Oxidative Stress leads to Altered, Nitric Oxide Levels

The biological rationale for the relationship between increased oxidative stress and altered NO levels is well-supported by the literature, validated by many studies presented in this area of research. The biological mechanism of this relationship is well-known and widely accepted. The reaction between NO and free radicals leads to the creation of peroxynitrite, which results in reduced NO bioavailability (Incalza et al., 2018; Mitchell et al., 2019; Nagane et al., 2021; Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). In addition, in vascular tissues, increased levels of O2- or peroxynitrite can oxidize BH4 and lead to uncoupled eNOS (Forstermann, 2010; Matsubara et al., 2015; Soloviev & Kizub, 2019). When uncoupled, eNOS transfers electrons to O2- rather than L-arginine, causing O2- production instead of NO production, further reducing NO bioavailability. Although much of the biological plausibility indicates that NO decreases with oxidative stress, ROS have been associated with increased NO as well (Nagane et al., 2021; Soloviev & Kizub, 2019). This is likely due to the complexity of NO regulation in signaling pathways, upregulation of inducible nitric oxide synthase (iNOS), as well as variations in stressors, doses, dose rates, models, duration of study and diseases present in studies (Nagane et al., 2021). 

The empirical data for this KER somewhat supports the relationship of increased oxidative stress eventuating in altered NO levels. The evidence was gathered from both in vivo and in vitro models. Gamma rays (Abdel-Magied & Shedid, 2020; Hasan, Radwan & Galal, 2019; Soucy et al., 2010), X-rays (Cervelli et al., 2017; Yan et al., 2020), altered gravity (Zhang et al., 2009) and heavy ions (Soucy et al., 2011) were used as stressors with dose levels ranging from 0.25 to 10 Gy. Direct and indirect measures of NO, including iNOS, eNOS and nitrite levels, were used as endpoints to investigate the effect of increased oxidative stress on NO levels. Evidence from radiation sources that induce oxidative stress in human umbilical vein endothelial cells (HUVECs) or rat aorta have demonstrated a change in NO levels (Abdel-Magied & Shedid, 2020; Cervelli et al., 2017; Hasan, Radwan & Galal, 2019; Soucy et al., 2010; Soucy et al., 2011; Yan et al., 2020; Zhang et al., 2009). 

Dose Concordance 

Moderate evidence is available within current literature that demonstrates dose-concordance between increased oxidative stress and altered NO levels. Elevations in the production of ROS have been reported with exposure to altered gravity and different doses (0.25 Gy, 1 Gy, 4 Gy, 5 Gy, and 10 Gy) of radiation in HUVECs and rat aorta/tissue models, and provide supporting evidence that an increase in oxidative stress can lead to subsequent alterations in NO levels (Abdel-Magied & Shedeed, 2020; Cervelli et al., 2017; Hasan, Radwan & Galal, 2019; Sakata et al., 2015; Soucy et al., 2010; Soucy et al., 2011; Yan et al., 2020; Zhang et al., 2009). For example, a study involving HUVEC X-ray irradiation at 0.25 Gy found increases in both ROS and nitrite/nitrate levels (Cervelli et al., 2017). As well, a study involving 1 Gy of iron ions demonstrated elevations in ROS production with accompanying reductions in NO levels using the DAF-FM DA fluorescent probe (Soucy et al., 2011).  

At high doses (>2 Gy), dose concordance between increased oxidative stress and altered nitric oxide can be observed. For example, 5 Gy gamma irradiation leads to increased ROS production with decreased NO levels measured using the DAF-FM DA fluorescent probe (Soucy et al., 2010) Gamma irradiation of rats with 6 Gy led to changes in oxidative stress indices that showed an increase in ROS, while iNOS levels were also increased (Hasan, Radwan & Galal, 2019). Rat cardiac tissue exposed to 8 Gy gamma irradiation also showed an increase in ROS through oxidative stress indices and an increase in nitrite/nitrate content (Abdel-Magied & Shedid, 2020). A study investigating HUVECs and rat aorta/tissue responses to 10 Gy and 4 Gy X-ray radiation, respectively, showed reduced eNOS dimerization and nitrite levels with elevations in ROS production in both models (Yan et al., 2020). Also in HUVECs, 10 Gy X-rays resulted in increased ROS, increased p-eNOS (Ser1177), decreased p-eNOS (Thr495), increased citrulline and increased NOx (nitrite and nitrate, NO proxies) (Sakata et al., 2015). NOx was also significantly increased after 20 Gy (Sakata et al., 2015). 

