Increased, Reactive oxygen species leads to Apoptosis

Reactive oxygen species (ROS) can be derived from exogenous sources or produced in vivo; these include the superoxide anion (O 2), the hydroxyl radical (OH), and hydrogen  peroxide (H 2O2). ROS at low levels participate in cell signaling while higher ROS concentrations are deleterious due to the oxidation of proteins, lipids, and DNA. Additionally,  persistent ROS production compromises the cellular antioxidant defense systems and results in oxidative stress and apoptosis (337). ROS can initiate apoptosis via the  mitochondrial and death receptor pathways. In the former, ROS have been shown to induce loss of the m, release of mitochondrial pro-apoptotic proteins, and activation of caspase 3 (49).

ROS signaling has been shown to mediate cytokine-induced apoptosis (Okouchi et al., 2007). TNF is a pro-inflammatory cytokine produced by macrophages and is the most studied cytokine in  apoptosis and the pathophysiology of various diseases, including neurodegenerative disorders (Jackson et al., 1999). Mechanistically, the binding of TNF to its receptor activates the NF-B and JNK signaling pathways believed to be mediated by ROS (Okouchi et al., 2007).  A role for ROS has also been implicated in death receptor-mediated apoptosis induced by apoptosis signal-regulating kinase 1 (ASK1), an ubquitiously expressed MAP kinase kinase kinase (MAPKKK), that activates JNK and p38 MAP kinase pathways (Okouchi et al., 2007).

Free radical scavenger or antioxidant N-acetyl-L-cysteine, a thiol-containing compound, has been shown to directly reduce the levels of ROS (Aruoma et al., 1989; Kim and Sharma 2004; Poliandri et al., 2003). To confirm that Cd-induced neuronal apoptosis is indeed due to its induction of ROS generation, PC12 and SH-SY5Y cells were pretreated with NAC (5mM) for 1h, and then exposed to Cd (10 and 20μM) for 24h (Long et al., 2008). Chen et al. (2008) found that NAC dramatically blocked Cd-induced ROS generation in PC12 cells and SH-SY5Y cells.  In addition, to further quantify the protective effect of NAC on Cd-induced apoptosis via blockage of ROS in a larger cell population, they performed annexin-V-FITC and propidium iodide staining followed by flow cytometry. They found NAC alone did not affect cell viability. However, it significantly blocked Cd-induced apoptosis.

Asit Rai et al. 2010 found that a metal mixture of arsenic, cadmium, and lead triggered ROS generation, reaching its peak after 1 hour of treatment.  They next investigated whether ERK1/2, JNK1/2, [Ca²⁺]_i and ROS signaling resulted in apoptosis by reating the MM-treated astrocytes with α-tocopherol (200 μg/ml), PD98059 (10μM), BAPTA-AM (5μM), or SP600125 (10μM).  They all suppressed apoptosis suggesting that activation of ERK1/2 and JNK1/2, followed by increased [Ca²⁺]_i and ROS generation, resulted in apoptosis in the MM-treated astrocytes.

When astrocytes were exposed to H2O2 for 30 min and then incubated without H2O2 for 1–5 days, cell toxicity including apoptosis was observed (Kazuhiro et al., 2004). Furthermore, the reperfusion injury induced by Ca²⁺ depletion or H2O2 exposure was exacerbated by the catalase inhibitor, 3-amino-1,2,4-triazole, and the GSH synthesis inhibitors, l-buthionine-S,R-sulfoximine and xanthine, while the injury was blocked by GSH, catalase and the iron chelators, 1,10-phenanthroline and deferoxamine (Takuma et al., 1999). These findings indicate that Ca²⁺ reperfusion-induced apoptosis is mediated by ROS production, especially by hydroxyl radical formation (Kazuhiro et al., 2004).

