzyme has the same cofactor and substrate requirements as other constitutive nitric-oxide synthases (**Figure 6**).

NOS1 is the neural (or brain) isoform, also known as *nNOS*. It plays an important role in neural communication via synaptic transmission from nerve to nerve across synapses, and from peripheral nerves to the brain.

NOS2 is also known as *iNOS*. It generates extremely elevated concentrations of NO, to participate in a host defense mechanism. It also takes several hours to be activated in response to an injury or infection. Unlike nNOS, which takes part in normal neural communication, an abnormal stimulus (a wound, tissue damage, hypoxia, bacterial infection, etc.) may induce iNOS.

**Figure 6.** Mechanism of NO production from NOS.

The third isoform is *eNOS* (or NOS3) which stands for "endothelial cell" NOS. This isoform is active at all times and is found in endothelial cells which are the cells that line the inner surface of all blood vessels and lymph ducts. The eNOS is activated by the pulsatile flow of blood through vessels and maintains the diameter of the blood vessels at an optimal level. In addition, it also promotes angiogenesis, a process of new blood vessels formation.

A fourth type, mitochondrial NOS (*mtNOS*), differentiated by its subcellular localization in the mitochondrial inner membrane, regulates various functions of mitochondria in liver, heart, kidney, breast, etc. The mitochondrial utilization of NO involves superoxide anion and H2 O2 , a species freely diffusible outside the mitochondria that participate in the modulation of cell proliferation and apoptosis and in cell transformation leading to cancer [24, 25].

#### **6. Endothelial nitric oxide synthase (eNOS)**

In mammals, under normoxic conditions, NO• is generated endogenously "on demand" when the guanidino nitrogen of L-arginine undergoes a five-electron oxidation to yield the gaseous free radical, nitric oxide, and citrulline in equimolar concentrations (**Figure 6**) [23].

The constitutive forms of NOS are endothelial NOS (eNOS; type III) and inducible NOS (iNOS; type II). Cofactors for NOS include oxygen, NADPH, tetrahydrobiopterin, and flavin

The inducible (calcium-independent) isoform (iNOS) produces much larger amounts of NO and is only expressed during inflammation. Whereas iNOS can produce injurious amounts of RNS (check), eNOS and nNOS produce beneficial amounts under physiological conditions. The constitutive (calcium-dependent) isoform and endothelial NOS (eNOS) produce small amounts of NO, which act as a vasodilator. The third form, neural NOS (nNOS; type I) serves as a transmitter in the brain and in different nerves of the peripheral nervous system to produce vasodilation. mtNOS constitutively expressed and membrane-bound nNOS isoform alpha, precluding a novel alternative spliced product. The mitochondrial production of nitric oxide is catalyzed nitric-oxide synthase (mtNOS). This enzyme has the same cofactor and

NOS1 is the neural (or brain) isoform, also known as *nNOS*. It plays an important role in neural communication via synaptic transmission from nerve to nerve across synapses, and from

NOS2 is also known as *iNOS*. It generates extremely elevated concentrations of NO, to participate in a host defense mechanism. It also takes several hours to be activated in response to an injury or infection. Unlike nNOS, which takes part in normal neural communication, an abnormal stimulus (a wound, tissue damage, hypoxia, bacterial infection, etc.) may induce iNOS.

substrate requirements as other constitutive nitric-oxide synthases (**Figure 6**).

adenine nucleotides.

186 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

peripheral nerves to the brain.

**Figure 6.** Mechanism of NO production from NOS.

Endothelial nitric oxide synthase enzyme, also known as nitric oxide synthase-3 (NOS-3) or constitutive NOS (cNOS), has been shown to be a critical regulator of carcinogenesis. It is a dimeric structure in their active form containing two identical monomers of 134 kD represented by *a reductase domain* containing the binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). While *an oxidase domain* comprises the binding sites for the heme group, zinc, the cofactor tetrahydrobiopterin (BH4), and the substrate L-arginine [26]. The reductase domain is attached to the oxidase domain via calmodulin-binding sequence [27].

The eNOS expression is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO•) in response to a number of stimuli. Constitutively expressed eNOS oxidizes L-arginine to generate L-citrulline and NO•. The essential cofactors for catalysis of this reaction include as calmodulin (CaM), flavin mononucleotide, flavin adenine dinucleotide, tetrahydrobiopterin (H4B) and NADPH.

Typically, the eNOS isoforms can be activated as a result of calmodulin (CaM) binding following a rise in intracellular calcium. They may also be activated and/or inhibited by phosphorylation via various protein kinases. Oxygen levels also regulate NOS levels in a cell type and isoform-specific manner by altering enzyme expression and by limiting the availability of oxygen, a key substrate for NO• synthesis.

Experimental and epidemiological evidence has shown the contributory role of eNOS induced NO• in tumor progression, suggesting a possible implication of endothelium expressed eNOS in tumors. The role of NO• in cancer biology is widespread, including its involvement in cellular transformation, malignant lesions, initiation, progression of the metastatic process, and induction of genotoxicity [28]. NO•-mediated DNA damage may be due to direct modification of DNA, or inhibition of DNA repair mechanisms [29]. Similarly, most of the RNS can cause DNA strand breaks and result in multiple mutations in DNA [30].

Breast carcinogenesis involves the transformation of a normal cell into a tumor cell mediated by a sequence of cellular and molecular events. These events consist of the attainment of precise characteristics, such as uncontrolled proliferation, avoidance of mitotic control, resistance to apoptosis, replicative immortality, escape from immune surveillance, progression by stimulating invasion and metastasis, angiogenesis, genomic instability, and deregulated metabolism [31].

Tumor-derived eNOS has shown to modulate cancer-related events (inflammation, apoptosis, cell cycle, angiogenesis, invasion, and metastasis) (**Figure 7**) and genetic studies showed that eNOS gene polymorphisms are associated with the development of multiple cancers [32].

**Figure 7.** Multiple roles of eNOS in tumor development.

#### **6.1. Inflammation**

Inflammation a localized protective response elicited by injury is known to cause DNA damage [33]. Chronic inflammation due to infection or injury is estimated to contribute to 25% of all cancers in the world [34]. A growing body of laboratory research has shown that inflammation is a key mediator in the promotion of malignant transformation, where pro-inflammatory cytokines can facilitate tumor growth and metastasis by altering tumor cell biology and activating stromal cells in the tumor microenvironment, such as vascular endothelial cells, tumor-associated macrophages, and fibroblasts.

NO• is closely related to inflammatory status and regarded as a critical inflammation mediator. Pro-inflammatory cytokines can modulate the expression of eNOS and can accelerate the growth and development of cancer. eNOS, for example, can regulate the expression of the pro-inflammatory molecules nuclear factor-κB (NF-κB) and cyclooxygenase-2 [32, 35, 36].

