Increase in RONS leads to Increase, DNA Damage

High. Reactive oxygen and nitrogen species from oxygen and respiratory activity are generally acknowledged to damage DNA under typical cellular conditions (Dickinson and Chang 2011; Aziz, Nowsheen et al. 2012; Tubbs and Nussenzweig 2017). Damage commonly occurs via oxidation of a nucleotide by the hydroxyl radical (or by radicals created by nitric oxide), or can occur indirectly in nearby nucleotides following the secondary reaction of a radical created in nucleotides (Cadet, Davies et al. 2017). Oxidative damage predominantly consists of DNA lesions (structural modifications to nucleotides) including single strand breaks, although double strand breaks can occur when transcription or translation machinery encounters damaged strands (Tubbs and Nussenzweig 2017).

High. Multiple studies show an increase in DNA damage with RONS treatment as well as dependent changes in both RONS and DNA damage in response to stressors. DNA damage increases with RONS dose, and temporal concordance between RONS and DNA damage events following ionizing radiation is consistent with a causative relationship, although few studies examine multiple doses and time points. A small number of studies do not find double strand breaks at physiological doses, or report an increase in one key event but not the other.

Treatment with H2O2 or other RONS inducers increase DNA damage and double strand breaks. H2O2 treatment reaches the nucleus where it can damage DNA (Ameziane-El-Hassani, Boufraqech et al. 2010; Ameziane-El-Hassani, Talbot et al. 2015). Oxidized nucleotides (including clusters) and single strand breaks are commonly reported following H2O2 treatment (Dahm-Daphi, Sass et al. 2000; Nakamura, Purvis et al. 2003; Yang, Durando et al. 2013; Sharma, Collins et al. 2016), and double strand breaks can occur when transcription and translation machinery encounters damaged strands (Berdelle, Nikolova et al. 2011; Yang, Durando et al. 2013; Tubbs and Nussenzweig 2017). However, it is less clear whether H2O2 or RONS cause a measurable increase in double strand breaks, particularly at physiologically relevant concentrations (in the range of 12 uM) (Liu and Zweier 2001; Ameziane-El-Hassani, Talbot et al. 2015). Studies report double strand breaks following treatment with 15 uM- 1mM H2O2 (Oya, Yamamoto et al. 1986; Driessens, Versteyhe et al. 2009; Seager, Shah et al. 2012; Werner, Wang et al. 2014; Ameziane-El-Hassani, Talbot et al. 2015; Sharma, Collins et al. 2016) as well as parallel increases in RONS and double strand breaks (Han, Chen et al. 2010; Berdelle, Nikolova et al. 2011; Stanicka, Russell et al. 2015). DNA damage including double strand breaks and mutations increase with H2O2 dose (Sandhu and Birnboim 1997; Dahm-Daphi, Sass et al. 2000; Driessens, Versteyhe et al. 2009; Seager, Shah et al. 2012; Lorat, Brunner et al. 2015; Sharma, Collins et al. 2016).

RONS is dose-dependently and reversibly associated with increased genomic instability (Dayal, Martin et al. 2008; Dayal, Martin et al. 2009; Buonanno, de Toledo et al. 2011; Pazhanisamy, Li et al. 2011; Datta, Suman et al. 2012; Bensimon, Biard et al. 2016) and with DNA damage in bystander cells (Azzam, De Toledo et al. 2002; Yang, Asaad et al. 2005; Yang, Anzenberg et al. 2007; Han, Chen et al. 2010; Buonanno, de Toledo et al. 2011) although other non-RONS factors such as telomere erosion and breakage-fusion-bridge events may be sufficient to maintain genomic instability (Suzuki, Kashino et al. 2009; Murnane 2012). To our knowledge no experiments have tested whether elevating intracellular RONS alone in one group of cells can cause DNA damage in nearby cells.

Antioxidants and other interventions to reduce RONS production also reduce or block the effect of RONS treatment on DNA base damage (Berdelle, Nikolova et al. 2011) and double-strand breaks (Ameziane-El-Hassani, Boufraqech et al. 2010; Ameziane-El-Hassani, Talbot et al. 2015; Stanicka, Russell et al. 2015). Similarly, nitric oxide scavengers can reduce DNA damage in cells treated with nitric oxide producers (Han, Chen et al. 2010) or in bystander cells.

Further support for a causative relationship between RONS and DNA damage comes from many studies showing that antioxidants and other interventions capable of reducing RONS can also reduce DNA damage following IR. Antioxidant reduction of nucleotide damage from IR occurs in isolated DNA (Winyard, Faux et al. 1992; Douki, Ravanat et al. 2006), and in vitro and in vivo antioxidants reduce nucleotide damage, double strand breaks, micronuclei, chromosomal damage, and mutations when added before (Azzam, De Toledo et al. 2002; Choi, Kang et al. 2007; Jones, Riggs et al. 2007; Ameziane-El-Hassani, Boufraqech et al. 2010; Ozyurt, Cevik et al. 2014; Ameziane-El-Hassani, Talbot et al. 2015; Fetisova, Antoschina et al. 2015; Manna, Das et al. 2015), or in the case of delayed (15 min to days) or bystander DNA damage, added after radiation (Yang, Asaad et al. 2005; Han, Chen et al. 2010; Pazhanisamy, Li et al. 2011; Ameziane-El-Hassani, Talbot et al. 2015). Interestingly, NO specific blockers reduce DNA damage and mutations in bystander cells but not in directly IR cells, suggesting that NO specifically contributes to the bystander effect (Zhou, Ivanov et al. 2008; Han, Chen et al. 2010).

