Oxidative Stress leads to Increase, Cell death

High concentrations of ROS induce cell death by activating apoptosis pathways and causing oxidative damage to lipids, proteins, and DNA, including base damage, strand breaks, and mutations. In addition, ROS cause damage to vital cellular components, including the mitochondria and cellular membrane, resulting in programmed cell death (Pacheco and Stock, 2013; Valko et al., 2007). When the hydroxyl radical interacts with DNA it can cause damage to both purine and pyrimidine bases, as well as the deoxyribose backbone. A common DNA lesion that has been extensively researched is the bonding of hydroxyl radicals to the guanine nucleotide base, known as the 8-hydroxyguanine (8-OH-G) bond (Glasauer & Chandel, 2013; Halliwell & Gutteridge, 1999; Valko et al., 2007; Valko et al., 2006). ROS can damage the cellular membrane by oxidizing the polyunsaturated fatty acids residues of the phospholipid bilayer, in a process known as lipid peroxidation. The final product of lipid peroxidation is malondialdehyde (MDA), a common marker of oxidative stress. Another aldehyde product of lipid peroxidation is 4-hydroxynonenal (4-HNE) (Siems, Grune, & Esterbauer, 1995; Valko et al., 2007). Proteins undergo oxidative damage through the interaction of ROS with its amino acid monomers. All amino acid side chains can be oxidized by RONS, with cysteine and methionine being particularly susceptible. A common measure of oxidative damage to proteins is the concentration of carbonyl groups (Stadtman, 2004; Valko et al., 2007).      

Programmed cell death is regulated by the balance of positive signals involved in cell survival, such as growth factors, and negative signals that can harm to the cell, including increased RONS concentration and oxidative damage to DNA (Hengartner, 2000; Valko et al., 2007). The redox environment of cells is regulated in large part by the intracellular concentration of the antioxidant, glutathione (GSH). When GSH drops below a certain level, the cellular environment becomes too oxidizing, and apoptosis occurs. Apoptosis begins to occur after moderate oxidation, with overwhelming oxidation resulting in necrosis (Cai & Jones, 1998; Evens, 2004; Valko et al., 2007; Voehringer et al, 2000). Intracellular damage to the cell via oxidative stress causes Bcl-2 to activate the pro-apoptotic Bcl-2 associated protein x (Bax) (Jezek et al., 2019; Memme et al., 2021; Pistilli, Jackson, & Alway, 2006; Philchenkov et al., 2004; Valko et al., 2007). Alternatively, ROS accumulation in the mitochondria can cause the mitochondrial permeability transition pore (mPTP) to open, allowing for an influx of solutes to enter the mitochondria, creating a hypotonic environment, and subsequently inducing apoptosis (Bauer & Murphy, 2020; Memme et al., 2021).  

Accumulation of ROS in the mitochondria can also lead to activation of the ion channel, transient receptor potential cation channel (TRPML1), which facilitates the release of Ca²⁺ from the lysosome into the cytosol, resulting in swelling of the endo-lysosomal structures and stimulation of transcription factor EB (TFEB)-mediated signalling cascade that culminates in increased autophagy (Erkhembaatar et al., 2017; Johnson et al., 2020; Todkar, Ilamathi, & Germain, 2017). Alternatively, an accumulation of NADPH oxidase (NOX)-generated ROS in endosomal compartments can lead to activation of autophagy. NOX2 enzymes, found in the endosome, induce oxidative damage to mitochondrial and nuclear DNA through reduction of NADPH, resulting in apoptosis. NOX-generated ROS can also increase signalling from endocytosed receptors that are responsible for inducing mitochondrial dysfunction induced-apoptosis (Davis Volk & Moreland, 2014; Harrison et al., 2018; Johnson et al., 2020; Karunakaran et al., 2019; Li et al., 2015; Ran et al., 2016; Tsubata, 2020).

The empirical evidence for this KER provides moderate support for a linkage between increased oxidative stress and increased cell death. Most of the evidence supporting this relationship come from studies that examine the effects of low linear energy transfer (LET) radiation, such as X-rays and gamma rays. However, one study examined the effects of high LET carbon ions and another exposed its model to simulated microgravity conditions. These studies observed dose and time concordant responses (Huang et al., 2019; Huang et al., 2018; Kondo et al., 2010; Li et al., 2020; Li et al., 2018; Liu et al., 2019; Liu et al., 2018; Yoo, Han & Kim, 2016). 

Incidence Concordance 

Few studies demonstrate a greater oxidative stress than cell death following a stressor. Human bone marrow-derived mesenchymal stem cells (hBMMSCs) irradiated with 8 Gy demonstrated greater increases to ROS levels than to apoptosis (Li et al., 2020). Similarly, rats irradiated with 35 Gy showed greater increases to ROS levels than to osteocyte apoptosis (Li et al., 2018). 

Dose Concordance 

Current literature on the impact of oxidative stress on cell death provides moderate evidence for a dose concordant link between the two key events. Studies that examined the effects of ionizing radiation (IR) and microgravity conditions on bone cells have observed both stressors induce significant increases in ROS and oxidative stress markers, as well as decreases in antioxidants, followed by subsequent increases in markers of cell death.