#### **6.2. Apoptosis**

precise characteristics, such as uncontrolled proliferation, avoidance of mitotic control, resistance to apoptosis, replicative immortality, escape from immune surveillance, progression by stimulating invasion and metastasis, angiogenesis, genomic instability, and deregulated

Tumor-derived eNOS has shown to modulate cancer-related events (inflammation, apoptosis, cell cycle, angiogenesis, invasion, and metastasis) (**Figure 7**) and genetic studies showed that eNOS gene polymorphisms are associated with the development of multiple

Inflammation a localized protective response elicited by injury is known to cause DNA damage [33]. Chronic inflammation due to infection or injury is estimated to contribute to 25% of all cancers in the world [34]. A growing body of laboratory research has shown that inflammation is a key mediator in the promotion of malignant transformation, where pro-inflammatory cytokines can facilitate tumor growth and metastasis by altering tumor cell biology and activating stromal cells in the tumor microenvironment, such as vascular endothelial cells,

NO• is closely related to inflammatory status and regarded as a critical inflammation mediator. Pro-inflammatory cytokines can modulate the expression of eNOS and can accelerate the growth and development of cancer. eNOS, for example, can regulate the expres-

metabolism [31].

188 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

cancers [32].

**6.1. Inflammation**

tumor-associated macrophages, and fibroblasts.

**Figure 7.** Multiple roles of eNOS in tumor development.

Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. Impaired apoptosis has been associated with initiation and development of cancer. The mechanism of apoptosis is regulated by an array of factors triggering and activating signaling cascade, subsequently leading to cellular death [33]. eNOS may be a molecular node in growth factor–mediated inhibition of apoptosis [37]. The antiapoptotic mechanism is understood on the basis of gene transcription of protective proteins and direct inhibition of the apoptotic executive effectors (caspase family protease).

The mechanisms of action that lead to the proneoplastic activity of NO• are via apoptosis inhibition by S-nitrosylation-inactivation of caspases- 1, 2, 4, 8 and 3, 6, 7 and disruption of the apoptotic protease activating factor 1/caspase-9 complex (Apaf-1/caspase-9 apoptosome is an essential initiator of caspase activation that initiates an apoptotic protease cascade) [38]. Other antiapoptotic effects of NO• depends on the interaction of NO•/cGMP, which inhibits the release of cytochrome C, stimulation of heat-shock protein (Hsp) 70 and Hsp 32, elevated Bcl-2 expression, repression of ceramide generation [39], and induces cyclooxygenase-2 activity [40]. In the animal model study, the lipopolysaccharide (LPS)-induced hepatic apoptosis was increased by the administration of NOS inhibitors [41]. Thus at high concentration, NO• has a potential role in cancer, i.e., inhibition of eNOS activity specific to tumor cells may be a viable option for the stimulation of apoptosis and treatment of cancer alone or in combination with chemotherapeutic agents.

#### **6.3. Angiogenesis**

Angiogenesis in mammary tumors can be stimulated by inflammation, which induces proliferation and morphogenesis of vascular endothelial cells in response to a large number of cytokines or angiogenic molecules produced by tumor and host cells [42]. Angiogenesis is essential for tumor growth and metastasis and has been considered the most important prognostic indicator for predicting overall survival [43]. eNOS strongly affects tumor growth by promoting angiogenesis [44]. Tumor growth–enhancing effects of vascular endothelial growth factor (VEGF) are associated with increased NOS activity and inhibition of apoptosis in human breast carcinoma xenografts [45].

Endogenous NO• promotes tumor blood flow via dilatation of arteriolar vessels. It decreases leukocyte-endothelial adhesive interactions and increases vascular permeability [46]. Several cancer treatment methods influence eNOS expression and activity. Low-dose irradiationinduced angiogenesis is believed to be mediated by NO• from eNOS [47]. Studies have shown that VEGF released as a purified protein or produced by tumor cells requires a functional NO•/cGMP pathway within the end compartment to promote neovascular growth. NO• also has an invasion-stimulating effect, which is mediated by upregulation of MMP-2 and MMP-9 (matrix metalloproteinases) and downregulation of TIMP-2 and possibly TIMP-3 (tissue inhibitors of MMP) [48].

#### **6.4. Tumor progression/invasion**

NO• has been investigated regarding its possible involvement in the promotion of breast carcinoma. Increased amounts of NO• have been observed in the blood circulation of advanced grade breast cancer patients [49] where the increased levels of NO• have shown to promote tumor angiogenesis. Nitrotyrosine, a marker derived from NO•, was correlated with expression of VEGF-C and has been associated with lymph node metastasis in breast carcinoma patients, implicating the role of NO• in the development and progression of breast cancer [50]. In relation to the link of eNOS with cell proliferation, the eNOS expression has been detected in tumor cells specifically in breast cancer [51].

Several studies have observed NO• released by eNOS, can stimulate cancer cell cycle progression and proliferation. More specific to eNOS, studies have shown that the eNOS/NO• pathway plays a role in cancer cell DNA/RNA synthesis and proliferation apart from promoting angiogenesis [52]. The eNOS gene plays an essential role in endothelial cell proliferation in cell culture models and is a central mediator of several endothelium growth stimulators, such as vascular endothelial growth factor (VEGF) and prostaglandin E2. In human breast cancer, eNOS appears to be expressed in tumor epithelial cells, and its presence is correlated with histological grade and lymph node status. Higher NOS activity has been found in invasive breast tumors when compared with benign or normal breast tissue carcinoma [53].

#### **6.5. Metastasis**

Metastasis is a complex process by which the malignant cancer cells from the breast expand into other regions of the body. Lymphatic metastasis is a critical determinant of cancer prognosis. Recent findings indicate that eNOS mediates VEGF-C-induced lymph-angiogenesis and, consequently, plays a critical role in lymphatic metastasis [54]. Investigational studies on tumors have provided substantial evidence of the contributory role of NO• in tumor development, where series of tests were performed on tumor-bearing mice using NOS inhibitors showed a delayed tumor growth through eNOS inhibition and barred metastasis, signifying the potential role of endothelium-derived eNOS in metastasis [55].

#### **7. Regulation OF** *NOS3* **gene expression**

The most fundamental level of NOS regulation is reflected in the tissue-specific expression of the different isoforms. The amount of NO produced results from the expression level and activity of eNOS. It is regulated by several interlinking mechanism such as transcriptional, posttranscriptional, and posttranslational modifications. The activity of NOS3 is also controlled by avid binding transcription factors namely Ets-1, Elf-1, Sp1, Sp3, and YY1 to the NOS3 promoter region. Posttranscriptionally, NOS3 activity is controlled by modifications of the primary transcript, stability of mRNA, its subcellular localization, and nucleocytoplasmic transport. Posttranslational modifications of NOS3 consist of fatty acid acylation, substrate, and cofactor availability, protein-protein interactions, and amount of phosphorylation. Another significant epigenetic mechanism for NOS3 gene expression is differential promoter methylation [47].

The gene-encoding eNOS located on chromosome 7q35-36 and is composed of 26 exons (coding sequences) and introns (sequences between exons) with an entire length of 21 kb and has more than 168 polymorphisms [56]. Over the last few years, polymorphisms of the gene have been identified, and their association with various diseases has been explored. Genetic comparison studies on healthy people and cancer patients have shown that polymorphisms in eNOS are associated with the development of cancers.

