Chemical nerve agents (CNAs) are a unique class of highly lethal neurotoxins and historically exploited in chemical warfare (Diauudin et al., 2019). Originating as a pesticide, organophosphate (OP) compounds were first synthesized as potent CNAs in the early 1930s, just in time for World War II, and have since been employed in the Persian Gulf War, Syria conflict, Matsumoto and Tokyo terrorist attacks, and political assassinations (Gupta, 2015; Chai et al., 2017). OPs, including tabun, sarin, soman, novichok, and diisopropylfluorophosphate (DFP), irreversibly inhibit acetylcholinesterase (AChE), an enzyme responsible for the breakdown of acetylcholine (ACh). The inhibition of AChE results in the accumulation of ACh, promoting acute muscarinic and nicotinic hyperexcitability in both peripheral and central nervous systems leading to respiratory depression, muscle fasciculation, status epilepticus (SE), and in extreme cases, death (Marrs, 1993; Holstege et al., 1997; Franca et al., 2019). Long-term consequences can be severe, resulting in neurodegeneration, neuroinflammation, behavioral deficits, and spontaneous recurrent seizures, i.e., epilepsy (Okumura et al., 1996; Miyaki et al., 2005).

The currently available medical countermeasures (MCMs) for OP toxicity control acute symptoms but are ineffective for SE-induced brain injury. While select military personnel carry MCMs on the field, it is unlikely that civilians will have early access in an exposure scenario. FDA-approved treatments for OP exposure include the cholinergic antagonist, atropine, the cholinesterase reactivator, 2-pralidoxime chloride (2-PAM), and the GABA allosteric modulator, Midazolam (Newmark, 2019). Atropine and 2-PAM can effectively reduce mortality by mitigating peripheral, but not central, effects of OP exposure; therefore, they do not offer long-term protection (Gao et al., 2016; Spampanato et al., 2019). Midazolam has been shown to terminate OP-induced behavioral SE following acute exposure, however, its neuroprotective effect depends on how quickly the drug is administered (Supasai et al., 2020). Moreover, the currently available MCMs fail to mitigate OP-induced oxidative stress, a critical underlying mechanism of neuroinflammation, neurodegeneration, and seizures that follow SE (Chang and Yu, 2010; Puttachary et al., 2015; Guignet et al., 2020).

Oxidative stress, or the imbalance between the generation of oxygen free radicals and antioxidants, is a consequence of OP intoxication in humans and rodents alike (Ranjbar et al., 2005; Pearson and Patel, 2016). Oxygen free radicals are oxygen molecules with an unpaired electron. They are highly reactive, causing lipid peroxidation, protein degradation, and DNA damage in their attempt to “steal” electrons (Lukaszewicz-Hussain, 2010; Walker, 2023). In the periphery, excess reactive oxygen species (ROS) increase proinflammatory cytokine production (Chen et al., 2018). In in vitro models of OP toxicity, oxidative stress was associated with lymphocytic death, nephrotoxicity, and gut dysbiosis (Gao et al., 2016; Ahmad et al., 2019; Sobolev et al., 2022; Gage et al., 2023). The central nervous system is especially vulnerable to ROS due to the brain’s high oxygen metabolic rate and concentration of polyunsaturated fatty acids (Vanova et al., 2018; Lee et al., 2021). The neuroinflammatory response to acute OP exposure, including astrogliosis and microgliosis, along with neuronal excitation, likely drives the initial production of ROS (Badr, 2020; Lee et al., 2021). Increased levels of ROS, gliosis, and neuronal loss create a chronic excitotoxic environment in the brain that lowers the seizure threshold and spurs cognitive and memory dysfunction (Chen, 2012).