Studies that apply IR to their experimental models provide the strongest support for dose concordance as they clearly demonstrate variances in oxidative stress and cell death following exposure to a range of doses. Oxidative stress was observed at the same or lower doses than cell death across all studies. Kondo et al. (2010) irradiated C57BL/6J mice with 1 or 2 Gy of ¹³⁷Cs gamma rays and observed significant increases to ROS production and the oxidative stress markers, MDA and 4-HNE, at 1 Gy, while apoptosis only experienced a significant increase at 2 Gy. Bai et al. (2020) irradiated the bone marrow derived mesenchymal cells (bmMSCs) of Sprague-Dawley rats with 2, 5, and 10 Gy of ¹³⁷Cs gamma rays and observed significant changes to levels of ROS, superoxide dismutase (SOD)1 and catalase (CAT)2, as well as cell viability at ≥2 Gy. The one study that applied high LET radiation, in this case 2 Gy of calcium ions, observed more significant increases to oxidative stress and cell death on average than studies that applied 2 Gy of a lower LET radiation type. Liu et al. (2019) observed ~2.2-, ~5.4-, and ~4.2-fold increases to ROS levels, early apoptosis, and late apoptosis/necrosis, respectively, after exposure to 2 Gy of carbon ions (LET=31.6 KeV/µm), while other studies that applied 2 Gy of lower LET radiation types, including X-rays and gamma rays, observed increases of ~1.2-fold to ~2.5-fold in ROS levels and increases of ~1.6-fold to 5.26-fold in apoptosis (Huang et al., 2019; Huang et al., 2018; Kondo et al., 2010; Liu et al., 2019). Furthermore, microgravity as a stressor also supports the relationship between oxidative stress and cell death. Yoo, Han & Kim (2016) did observe significant increases to both oxidative stress and cell death after exposing MC3T3-E1 murine pre-osteoblast cells to microgravity conditions.

Time Concordance 

There is moderate evidence in the current literature to support a time concordant relationship between oxidative stress and cell death. All of the studies that measured oxidative stress and cell death endpoints at multiple time points observed significant changes to oxidative stress earlier or at the same time as changes to cell death (Huang et al., 2018; Kondo et al., 2010; Li et al., 2020; Li et al., 2018). Huang et al. (2018) irradiated murine RAW264.7 osteoclast precursor cells with 2 Gy of gamma rays and observed a significant increase in ROS levels at 2 hours post-irradiation, while increases to apoptosis were not reported until 24 hours. Kondo et al. (2010) irradiated C57BL/6J mice with 1 and 2 Gy of ¹³⁷Cs gamma rays and observed significant increases to both ROS levels and apoptosis by day 3 post-irradiation. Li et al. (2020) irradiated hBMMSCs with 8 Gy of radiation and observed significant increases to both ROS levels and cell apoptosis by 24 hours post-exposure. Li et al. (2018) observed significant increases to ROS activity, as well as significant decreases to SOD activity, at 1 day post-irradiation, while significant increases to empty lacunae were not reported until 4 months post-irradiation. Lastly, Wang et al. (2016) irradiated murine MC3T3-E1 cells with 6 Gy of X-rays and observed significant increases to ROS production at 24 hours post exposure and extracellular hydrogen peroxide levels at 3 hours post exposure, while significant decreases to cell viability did not occur until day 4.

Essentiality 

Several studies have investigated the essentiality of the relationship, where the blocking or attenuation of the upstream KE causes a change in frequency of the downstream KE. The increase in oxidative stress can be modulated by certain drugs and antioxidants. Treatment with α-2-macroglobulin (α2M) decreased SOD activity and reduced the rate of apoptosis and autophagy in human bone marrow mesenchymal stem cells hBMMSCs (Liu et al., 2018). This countermeasure also showed the same influence on SOD activity and a decrease in osteocyte apoptosis (Li et al., 2018). Sema3a was found to reduce ROS and promote the apoptosis of the Raw264.7 cells post-adiation (Huang et al., 2018). Treatment with Amifostine (AMI) reversed the radiation-induced effects on ROS levels and reduced the percentage of apoptotic cells and DNA damage (Huang et al., 2019; Zhang et al., 2020). Cerium oxide (CeO₂) nanoparticles significantly reduced increases to ROS production and hydrogen peroxide, as well as causing cell viability to recover significantly by day 4 post-irradiation (Wang et al., 2016). Lastly, the antioxidant melatonin was shown to reverse the effect of microgravity on Bcl-2, Bax, Cu/Zn-SOD and manganese superoxide dismutase (Mn-SOD) to control levels (Yoo, Han & Kim, 2016).

• When MC3T3-E1 murine preosteoblast cells underwent microgravity conditions in a 3D clinostat, CAT expression increased by ~1.25-fold. This response was the opposite of the other antioxidants that were measured and is contrary to the decrease in antioxidant expression normally seen after microgravity exposure (Yoo, Han & Kim, 2016).

• Kondo et al. (2010) did not observe any significant effects to MDA+4-HNE levels or apoptosis after subjecting their C57BL/6J mice to hindlimb unloading. 

Dose/Incidence Concordance 

Time Concordance 

None identified