A single nucleotide polymorphism (SNP), T-786C, was identified in the 5′ flanking region involving a substitution of thymine (T) to cytosine (C) at a locus 786 base pairs upstream [57]. Another common variant of eNOS with a G to T transversion at nucleotide position 894 (G894T) leading to a change in amino acid at 298 (Glu298Asp) has been reported [58], and a 27-bp variable number of tandem repeats (VNTR) polymorphism in intron 4 (intron 4b/4a) [59] and high numbers of CA, which have been repeated in intron 13 of eNOS gene, are also known to be associated with complex disorders. These polymorphisms seem to be functional and have been widely investigated for their associations with cancer risk [60, 61]. Molecular studies of eNOS −786T > C, intron 4b/4a, and 894G > T polymorphisms (**Figure 8**) if performed in large and unbiased can provide valuable insights into the association between the eNOS gene and breast cancer risk.

**Figure 8.** Organization of eNOS gene.

#### **8. Conclusion**

**6.4. Tumor progression/invasion**

190 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

carcinoma [53].

**6.5. Metastasis**

detected in tumor cells specifically in breast cancer [51].

NO• has been investigated regarding its possible involvement in the promotion of breast carcinoma. Increased amounts of NO• have been observed in the blood circulation of advanced grade breast cancer patients [49] where the increased levels of NO• have shown to promote tumor angiogenesis. Nitrotyrosine, a marker derived from NO•, was correlated with expression of VEGF-C and has been associated with lymph node metastasis in breast carcinoma patients, implicating the role of NO• in the development and progression of breast cancer [50]. In relation to the link of eNOS with cell proliferation, the eNOS expression has been

Several studies have observed NO• released by eNOS, can stimulate cancer cell cycle progression and proliferation. More specific to eNOS, studies have shown that the eNOS/NO• pathway plays a role in cancer cell DNA/RNA synthesis and proliferation apart from promoting angiogenesis [52]. The eNOS gene plays an essential role in endothelial cell proliferation in cell culture models and is a central mediator of several endothelium growth stimulators, such as vascular endothelial growth factor (VEGF) and prostaglandin E2. In human breast cancer, eNOS appears to be expressed in tumor epithelial cells, and its presence is correlated with histological grade and lymph node status. Higher NOS activity has been found in invasive breast tumors when compared with benign or normal breast tissue

Metastasis is a complex process by which the malignant cancer cells from the breast expand into other regions of the body. Lymphatic metastasis is a critical determinant of cancer prognosis. Recent findings indicate that eNOS mediates VEGF-C-induced lymph-angiogenesis and, consequently, plays a critical role in lymphatic metastasis [54]. Investigational studies on tumors have provided substantial evidence of the contributory role of NO• in tumor development, where series of tests were performed on tumor-bearing mice using NOS inhibitors showed a delayed tumor growth through eNOS inhibition and barred metastasis, signifying

The most fundamental level of NOS regulation is reflected in the tissue-specific expression of the different isoforms. The amount of NO produced results from the expression level and activity of eNOS. It is regulated by several interlinking mechanism such as transcriptional, posttranscriptional, and posttranslational modifications. The activity of NOS3 is also controlled by avid binding transcription factors namely Ets-1, Elf-1, Sp1, Sp3, and YY1 to the NOS3 promoter region. Posttranscriptionally, NOS3 activity is controlled by modifications of the primary transcript, stability of mRNA, its subcellular localization, and nucleocytoplasmic transport. Posttranslational modifications of NOS3 consist of fatty acid acylation, substrate, and cofactor

the potential role of endothelium-derived eNOS in metastasis [55].

**7. Regulation OF** *NOS3* **gene expression**

All these findings suggest that the expression of eNOS in breast cancer may be a critical event in carcinogenesis. Understanding different actions of NO• induced by eNOS in breast cancer at the molecular level can help in providing diagnostic or prognostic markers and also in devising potential strategies for prevention of breast cancer. The ability of many tumors to exploit eNOS/NO for a survival, proliferative, and metastatic advantage suggests that pharmacological use of eNOS inhibitors might attenuate these effects. Therefore, selective targeting of eNOS may prove a useful therapeutic or chemopreventive measure. However, further careful studies are needed to confirm the potential therapeutic role of eNOS as a novel target for breast cancer therapy.

### **Author details**

Tupurani Mohini Aiyengar, Padala Chiranjeevi and Hanumanth Surekha Rani\*

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Genetics, University College of Science, Osmania University, Hyderabad, Telangana State, India

#### **References**

at the molecular level can help in providing diagnostic or prognostic markers and also in devising potential strategies for prevention of breast cancer. The ability of many tumors to exploit eNOS/NO for a survival, proliferative, and metastatic advantage suggests that pharmacological use of eNOS inhibitors might attenuate these effects. Therefore, selective targeting of eNOS may prove a useful therapeutic or chemopreventive measure. However, further careful studies are needed to confirm the potential therapeutic role of eNOS as a novel target

Tupurani Mohini Aiyengar, Padala Chiranjeevi and Hanumanth Surekha Rani\*

Department of Genetics, University College of Science, Osmania University, Hyderabad,

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## **Nitric Oxide Synthase and Nitric Oxide Involvement in Different Toxicities**

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#### **Abstract**

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Nitric oxide (NO) is known to have a very short half‐life, and it is oxidized to nitrate (NO3 **−** ) and nitrite (NO2 **−** ). The activity and/or expression of nitric oxide synthases (NOSs) can change in response to toxins or therapeutic medications. For example, in recent studies in our laboratory and others, it has been reported that the amount of NO was increased in the serum of N‐nitroso compounds‐treated animals. N‐nitroso com‐ pounds, which are found in different types of foodstuffs, including meat, salted fish, alcoholic beverages, agricultural drugs, insecticides, cigarettes, and several vegetables, are known to have carcinogenic effects. In addition, it is experimentally used to induce liver carcinoma to study the mechanisms of liver cytotoxic injury. Uncontrolled, pro‐ longed, and/or massive production of NO by inducible NOS may cause liver damage, inflammation, and even tumor development during N‐nitroso compound toxicity. In this chapter, we explain the roles of NOS and NO in various toxicity conditions, such as toxicity in environment pollutant or food additive, and present the evaluation of the toxicity and the importance of NOSs in human health.

**Keywords:** nitric oxide synthase, nitrate, nitrite, nitric oxide, N‐nitroso compounds, toxicity

#### **1. Introduction**

Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) (EC: <IP_ADDRESS>) through oxidation of L‐arginine to L‐citrulline [1–5]. NO is a biologically significant molecule for many species from bacteria to mammals. Mechanisms for NO synthesis in an organism are extremely limited. Nitric oxide synthase enzyme is the only source of endogenous NO, except NO formed by metabolism of the nitro compounds entering the organism [6]. Three

© 2017 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.

 different types of NOS isoforms have been isolated from different tissues, such as vascular endothelium, brain, macrophage, and urinary system, of mammals, including (a) neuronal NOS (nNOS), (b) inducible NOS (iNOS), and (c) endothelial NOS (eNOS) [5, 7]. Neuronal NOS (nNOS, NOS1) is a Ca+2‐dependent, ~160 kDa enzyme that is found in the central and peripheral nervous system cells and striated muscle [4, 8]. Inducible NOS (iNOS, NOS2) is a calcium‐insensitive, ~130 kDa enzyme that was first isolated from activated macrophages and that can be activated by some cytokines (IL‐1, TNF, IF‐γ) or bacterial endotoxins [4, 9, 10]. Endothelial NOS (eNOS, NOS3) is a Ca+2/calmodulin‐dependent, ~135 kDa enzyme that is localized in vascular endothelial cells, hippocampal neural cells, pulmonary and renal epithe‐ lial cells, and cardiac myocytes [4, 9, 11].