Simulated microgravity showed increased eNOS and iNOS in rats, while superoxide levels, although not quantitatively shown, increased following altered gravity (Zhang et al., 2009). Although, the authors state that the increase in NOS would likely cause decreased NO production because of NOS uncoupling and peroxynitrite production (Zhang et al., 2009). 

Time Concordance 

Very few studies demonstrate time concordance of this relationship. In HUVECs irradiated with 0.25 Gy of X-rays, ROS levels increased after 45 minutes, while nitrite and nitrate levels increased after 24 hours. In rats exposed to 6 Gy of gamma rays, both oxidative stress indices and iNOS increased 4 weeks post-irradiation (Hasan, Radwan & Galal, 2019). 

Incidence concordance 

There is moderate evidence of incidence concordance for this relationship. Yan et al. (2020) showed both in vivo in a rat model irradiated with 4 Gy of X-rays and in vitro in HUVECs irradiated with 10 Gy of X-rays that superoxide levels increased more than NO and eNOS were decreased following irradiation. Rats irradiated with 1 Gy of 56Fe ions showed a 1.8-fold increase in ROS and just a 0.8-fold decrease in NO levels (Soucy et al., 2011). Similarly, gamma irradiation of rats at 5 Gy resulted in a 1.7-fold increase in ROS and a 0.7-fold decrease in NO levels (Soucy et al., 2010). In HUVECs irradiated with 10 Gy of X-rays, ROS increased 15.5-fold while NOx levels increased just 10-fold (Sakata et al., 2015). 

Essentiality    

Studies scrutinizing the usefulness of antioxidants in inhibiting oxidative stress show a moderately supported relationship between elevated oxidative stress and altered NO levels. Antioxidants have been implicated in the activation of the NOS family of enzymes by preventing BH4 oxidation, thereby increasing NO bioavailability (Kojsova et al., 2006). One study observed that treatment with bradykinin potentiating factor (BPF), which can affect eNOS regulation through the renin aldosterone angiotensin system, contributed to elevations in the antioxidant glutathione (GSH) and a marker of antioxidant potential, ferric reducing antioxidant power (FRAP) (Hasan, Radwan & Galal, 2019). BPF contributed to reductions in malondialdehyde (MDA), a biomarker for oxidative stress, while also increasing iNOS levels (Hasan, Radwan & Galal, 2019). 

Another study demonstrated that a composition of antioxidants (resveratrol, extramel, seleno-L-methionine, Curcuma longa, reduced glutathione, and vitamin C (RiduROS) inhibited increases in ROS while restoring NO levels (Cervelli et al., 2017). Furthermore, rat mesenteric arteries and HUVECs treated with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of Gch1 that is important for BH4 synthesis, lowered both the dimer:monomer eNOS ratio and the nitrite (measure of NO) concentration, and increased superoxide following irradiation (Yan et al., 2020). The notable elevation in O2- demonstrates that inhibiting Gch1 and limiting BH4 activity can prevent eNOS activity and elevate ROS Levels (Yan et al., 2020). 

In addition, two studies demonstrated that treatment with oxypurinol (OXP), a xanthine oxidase (XO) inhibitor where XO is responsible for generating cardiac ROS, led to reductions in ROS well under the control level whereas NO increased to control levels demonstrating that oxidative damage contributes to a decrease in NO bioavailability (Soucy et al., 2010; Soucy et al., 2011). 

Zinc oxide nanoparticles (ZnO-NPs) can act as antioxidants; Abdel-Magied & Shedid (2020) found that low concentrations (10 mg/kg ZnO-NPs) decreased oxidation and NO levels in rats after gamma irradiation. Angiotensin II type 1 (AT1) receptors are able to regulate NOS levels. Treatment with losartan, an AT1 receptor antagonist, led to a decrease in O2- , iNOS and eNOS levels, demonstrating that the increase in NOS and ROS can be prevented by blocking AT1 receptors (Zhang et al., 2009). Biotin was found to increase GSH content, superoxide dismutase (SOD), catalase (CAT) activity and return NOx levels to near control values following irradiation in hippocampus (Abdel-Magied & Shedid, 2020). 

• The directionality of changes to NO is inconsistent between studies, as some studies show increased NO levels and other studies show decreased NO levels. Improved methods are needed to assess NO levels directly, which may facilitate an understanding of the relationship (Cervelli et al., 2017, Hasan, Radwan & Galal, 2019). This, along with variation in experimental conditions, can account for the inconsistencies in NO changes between studies. 

Dose/Incidence Concordance 

Time Concordance 

Not Identified.