Although mitochondria are traditionally considered the primary contributors to oxidative stress because of high ATP consumption during OP-induced SE (Milatovic et al., 2006; Lukaszewicz-Hussain, 2010), emerging evidence suggests the NADPH oxidase (NOX) is also a significant primary producer of free radicals (Walker, 2023). NOX activation creates a feed-forward mechanism of ROS generation in concurrence with the mitochondria (Dikalov, 2011). Thus, both NOX and mitochondria are highly implicated in oxidative stress. Increased concentrations of the oxidative stress markers ROS, oxidized glutathione (GSSG), nitrite, and the NOX2 subunit GP91^phox, support mitochondrial dysfunction and NOX upregulation following OP exposure (Mostafalou and Abdollahi, 2018; Putra et al., 2020a). Therefore, targeting NOX activity can be an option for attenuating OP toxicity-induced oxidative stress.

Apocynin is a broad NOX inhibitor, though it has been suggested to have an affinity for NOX2 inhibition (Weston et al., 2013). NOX2 is one of seven in the NOX family, and is expressed in phagocytic cells such as microglia and abundantly upregulated in neurological disorders (Sumimoto et al., 2005). Furthermore, apocynin has been found to reduce mitochondrial dysfunction (Du et al., 2019). In neurodegenerative disease models such as Parkinson’s Disease and epilepsy, as well as diabetic cardiomyopathy and hypertension, apocynin reduced neuronal death, microglial and astroglial activation, inflammation, and excessive ROS production (Costa et al., 2009; Roe et al., 2011; Ghosh et al., 2012; Jaiswal and Kumar, 2022). Previously, we discovered that diapocynin (DPO), a dimer of apocynin, suppressed astrogliosis, neurodegeneration, and GP91^phox expression in rat brains 6 weeks post-DFP exposure (Putra et al., 2020a). Due to their low bioavailability and high dosages required for efficacy (Putra et al., 2020a; Savla et al., 2021), apocynin and DPO are unlikely to be feasible for human use. To circumvent this problem, we tested mitoapocynin (MPO, apocynin TPP⁺ conjugated to enable mitochondrial uptake), which has shown excellent efficacy at a very low dose (3 mg/kg, oral) in a mouse Parkinson’s disease model (Ghosh et al., 2016; Langley et al., 2017). Notably, MPO (10 mg/kg, oral) reduced brain oxidative stress in rats after repeated exposure to a pesticide OP, chlorpyrifos (Singh et al., 2018). We predicted that treatment with 10 mg/kg MPO (oral) will also reduce oxidative stress markers in the peripheral and central nervous systems in response to acute exposure to DFP.

Experimental design is illustrated in Figure 1A.

We have previously shown that DFP-induced SE increases oxidative stress and exacerbates brain pathology. NOX inhibitors such as DPO (300 mg/kg), an apocynin-derivative, significantly mitigated the DFP-induced effects in a rat DFP model (Putra et al., 2020a). In this study, we hypothesized that a mitochondrial-targeted NOX inhibitor, mitoapocynin (MPO), will yield better outcomes at a lower dose by reducing NOX production of free radicals and, further, oxidative stress in the periphery and the brain due to DFP exposure. Based on the serum kinetics of MPO (10 mg/kg, oral), we tested a treatment regimen of twice a day for the first 3 days, followed by once a day for the next 4 days in a rat DFP model. This dosing regimen reduced DFP-induced oxidative stress and inflammatory markers in the periphery (serum) but had a limited effect in preventing DFP-induced neurodegeneration and gliosis in the brain. Pharmacokinetic studies confirmed that MPO crossed the blood-brain-barrier in a mouse model (Ghosh et al., 2016). In a rat model, we detected 18 ± 9 pg/g MPO in the brain 3 h post-treatment with 10 mg/kg in 2% ethanol. Since MPO is a mitochondrial-targeted drug, we hypothesized that such low brain concentrations of MPO may be enough to mitigate the DFP-induced brain pathology. However, the results did not support the hypothesis. A better formulation and higher concentrations of MPO may yield better results.