Level of NO can be determined indirectly by measuring the concentration of nitrate (NO3 **−** ) and nitrite (NO2 **−** ) using an acidic Griess reaction. In recent studies in our laboratory and oth‐ ers, it has been reported that the amount of NO can change in response to various toxicity conditions [12–18] which are closely associated with animal and human disease conditions. In this chapter, we mention NO3 **−** and NO2 **−** , the molecules which are naturally found in foods as NO sources, agricultural activities such as the use of artificial fertilizers, polluted water, curing process to give a natural smell and taste to meat. The conversion of NO3 **−** and NO2 **−** into NO in the gastrointestinal tract, the effect of NOS, and possible mechanisms for how it is converted back into NO3 **−** and NO2 **−** in the bloodstream will also be covered. Studies on N‐nitrosamines, which are formed by reaction of NO3 **−** and NO2 **−** with amines, and which can be seen in cured meats, cigarette smoke, and rubber industry, reveal the carcinogenic effects of these molecules (**Figure 1**).

**Figure 1.** Formation of N‐nitroso compounds from NO3 − , NO2 − , NO, and their effects on human health.

#### **2. NO3 − and NO2 − as environment pollutants and food additives**

 different types of NOS isoforms have been isolated from different tissues, such as vascular endothelium, brain, macrophage, and urinary system, of mammals, including (a) neuronal NOS (nNOS), (b) inducible NOS (iNOS), and (c) endothelial NOS (eNOS) [5, 7]. Neuronal NOS (nNOS, NOS1) is a Ca+2‐dependent, ~160 kDa enzyme that is found in the central and peripheral nervous system cells and striated muscle [4, 8]. Inducible NOS (iNOS, NOS2) is a calcium‐insensitive, ~130 kDa enzyme that was first isolated from activated macrophages and that can be activated by some cytokines (IL‐1, TNF, IF‐γ) or bacterial endotoxins [4, 9, 10]. Endothelial NOS (eNOS, NOS3) is a Ca+2/calmodulin‐dependent, ~135 kDa enzyme that is localized in vascular endothelial cells, hippocampal neural cells, pulmonary and renal epithe‐

Level of NO can be determined indirectly by measuring the concentration of nitrate (NO3

ers, it has been reported that the amount of NO can change in response to various toxicity conditions [12–18] which are closely associated with animal and human disease conditions.

as NO sources, agricultural activities such as the use of artificial fertilizers, polluted water,

into NO in the gastrointestinal tract, the effect of NOS, and possible mechanisms for how

be seen in cured meats, cigarette smoke, and rubber industry, reveal the carcinogenic effects

− , NO2 −

**−**

and NO2

**−**

N‐nitrosamines, which are formed by reaction of NO3

**Figure 1.** Formation of N‐nitroso compounds from NO3

and NO2

**−**

curing process to give a natural smell and taste to meat. The conversion of NO3

**−**

) using an acidic Griess reaction. In recent studies in our laboratory and oth‐

**−**

and NO2

, the molecules which are naturally found in foods

in the bloodstream will also be covered. Studies on

**−**

, NO, and their effects on human health.

**−** )

**−**

**−**

with amines, and which can

and NO2

lial cells, and cardiac myocytes [4, 9, 11].

198 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**−**

In this chapter, we mention NO3

it is converted back into NO3

of these molecules (**Figure 1**).

and nitrite (NO2

Nitrogen is the basic element for essential micromolecules, such as amino acids, proteins, and nucleic acids. Nitrogen is the most abundant gas in the atmosphere; however, it has to be fixated before it is taken by plants and animals. Fixation is an important part of the nitrogen cycle. In this cycle, N2 is converted into ammonium and various nitrogen oxides. These higher nitrogen oxides are eventually gradually reduced. The nitrogen is then freed into the atmosphere and the cycle is completed. Bacteria play an important role in the cycle as they can catalyze each step, including the interconversion of different nitrogen oxides. NO3 − , NO2 − , and NO are all necessary intermediate products in the denitrification process and are catalyzed by NO3 − , NO2 − , and NO reductases, respectively [19]. Bacteria use these molecules as terminal electron acceptors in the absence of oxygen. The production and metabolism of nitrogen oxides also occur in mammals. NO3 − is easily converted into NO2 − in mammals by the activity of enzymes in both bacteria and mammals. The NO2 − then later can react with different molecules, such as amines, amides, and to form N‐nitroso compounds which can be carcinogenic [20]. Potentially carcinogenic or inert oxidizing molecules, such as NO3 − and NO2 − , occur as a result of endogenous NO metabolism during the food chain (**Figure 1**) [21].

Although nitrogen is found naturally in surface waters, its amount increases in many parts of the world. The reason for this is the pollution caused by commonly used inorganic fertilizers, soil drainage, or contamination of water resources by sewage [22]. The main causes of water pollution are pollution from industrial and agricultural activities. Chemical fertilizers used in agricultural production have an important role. NO3 **−** is applied in increasing amounts in the fertilizers for agricultural production, and they accumulate in the soil. This accumulated NO3 **−** , in varying amounts depending on the conditions, moves toward the deeper parts of the ground especially with rainwater, and some of it reaches underground and some to surface waters. High NO3 **−** concentrations in water resources pose a potential risk to human health, because sunlight and some bacteria can easily convert NO3 **−** into NO2 **−** [23]. NO3 **−** and NO2 **−** can also occur spontaneously in vegetables and fruits consumed by humans and especially in animal feed [24]. Vegetables and fruits usually receive NO3 **−** and NO2 **−** from the soil [25]. As a result of nitrogenous fertilizers being used in excess to increase appearance and yield in the plants, plants store NO3 **−** in excess of their need. When the amount of received NO3 **−** is high, the reduction to ammonia is limited and NO2 **−** accumulates as an intermediate metabolism product [26, 27].

Excess NO3 **−** and NO2 **−** can also be utilized to cure meats. In order to improve the taste, pro‐ tection, appearance, and quality of the meat, NO3 **−** and NO2 **−** are used for curing purposes. NO2 **−** can also be used as a preservative against the proliferation of microorganisms, especially *Clostridium botulinum*. It also inhibits lipid peroxidation and prevents putrefaction [28].

Dietary NO3 **−** and NO2 **−** , which are taken by the organisms, could cause various physiological and pathological outcomes.