Exposure of cells to 5 M iAs significantly triggered the expression of ER stress-related molecules, including: the proteins and mRNAs expression of GRP 78, CHOP, XBP-1 in a time-dependent manner (for 6–24 h) as well as the degradation of full-length (55 kDa) caspase-12 (downstream ER stress molecule). However, GRP 94 was not affected by iAs treatment. These effects of iAs-induced ER stress protein responses could be reversed by pre-treatment with NAC. Furthermore,  transfection of Neuro-2a cells with GRP 78- and CHOP-specific si-RNA, respectively, markedly reduced the protein expression levels of GRP 78 and CHOP in the cells treated with iAs and significantly attenuate the iAs-induced caspase-3, -7, and -12 activations. These results indicate that oxidative stress-mediated ER stress activation pathway is also involved in iAs-induced neuronal cell apoptosis (Tien-Hui, et al. 2014).

Recent studies have shown that ROS generation induced by toxic metals (including arsenic) causes neuronal apoptosis, which is closely associated with the progression of neurodegenerative diseases (Bharathi and Jagannathan 2006; Flora et al., 2009; Gharibzadeh 2008).

Okouchi et. al. (2007) found that peroxide-induced apoptosis in undifferentiated PC12 cells was mediated by an early loss of the cellular glutathione–glutathione disulfide (GSH/GSSG) redox balance that preceded an increase in Bax expression, mitochondrial-to-cytosol cytochrome c translocation, and activation of caspase 3 (Pias and Aw 2002; Pias and Aw 2002; Pias et al., 2003). Apoptosis was  ameliorated by the overexpression of mitochondrial superoxide dismutase, MnSOD (SOD2), and by pretreatment of cells with the antioxidant, N-acetyl cysteine (NAC) ^(23-25).

As first demonstrated in mouse fibrosarcoma cells, TNF treatment disrupts mitochondrial electron transport and  enhances ROS production (Schulze–Osthoff et al., 1992). Recent studies by Han et al. (2006) showed that modulation of the hepatocyte redox environment by ROS interfered with NF-B signaling in TNF-induced apoptosis. Notably, cell apoptosis occurred within a certain redox window in which mild redox imbalance inhibited NF-B activation, but not caspase activity (Okouchi et al., 2007).

ROS and/or oxidative damage can activate gene transcription and transcribed genes may be implicated in either cell survival or cell death (Klein and Ackerman 2003).

The increase in reactive oxygen species at As(III) concentrations of 0.5 mg/l or more may play an apoptogenic role and/or be a consequence of events occurring during apoptosis (Rocha et al. 2011). It is generally reported that ROS cause an increase in [Ca²⁺]i of various cell types, which might be one of the causes for the C17.2 cells to enter apoptosis (Rocha et al. 2011). According to Hool and Corry (2007), the redox control of Ca²⁺ transport is due to the fact that ROS can react with the thiol groups of protein that form part of the Ca²⁺ transporters or channels. Alternatively, mitochondrial matrix Ca²⁺ overload can lead to enhanced generation of reactive oxygen species, triggering the permeability transition pore, dissipation of transmembrane mithocondrial potential, and cytochrome c release (Brookes et al., 2004). In any case, the fact that treatment with various antioxidants (vitamin E, tocopherol, and quercetin) did not rescue the cells from death by apoptosis indicates that oxidative stress was not the main cause of the observed cell death (Rocha et al. 2011).

Superoxides and lipid peroxidation are increased during apoptosis induced by myriad stimuli (Bredesen 1995). However, generation of ROS may be a relatively late event, occurring after cells have embarked on a process of caspase activation (Green and Reed 1998). In this regard, attempts to study apoptosis under conditions of anoxia have demonstrated that at least some proapoptotic stimuli function in the absence or near absence of oxygen, which implies that ROSs are not the sine qua non of apoptosis (Jacobson and Raff 1995). However, ROSs can be generated under conditions of virtual anaerobiosis (Degli Esposti and McLennan 1998), and thus their role in apoptosis cannot be excluded solely on this basis (Green and Reed 1998).

Okouchi et. al. (2007) found that PC12 apoptosis can be initiated by GSH/GSSG redox imbalance alone independently of ROS generation (Pias et al., 2003), suggesting that a loss of cellular  redox homeostasis is downstream of ROS signaling in neuronal cell apoptosis.