DFP, a potent OP, is a surrogate for nerve agents. Nerve agents such as VX and sarin have been used in chemical warfare and terrorist attacks (Chai et al., 2017). The current MCMs effectively prevent nerve agent-induced acute symptoms and mortality only if administered within <20 min of exposure, which is impractical. Importantly, no effective countermeasures are available to mitigate the long-term effects of brain injury in survivors. Nerve agent exposure induces oxidative stress and long-term behavioral deficits in exposed subjects (Gao et al., 2016; Spampanato et al., 2019). Long-lasting neurodegeneration, neuroinflammation, cognitive dysfunction, and other comorbidities will severely impact the quality of life (Okumura et al., 1996; Miyaki et al., 2005). Therefore, preventing the long-term effects of brain injury after exposure to OP nerve agents is an unmet obligation.

Acute exposure to DFP induces SE within minutes of exposure (Rao et al., 2022). SE severity, measured by the number of minutes spent in SE, predicts long-term brain injury and peripheral pathology. Animals that spent <20 min in CS (≥stage 3) during SE presented with moderate gliosis and neurodegeneration in discrete brain regions compared to those that spent > 20 min in CS, in which the brain damage was more widespread (Gage et al., 2021). Therefore, animals must maintain sustained SE severity for more than 20 min to investigate the real therapeutic effects of an interventional drug. Furthermore, it is necessary to balance SE severity between treatment groups (vehicle vs. test drug) to determine the mitigating effects of a test drug. In this study, we achieved SE severity > 30 min and balanced SE severity between vehicle and MPO groups in the age-matched mixed-sex cohort, thus eliminating the potential SE severity variable.

Oxidative and nitroxidative stress, induced by excess ROS and reactive nitrogen species (RNS), occur concurrently and serve as peripheral biomarkers (Davies et al., 2003; Cipak Gasparovic et al., 2017). We found significantly increased serum nitrite, ROS, and GSSG levels at 8 days post-DFP challenge. Treating with MPO significantly reduced nitrite concentrations in DFP-challenged animals. MPO also reduced increased ROS levels in DFP-exposed animals. High oxidized glutathione (GSSG) levels imply increased scavenging of excess ROS by glutathione (Owen and Butterfield, 2010). The significant reduction in GSSG in MPO-treated DFP-challenged animals suggests rescue of the altered redox potential. In our previous DFP studies, increased serum nitrite levels were also observed at 6 weeks post-exposure (Putra et al., 2020a,b). DPO treatment significantly reduced DFP-induced nitrite levels (Putra et al., 2020a). These findings demonstrate the beneficial effects of MPO against DFP-induced oxidative stress in the peripheral circulation.

OP-induced nitroxidative or oxidative stress can promote proinflammatory cytokines or vice versa. For example, RNS activation of TNF-α via the NFkB signaling pathway promotes excessive generation of ROS (Davies et al., 2003; Yang et al., 2018). Therefore, it is unsurprising that DFP exposure significantly increased proinflammatory cytokines IL-1β, IL-6, and TNF-α. Previous studies reported similar findings in the DFP model (Putra et al., 2020b). In this study, we demonstrated a significant reduction of these cytokines in MPO-treated animals exposed to DFP. Neither DFP nor MPO significantly altered IL-10 or MCP1 levels in the serum. This is likely attributable to the functionality of cytokine IL-10 as both anti- and proinflammatory, and MCP1, which is a monocyte chemoattractant produced by circulating monocytes and astrocytes (Gao et al., 2009; Yadav et al., 2010).

Microglia, the resident macrophages of the brain, are known to proliferate after exposure to OP nerve agents (Kettenmann et al., 2011; Gage et al., 2022a). Activated/reactive microglia can become maladaptive and release excess amounts of ROS, RNS, and cytokines. Additionally, they may change in morphology indicative of a phagocytic nature (Lee et al., 2021). The Cluster of Differentiation 68 (CD68) is a lysosomal transmembrane glycoprotein expressed in high levels in phagocytic microglia and widely used as a marker for reactive microglia (Kettenmann et al., 2011). In our DFP model, CD68 expressing microglia were significantly increased in both DFP + VEH and DFP + MPO groups in the hippocampus, amygdala, and piriform cortex. MPO marginally reduced the presence of microglia in extrahippocampal and hippocampal regions and CD68 in the PC in DFP-challenged animals. Interestingly, we did not observe a reduction in DFP-induced microgliosis or CD68-positive microglia in DPO-treated animals in the 6-week DFP study (Putra et al., 2020a). It is likely that MPO may attenuate microgliosis at a higher dose and could produce a potent anti-inflammatory effect in the brain.