#### **3. Conversion of NO3 − and NO2 − into NO in the gastrointestinal system: possible role of NOSs**

In an organism, NO2 **−** is converted into NO in three ways:

In this section, the conversion of NO3 **−** and NO2 **−** , taken from foods in the gastrointestinal tract, into NO will be mentioned. At the end of this pathway, NO3 **−** and NO2 **−** are synthesized again from NO. This synthesis in the tissues is catalyzed by NOSs. With the identification of the NO3 **−** ‐NO2 **−** ‐NO pathway, the importance of the diet, rather than the biological significance of systemic NO3 **−** and NO2 **−** , in the physiological regulation of NO has arisen. NO3 **−** , rich in green leafy vegetables such as beetroot, is reduced to NO2 **−** by bacterial NO3 **−** reductase in the com‐ mensal anaerobic microflora in the oral cavity by saliva secretion and is reduced to NO in the stomach [29–31]. The highest NO concentration is obtained from an acidic stomach pH after a NO3 **−** ‐rich meal (**Figure 1**) [29].

The rapid postprandial increase in gastric NO is directly proportional to many actions, such as mucus production in the gastrointestinal tract, increased vascular tone, antimicrobial effect, and immunomodulation. It has also been shown that this increased NO is related to many physiological mechanisms, such as the prevention of ischemia‐reperfusion injury and increased cerebral blood flow [21, 29].

How endogenous NO3 **−** and NO2 **−** can be synthesized in the body if NO3 **−** and NO2 **−** are not taken into the body with nutrients? The inorganic NO3 **−** and NO2 **−** , which cannot be taken up with nutrients in starving mammals, are mainly derived from NOSs. These enzymes form NO by using l‐arginine and oxygen, and then this NO is rapidly degraded to NO3 **−** and NO2 **−** . Endogenous NO3 **−** and NO2 **−** are synthesized in this way [30]. Endogenous NO can also be produced by NOSs using NO3 **−** and NO2 **−** in our daily nutrients [30, 32].

NO is a highly diffusible free radical that participates in various in vivo signal pathways and is involved in critical physiological events, such as regulation of vascular tone and immune response [31]. NO also exhibits antimicrobial activity [33] other than its regulatory role in vascular tone. Numerous intracellular pathogenic parasites [34] and bacteria [35] are susceptible to NO.

NO2 **−** in saliva is converted non‐enzymatically into NO and some other nitrogen oxide species when it enters into the stomach with low pH [30]. Increasing number of studies on cardio‐ vascular, inflammatory, and gastrointestinal diseases reported that the NO‐related effects of dietary NO3 **−** and NO2 **−** are protective and preventive. Recent data suggest that these anions are beneficial to gastrointestinal cancer [36] and cardiovascular diseases rather than having harmful effects. Dietary NO3 **−** and salivary NO2 **−** have been shown to protect gastric mucus from experimentally induced gastric damage by increasing gastric mucus thickness and mucosal blood flow [30].

NO plays an important role for the intestine. It is produced from arginine by eNOS and iNOS in a reaction catalyzed in the intestine. eNOS is structurally expressed in low levels in intestinal microcapillaries and is responsible for the initial levels of NO. Low NO lev‐ els regulate vascular tone and mucosal blood flow in cyclic guanosine monophosphate and neuron‐dependent manner and are also crucial for mucosal homeostasis. Additionally, NO can protect from oxidative stress by diminishing oxygen radicals. eNOS‐derived NO facilitates leucocyte uptake by supporting endothelial adhesion of leukocytes. iNOS is upregulated during inflammation and increases NO synthesis. NO also allows dilatation of capillary vessels. Excess NO secreted during inflammation has harmful effects on the intestinal barrier [37, 38].

**3. Conversion of NO3**

**possible role of NOSs**

**−**

200 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

(c) NO can be produced by acidification of NO2

**−**

leafy vegetables such as beetroot, is reduced to NO2

into NO will be mentioned. At the end of this pathway, NO3

In this section, the conversion of NO3

and NO2

‐rich meal (**Figure 1**) [29].

increased cerebral blood flow [21, 29].

**−**

produced by NOSs using NO3

**−**

and NO2

and NO2

**−**

**−**

**−**

taken into the body with nutrients? The inorganic NO3

**−**

and NO2

How endogenous NO3

Endogenous NO3

NO2 **−**

dietary NO3

**−**

mucosal blood flow [30].

harmful effects. Dietary NO3

and NO2

**−**

**−**

In an organism, NO2

NO3 **−** ‐NO2 **−**

a NO3 **−**

systemic NO3

**−**

 **and NO2**

**−**

 is converted into NO in three ways: (a) It is enzymatically (via NOSs) reduced to NO in the circulation and tissues.

at low pH and hypoxic conditions that occur during intense exercise.

**−**

and NO2

(b) It is non‐enzymatically reduced to NO in acidic stomach environment, capillary beds, and

**−**

from NO. This synthesis in the tissues is catalyzed by NOSs. With the identification of the

mensal anaerobic microflora in the oral cavity by saliva secretion and is reduced to NO in the stomach [29–31]. The highest NO concentration is obtained from an acidic stomach pH after

The rapid postprandial increase in gastric NO is directly proportional to many actions, such as mucus production in the gastrointestinal tract, increased vascular tone, antimicrobial effect, and immunomodulation. It has also been shown that this increased NO is related to many physiological mechanisms, such as the prevention of ischemia‐reperfusion injury and

with nutrients in starving mammals, are mainly derived from NOSs. These enzymes form

NO is a highly diffusible free radical that participates in various in vivo signal pathways and is involved in critical physiological events, such as regulation of vascular tone and immune response [31]. NO also exhibits antimicrobial activity [33] other than its regulatory role in vascular tone.

 in saliva is converted non‐enzymatically into NO and some other nitrogen oxide species when it enters into the stomach with low pH [30]. Increasing number of studies on cardio‐ vascular, inflammatory, and gastrointestinal diseases reported that the NO‐related effects of

are beneficial to gastrointestinal cancer [36] and cardiovascular diseases rather than having

from experimentally induced gastric damage by increasing gastric mucus thickness and

**−**

NO by using l‐arginine and oxygen, and then this NO is rapidly degraded to NO3

**−**

Numerous intracellular pathogenic parasites [34] and bacteria [35] are susceptible to NO.

and salivary NO2

‐NO pathway, the importance of the diet, rather than the biological significance of

**−**

can be synthesized in the body if NO3

**−**

in our daily nutrients [30, 32].

are protective and preventive. Recent data suggest that these anions

and NO2

are synthesized in this way [30]. Endogenous NO can also be

**−**

, in the physiological regulation of NO has arisen. NO3

**−**

in the oral cavity.

**−**

by bacterial NO3

and NO2

 **into NO in the gastrointestinal system:**

, taken from foods in the gastrointestinal tract,

**−**

**−**

**−**

have been shown to protect gastric mucus

and NO2

, which cannot be taken up

**−**

**−** are not

and NO2

**−** .

are synthesized again

reductase in the com‐

, rich in green

**−**

NO reacts with the superoxide ion to form a reactive oxygen and nitrogen type, peroxynitrite, which can be harmful for epithelial cells. It can induce enterocyte cell apoptosis and inhibit proliferation. iNOS is expressed in intestinal smooth muscle cells, endothelial and epithelial cells [38]. During inflammatory conditions in the intestine, such as necrotizing enterocolitis [39], ulcer [29], and colon cancer [40], expression of iNOS mRNA increases.