Temporal concordance between RONS and DNA damage events following a stressor (ionizing radiation) is consistent with a causative relationship between RONS and DNA damage. Following ionizing radiation, an increase in RONS typically occurs coincident with DNA damage. Few studies examine multiple doses and time points, and detection methods have differing sensitivities. However, both RONS and double strand breaks appear rapidly after IR (Ameziane-El-Hassani, Boufraqech et al. 2010; Denissova, Nasello et al. 2012; Martin, Nakamura et al. 2014), and in several studies RONS and DNA single and double strand breaks, chromosomal damage, and micronuclei appear at the same time points over several days following IR (Choi, Kang et al. 2007; Jones, Riggs et al. 2007; Du, Gao et al. 2009; Saenko, Cieslar-Pobuda et al. 2013; Ameziane-El-Hassani, Talbot et al. 2015; Manna, Das et al. 2015). RONS also appears coincident with longer term DNA damage including nucleotide damage, double strand breaks, and micronuclei, both in IR exposed (Dayal, Martin et al. 2008; Pazhanisamy, Li et al. 2011; Datta, Suman et al. 2012; Werner, Wang et al. 2014; Ameziane-El-Hassani, Talbot et al. 2015) and in bystander cells not directly exposed to IR (Buonanno, de Toledo et al. 2011).

While the bulk of the evidence support a mechanism where RONS increases DNA damage, including double strand DNA breaks, not all studies report these effects. Some studies report the induction of single strand breaks by H2O2, but only show double strand breaks with H2O2 doses at or above 1 mM H2O2 (Dahm-Daphi, Sass et al. 2000; Lorat, Brunner et al. 2015) or do not find an effect of H2O2 on double strand breaks at any concentration (Gradzka and Iwanenko 2005; Ismail, Nystrom et al. 2005). These conflicting results may be partially explained by experimental variations including temperature (two of the studies showing reduced or no effect were exposed to H2O2 at 4C or colder) or other factors including catalysts required to transform H2O2 into DNA damaging OH radicals (Nakamura, Purvis et al. 2003). The reduction of IR-induced DNA damage (including double strand breaks) by antioxidants is strong evidence for an essential role of RONS in DNA damage, but antioxidants don’t reduce all DNA damage from IR and anti-oxidants that reduce double strand breaks and chromosomal aberrations after IR don’t necessarily reduce baseline DNA damage (Fetisova, Antoschina et al. 2015). This incomplete effect suggests either that antioxidants are unable to fully reduce endogenous RONS, or that additional sources of DNA damage are also at work. Furthermore, RONS can be observed following IR in the absence of DNA nucleotide damage (Yoshida, Goto et al. 2012) and counter to expectations lower (10 uM) doses of H2O2 applied six days after IR were associated with a decrease in detectable micronuclei (Werner, Wang et al. 2014), suggesting that additional factors (such as repair and apoptosis or changes in endogenous antioxidants) may influence the effect of RONS on IR-induced DNA damage. Finally, double strand breaks and chromosomal damage can be observed following IR in the absence of measured RONS (Suzuki, Kashino et al. 2009), although since antioxidants are still capable of reducing DNA damage in the absence of measurable RONS, such a discrepancy might be attributable to a lack of sensitivity in RONS detection methods (Yang, Asaad et al. 2005).

RONS activates or is essential to many inflammatory pathways including TGF-β  (Barcellos-Hoff and Dix 1996; Jobling, Mott et al. 2006), TNF (Blaser, Dostert et al. 2016), Toll-like receptor (TLR) (Park, Jung et al. 2004; Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006; Miller, Goodson et al. 2017; Cavaillon 2018), and NF-kB signaling (Gloire, Legrand-Poels et al. 2006; Morgan and Liu 2011). These interactions principally involve ROS, but RNS can indirectly activate TLRs and possibly NF-kB. Since inflammatory signaling and activated immune cells can also increase the production of RONS, positive feedback and feedforward loops can occur (Zhao and Robbins 2009; Ratikan, Micewicz et al. 2015; Blaser, Dostert et al. 2016).

Damage inflicted by RONS on cells activate TLRs and other receptors to promote release of cytokines (Ratikan, Micewicz et al. 2015). For example, oxidized lipids or oxidative stress-induced heat shock proteins can activate TLR4 (Miller, Goodson et al. 2017; Cavaillon 2018).

ROS is essential to TLR4 activation of downstream signals including NF-kB. Activation of TLR4 promotes the surface expression and movement of TLR4 into signal-promoting lipid rafts (Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006). This signal promotion requires NADPH-oxidase and ROS (Park, Jung et al. 2004; Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006). ROS is also required for the TLR4/TRAF6/ASK-1/p38 dependent activation of inflammatory cytokines (Matsuzawa, Saegusa et al. 2005). ROS therefore amplifies the inflammatory process.

RONS can also fail to activate or actively inhibit inflammatory pathways, and the circumstances determining response to RONS are not well known (Gloire, Legrand-Poels et al. 2006).

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