Numerous studies have shown that NO3 **−** and NO2 **−** obtained from pharmacologic supple‐ ments or diet have obvious effects on gastrointestinal function [29, 39, 40]. However, it is still unclear whether endogenous NO3 **−** and NO2 **−** derived from NOSs in the endothelium and elsewhere affect gastric function. This situation has been tried to be illuminated in germ‐free and starved animals [30, 41].

The gastric NO levels are very low in germ‐free animals lacking microflora even after dietary NO3 **−** load. A significant amount of NO2 **−** is produced in the saliva even in the case of fasting, indicating NO3 **−** production due to endogenous NOS production [30]. Petersson et al. [30] reported that three doses of NO2 **−** given to germ‐free rats not only increased stomach mucus thickness by more than fourfold but also unexpectedly had an effect in the non‐NO2 **−** group. This suggests that endogenous NO3 **−** from NOSs also plays a role in the regulation of gastric physiology. In a similar study in humans, individuals were given a low NO3 **−** diet with an antibacterial mouthwash containing chlorhexidine to lower the reduction of oral NO3 **−** , and it was determined that the levels of circulating NO2 **−** lowered, and this then increased the blood pressure [42]. These studies show that NO3 **−** and NO2 **−** have a NO3 **−** ‐NO2 **−** ‐NO pathway that starts from the mouth and ends in the mouth through digestive and circulatory system. It is possible that NO3 **−** and NO2 **−** in the circulation and saliva may originate from the endogenous NOS pathway.

In addition to NO3 **−** , NO2 **−** , NO, sodium nitrite (NaNO2 ) is an inorganic compound taken by endogenous sources. NaNO2 may have some beneficial and undesirable effects. NaNO2 is a preservative used in processed meats, such as salami and bacon. NaNO2 is synthesized by several chemical reactions, including the reduction of sodium nitrate. NaNO2 is used as an additive in foods. There are some suspects about the safety of use in foods, but it is still being used, and, on the contrary, there is information that NaNO2 may actually be healthy [43]. There are studies on the effects of NaNO2 on human health from 1945 [44] to present date [45].

NO3 **−** salts are used as a cheap nitrogen source in fertilizers. Therefore, with the widespread use of nitrogenous fertilizers in agriculture and inappropriate disposal of nitrogenous wastes, humans are exposed to high NO3 **−** /NO2 **−** levels at an alarming rate, especially through contam‐ inated food and water [46]. Infants and individuals with deficiency of glucose‐6‐phosphate dehydrogenase are particularly sensitive to high levels of NO3 **−** /NO2 **−** [45]. The digestive sys‐ tem of newborn babies convert NO3 **−** to NO2 **−** , and NO2 **−** reacts with hemoglobin and prevents oxygen transport to the tissues. As a result, "methemoglobinemia" known as "blue baby syn‐ drome" occurs in infants [47].

Prolonged non‐lethal exposure to high levels of NO3 **−** /NO2 **−** may cause respiratory failure, growth failure, diabetes, neurological disorders, and cancer [48]. NaNO2 causes oxidative stress in human erythrocytes in vitro by increasing lipid and protein oxidation, osmotic fra‐ gility, and membrane damage [49].

Despite the fact that NaNO2 has not been reliable in the past and has the potential to cause many cancers, it has recently been reported that it can prevent myocardial ischemia‐reperfu‐ sion injury in diabetic rats by regulating eNOS and iNOS expression and inhibiting lipid peroxidation in the heart [50]. It also prevents hypertension and increases endothelium‐ dependent relaxation and total NO by regulating eNOS activity [51].

Serum malondialdehyde, NO, arginase, and glutathione S‐transferase activities were incre‐ ased, and glutathione and catalase activities were decreased in NaNO2 ‐treated rats [52]. In another study, decrease in GSH and catalase activity was reported in NaNO2 ‐intoxicated rats [53]. In their study investigating the histopathological, biochemical, and genotoxic effects of low dose NaNO2 administration for 8 months, Ozen et al. [54] reported that the liver and kid‐ ney NO levels were decreased in rats. The reduction in NO levels may be explained by the rapid and/or efficient removal of this molecule from these tissues, resulting in an increase in serum levels due to reduced NO by metabolic depletion. Therefore, the investigators reported that the increased serum NO level did not contradict with the decreasing NO level in the tis‐ sues [54]. In addition, these and other investigators indicated that chronic administration of NaNO2 increased iNOS activity in experimental animals [54, 55].

Peroxynitrite can interact with tyrosine residues to form nitrotyrosine. Ozen et al. [54] showed that the expression of iNOS and nitrotyrosine was increased in the liver and kidney tissues of NaNO2 ‐treated mice, and it caused tissue degeneration in both organs. Peroxynitrite can be decomposed to form NO3 **−** and NO2 **−** which can cause DNA damage, as well [56, 57].

The mechanisms of NaNO2 are still not fully understood; this suggests that further work needs to be performed in the future.

#### **4. N‐nitroso compounds**

#### **4.1. The chemical structure and sources of N‐nitroso compounds**

NO2 **−** is the precursor of N‐nitroso compounds that have carcinogenic effect [58, 59]. NO2 **−** is converted into nitrous acid in acidic environment, and nitrous acids react with secondary amines to form nitrosamine compounds (**Figure 2**) [60].

Nitric Oxide Synthase and Nitric Oxide Involvement in Different Toxicities http://dx.doi.org/10.5772/intechopen.68494 203

**Figure 2.** Formation of nitrosamine from NO3 − .

humans are exposed to high NO3

202 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

tem of newborn babies convert NO3

gility, and membrane damage [49].

Despite the fact that NaNO2

low dose NaNO2

NaNO2

NaNO2

NO2 **−**

decomposed to form NO3

The mechanisms of NaNO2

**4. N‐nitroso compounds**

needs to be performed in the future.

drome" occurs in infants [47].

**−** /NO2 **−**

> **−** to NO2 **−**

growth failure, diabetes, neurological disorders, and cancer [48]. NaNO2

dependent relaxation and total NO by regulating eNOS activity [51].

ased, and glutathione and catalase activities were decreased in NaNO2

increased iNOS activity in experimental animals [54, 55].

**−**

**−**

and NO2

**4.1. The chemical structure and sources of N‐nitroso compounds**

amines to form nitrosamine compounds (**Figure 2**) [60].

another study, decrease in GSH and catalase activity was reported in NaNO2

dehydrogenase are particularly sensitive to high levels of NO3

Prolonged non‐lethal exposure to high levels of NO3

inated food and water [46]. Infants and individuals with deficiency of glucose‐6‐phosphate

oxygen transport to the tissues. As a result, "methemoglobinemia" known as "blue baby syn‐

stress in human erythrocytes in vitro by increasing lipid and protein oxidation, osmotic fra‐

many cancers, it has recently been reported that it can prevent myocardial ischemia‐reperfu‐ sion injury in diabetic rats by regulating eNOS and iNOS expression and inhibiting lipid peroxidation in the heart [50]. It also prevents hypertension and increases endothelium‐

Serum malondialdehyde, NO, arginase, and glutathione S‐transferase activities were incre‐

[53]. In their study investigating the histopathological, biochemical, and genotoxic effects of

ney NO levels were decreased in rats. The reduction in NO levels may be explained by the rapid and/or efficient removal of this molecule from these tissues, resulting in an increase in serum levels due to reduced NO by metabolic depletion. Therefore, the investigators reported that the increased serum NO level did not contradict with the decreasing NO level in the tis‐ sues [54]. In addition, these and other investigators indicated that chronic administration of

Peroxynitrite can interact with tyrosine residues to form nitrotyrosine. Ozen et al. [54] showed that the expression of iNOS and nitrotyrosine was increased in the liver and kidney tissues of

is the precursor of N‐nitroso compounds that have carcinogenic effect [58, 59]. NO2

converted into nitrous acid in acidic environment, and nitrous acids react with secondary

‐treated mice, and it caused tissue degeneration in both organs. Peroxynitrite can be

which can cause DNA damage, as well [56, 57].

are still not fully understood; this suggests that further work

administration for 8 months, Ozen et al. [54] reported that the liver and kid‐

, and NO2

**−**

**−** /NO2 **−**

levels at an alarming rate, especially through contam‐

[45]. The digestive sys‐

causes oxidative

‐treated rats [52]. In

‐intoxicated rats

**−** is

reacts with hemoglobin and prevents

may cause respiratory failure,

**−** /NO2 **−**

has not been reliable in the past and has the potential to cause

**Figure 3.** Chemical formula for nitrosamine.

Nitrosamines are chemical compounds with general formulas as shown in (**Figure 3**).

Nitrosamines are used in the manufacture of some cosmetics, pesticides, and most rubber products [61, 62]. Nitrosamines are found in latex products, cereal, tea, many foods, ciga‐ rettes, and cigarette smoke [60]. They are also formed by the reduction of NO3 **−** , which is abundant in nature, into NO2 **−** by bacteria [27].

The most commonly used N‐nitroso compounds for the purpose of toxicity are N‐nitrosodi‐ methylamine, N‐nitrosodiethylamine, N‐nitrosopyrrolidine, and N‐nitrosopiperidine [63, 64].

Dimethylnitrosamine (also called N‐nitrosodimethylamine, DMN, DMNA, NDMA—C2 H6 N2 O) is found in wheat flour, cheese, smoked meat, fish, and other food products (**Figure 4**) [65]. It can be formed by reaction of dimethylamine with NO2 **−** . In addition, it can be formed by nitro‐ zation and decarboxylation of amino acids, such as glycine and alanine [66].

**Figure 4.** Chemical formula for dimethylnitrosamine.

Diethylnitrosamine (also called N‐nitrosodiethylamine, DEN, DENA, NDEA—C4 H10N2 O) is found in chemicals used in agriculture and rubber industry, cigarette smoke, alcoholic bever‐ ages, and processed meat products (**Figure 5**) [67]. It can be formed by reaction of diethyl‐ amine with NO2 **−** . Additionally, it can be formed by nitrozation and decarboxylation of amino acids, such as glycine alanine [66].

**Figure 5.** Chemical formula for diethylnitrosamine.

N‐nitrosopyrrolidine (also called NPYR—C4 H8 N2 O) is found in cigarette smoke, meat and fish products (**Figure 6**) [68]. In meat products, this compound is formed by the nitrozation and decarboxylation of l‐proline [69].

**Figure 6.** Chemical formula for N‐nitrosopyrrolidine.

N‐nitrosopiperidine (also called NPIP—C5 H10N2 O) formation follows these steps: decarbox‐ ylation of lysine results in cadaverine; cadaverine is converted into piperidine by heat and the reaction of the resulting piperidine with NO2 **−** (**Figure 7**) [69, 70].

**Figure 7.** Chemical formula for N‐nitrosopiperidine.

N‐nitrosamines can be found in food products and might cause serious health problems for humans.

#### **4.2. N‐nitrosamines in meat and dairy products**

N‐nitrosamines taken with food and found in the environment have been found to cause serious health risks above a certain level, even though they have both food processing and protection functions as additives.

Nitrosamines occur mostly in meat and dairy products [71]. Since meat, an important nutri‐ ent, is easily decomposed by different factors, it is necessary to protect it with various meth‐ ods and to increase its durability. For this purpose, some ingredients are added to meat and meat products. Cured meat products, unlike fresh meat or salted meat with only table salt, have a pleasant smell, flavor, and a natural looking but a heat‐resistant color. Today, this process is applied to most of the meat products consumed [72, 73]. It has been reported that the degradation products of NO3 **−** and NO2 **−** result in the formation of carcinogenic nitrosa‐ mines by combining with amino acids, such as putrescine, thiamine, piperidine, pyrrolidine, histamine, cadaverine, trimethylamine, β‐phenylethylamine, n‐propylamine, and isopropyl‐ amine [74–77].

The most important sources of nitrosamines in dairy products, such as cheese and butter, are NO3 **−** , NO2 **−** , and amine compounds [78]. The metabolic activities of some microorganisms in milk and dairy products result in the formation of histamine and tyramines, and nitrosamines are formed by the reaction of the secondary amines, which are the degradation products of these biogenic amines in various ways, with NO2 **−** [65, 69].

The first formation of these nitrosamines in meat and dairy products is seen in the oral cavity [79]. The salivary secretion contains abundant NO3 **−** and this NO3 **−** is reduced to NO2 **−** by nitrate reductase enzyme [72]. This NO2 **−** causes the formation of nitrosamines [80]. These compounds can be taken into the stomach in various ways, such as ingestion or smoking, or can also be formed by the reaction of NO2 **−** and amines in acidic conditions [81]. Some bacteria in the stomach and intestines increase the formation of nitrosamines by facilitating the conversion of NO3 **−** into NO2 **−** . NO2 **−** transforms into nitrous acid in the acidic environment of the stom‐ ach, and nitrous acid reacts with amines in the environment to form nitrosamines [60, 82]. Nitrosamines are usually excreted through urine [83, 84].

### **5. The roles of NOS isoforms and** *N***‐nitrosamine compounds in liver toxicity or carcinogenesis**

NO3 **−** can easily be formed in mammalian systems through bacterial and mammalian enzymes. The resulting NO3 **−** can then react with amines, amides, and amino acids to form N‐nitroso compounds. While NO3 **−** has relatively low toxicity, NO2 **−** and N‐nitroso compounds have higher toxicity in mammals. For this reason, there are many studies investigating the toxic‐ ity of these two molecules, as well as studies investigating ways to decrease the detrimental effects of these two molecules [20]. It has been suggested in long‐term experimental animal studies that nitrosamines cause cancer in many tissues, but the role of nitrosamines in the formation of cancers is still being investigated.

#### **5.1. NOS isoforms in carcinogenesis**

N‐nitrosopyrrolidine (also called NPYR—C4

N‐nitrosopiperidine (also called NPIP—C5

**Figure 6.** Chemical formula for N‐nitrosopyrrolidine.

reaction of the resulting piperidine with NO2

**Figure 7.** Chemical formula for N‐nitrosopiperidine.

**4.2. N‐nitrosamines in meat and dairy products**

protection functions as additives.

humans.

and decarboxylation of l‐proline [69].

**Figure 5.** Chemical formula for diethylnitrosamine.

204 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

H8 N2

fish products (**Figure 6**) [68]. In meat products, this compound is formed by the nitrozation

H10N2

**−**

ylation of lysine results in cadaverine; cadaverine is converted into piperidine by heat and the

N‐nitrosamines can be found in food products and might cause serious health problems for

N‐nitrosamines taken with food and found in the environment have been found to cause serious health risks above a certain level, even though they have both food processing and

Nitrosamines occur mostly in meat and dairy products [71]. Since meat, an important nutri‐ ent, is easily decomposed by different factors, it is necessary to protect it with various meth‐ ods and to increase its durability. For this purpose, some ingredients are added to meat and

(**Figure 7**) [69, 70].

O) is found in cigarette smoke, meat and

O) formation follows these steps: decarbox‐

Various studies have indicated that three NOS isoforms both trigger and prevent cancer etiol‐ ogy. Nitric oxide synthase activity has been detected in a variety of tumor cells, and it has been shown to be closely related to tumor grade, proliferation rate, and cancer development. High NOS expression can be cytotoxic for cancer cells. On the other hand, low NOS expression may have an adverse effect and may increase tumor development [85, 86]. For this reason, NOS can be both genotoxic and angiogenic. High NO production leads to angiogenesis by increas‐ ing the VEGF gene, especially in p53 mutant cells. In addition, NO can alter the expressions of DNA repair proteins, such as poly (ADP‐ribose) polymerase and DNA‐protein kinase in tumor cells. NO may exhibit carcinogenic effect by the production of different NO metabo‐ lites. For example, NO may rapidly react with intracellular environment to form N‐nitroso compounds. These metabolites, for example, can cause genotoxic effects by creating DNA damage [86]. In some other studies, N‐nitroso compounds have been reported to alter the activity of creatine kinase, lactate dehydrogenase [87], pyruvate kinase [88], and Na/K‐ATPase [89] enzymes.

#### **5.2.** *N***‐nitrosamine compounds in liver toxicity and cancer**

Certain levels of nitrosamines taken in the body with any food ingredient are less likely to cause cancer in the human body alone. However, different types of nitrosamines, which are continuously taken from different sources, such as air and cigarettes, increase the risk of developing cancer [65, 90]. Although NO3 **−** and NO2 **−** create a toxicological problem, the main problem is that they turn into nitrosamines, which are carcinogenic. Nitrosamines are known to exhibit carcinogenic effect through binding to proteins and nucleic acids [91]. Nitrosamines also have mutagenic and teratogenic effects [61]. Because many organ‐specific nitrosamines are metabolized in the same way in human and animal tissues, humans are very sensitive to the carcinogenic properties of nitrosamines [67]. N‐nitroso compounds are potent alkylat‐ ing agents that can form endogenously and can cause cancer in surrounding animals [64]. Bacterial decarboxylation of amino acids in NO3 **−** taken with nutrients results in amines and amides [58]. There is a relationship between the formation of N‐nitroso compounds by bacte‐ rial catalysis and increased risk for liver, stomach, esophagus, nasopharynx, chronic urinary tract infections, and bladder squamous cell carcinoma [77, 92].

Metabolic activations of the nitrosamines occur primarily in the liver, and this transfor‐ mation can occur in all cells. Dimethylnitrosamine is a potent carcinogen that can induce malignant tumors in various animal species in various tissues, including the liver, lungs, and stomach [79]. Various studies on different species of mice have shown that adenomas and adenocarcinomas in the lungs and hepatocarcinoma in the liver are formed by dimeth‐ ylnitrosamine. The target organs of dimethylnitrosamine are the liver, lungs, and kidneys [65, 79].

It is suggested that diethylnitrosamine metabolism is catalyzed by the enzymes of the multi‐ functional cytochrome P‐450 monooxygenase system and toxic effects are initiated by its meta‐ bolic activation, and that the resulting reactive intermediate products have little affinity for the catalytic domains of the binding enzymes, so that instead of being excreted with urine, they stimulate the onset of mutation, cancer, and necrosis by forming covalent bonds with impor‐ tant cellular components [93, 94]. Low concentrations of diethylnitrosamine cause mutations and cancer which was shown by the Ames assay [94]. Many studies suggest that the harmful effects of diethylnitrosamine may be reduced by various antioxidant molecules. The adminis‐ tration of molecules such as α‐lipoic acid [95], omega‐3 [96], blueberry [97], and beta‐carotene [98] were stated to reduce the carcinogenic effect of nitrosamines in experimental animals.

N‐nitrosopiperidine and N‐nitrosopyrrolidine are structurally cyclic nitrosamines with dif‐ ferent carcinogenic activities. Comparative carcinogenicity studies of these two nitrosamines in rats revealed that N‐nitrosopiperidine caused esophagus, liver, and stomach tumors, and N‐nitrosopyrrolidine caused tumors mainly in the liver [63].

#### **6. Conclusion**

have an adverse effect and may increase tumor development [85, 86]. For this reason, NOS can be both genotoxic and angiogenic. High NO production leads to angiogenesis by increas‐ ing the VEGF gene, especially in p53 mutant cells. In addition, NO can alter the expressions of DNA repair proteins, such as poly (ADP‐ribose) polymerase and DNA‐protein kinase in tumor cells. NO may exhibit carcinogenic effect by the production of different NO metabo‐ lites. For example, NO may rapidly react with intracellular environment to form N‐nitroso compounds. These metabolites, for example, can cause genotoxic effects by creating DNA damage [86]. In some other studies, N‐nitroso compounds have been reported to alter the activity of creatine kinase, lactate dehydrogenase [87], pyruvate kinase [88], and Na/K‐ATPase

Certain levels of nitrosamines taken in the body with any food ingredient are less likely to cause cancer in the human body alone. However, different types of nitrosamines, which are continuously taken from different sources, such as air and cigarettes, increase the risk of

and NO2

problem is that they turn into nitrosamines, which are carcinogenic. Nitrosamines are known to exhibit carcinogenic effect through binding to proteins and nucleic acids [91]. Nitrosamines also have mutagenic and teratogenic effects [61]. Because many organ‐specific nitrosamines are metabolized in the same way in human and animal tissues, humans are very sensitive to the carcinogenic properties of nitrosamines [67]. N‐nitroso compounds are potent alkylat‐ ing agents that can form endogenously and can cause cancer in surrounding animals [64].

**−**

amides [58]. There is a relationship between the formation of N‐nitroso compounds by bacte‐ rial catalysis and increased risk for liver, stomach, esophagus, nasopharynx, chronic urinary