We investigated NOX2 because of its role in neuroinflammation and high expression in microglial cells (Ma et al., 2017). At rest, NOX2 has both cytosolic and membrane-bound subunits. In an in vitro study, MPO treatment of cultured microglia cell lines restricted the translocation of the cytosolic subunits of NOX to its membrane-bound subunits, preventing enzyme activation and the production of free radicals (Langley et al., 2017). Following brain injury, its cytosolic subunits translocate to membrane-bound GP91^phox to form a functional complex, initiating free radical production by NOX2 (Gao et al., 2012; Ma et al., 2017). Similar to our previous 6-week DFP study (Putra et al., 2020a), we observed a significant increase in the number of GP91^phox-positive cells in the brain as early as 1-week post-exposure in this study. While increased GP91^phox level is associated with ROS production, its presence alone does not necessarily indicate NOX activation (Ma et al., 2017). A limitation of the current study is the lack of investigation of cytosolic subunits translocation to the membrane, which is challenging in an in vivo model. Other NOX enzymes may likely be involved in DFP-induced toxicity, and NOX inhibitors DPO and MPO may also suppress ROS production through these pathways, which is not invested in this study. Astroglia expresses NOX1, NOX4, or both (Gao et al., 2012). An increase in astrocytes may imply the upregulation of NOX and ROS production (Chen et al., 2020). Future studies will investigate the direct effect of MPO on the astroglial NOX1 and NOX4 responses to DFP. Astrogliosis, as such, is not necessarily harmful; however, complement 3 (C3) expression by astrocytes may suggest their reactivity in response to an injury (Wei et al., 2021). C3 is a mediator of innate immunity and interacts with microglia, and promotes synaptic pruning (Stevens et al., 2007; Shinjyo et al., 2009). We observed a significant increase in reactive astrogliosis in previous DFP studies at 8 days and 6 weeks post-DFP exposure (Putra et al., 2020a,b). DPO significantly mitigated both reactive astrogliosis and C3-positive astrocytes in a 6-week DFP study (Putra et al., 2020a). In the present study, we did observe a significant increase in C3-positive astrocytes in hippocampal and extrahippocampal brain regions of the DFP-exposed vehicle-treated group, but MPO had no effect. In the PC, there was an unexpectedly significant increase of C3-positive astroglia in non-DFP animals. In an in vitro manganese exposure model, MPO reduced the astroglial inflammatory response by reducing IL-1β and IL-18 expression (Sarkar et al., 2017). We have shown a reduction of C3 in a DFP model with a high dose of DPO (300 mg/kg) (Putra et al., 2020a). Therefore, the prospect of a differential impact of MPO on NOX or cell-specific ROS generation in a dose-dependent manner requires further investigation.

Cortical glial scars in the DFP model are characterized by reactive astroglia lining the scar with clusters of reactive microglia and degenerating neurons at its core (Gage et al., 2022a). In our study, one-third of animals with glial scars were observed in DFP + VEH and DFP + MPO groups. MPO at 10 mg/kg did not impact the occurrence of glial scars in this study. In DFP-exposed animals, neurodegeneration was observed in the hippocampus and was especially distinct in the amygdala and piriform cortex, where glial scars typically occur in DFP models. In a previous study, neurons in the CA1 region were found to be vulnerable to cell death and had a higher mitochondrial production of ROS than neurons in the CA3 region (Wang and Michaelis, 2010). In DFP + MPO-treated animals in this study, we found a trend in this direction; however, it did not reach statistical significance. MPO at 10 mg/kg did not reduce DFP-induced neurodegeneration. A dose optimization study is required in future investigations.

Overall, DFP exposure significantly increased oxidative stress and proinflammatory cytokines in the serum and propagated reactive microgliosis and astrogliosis, and neurodegeneration in key brain regions. MPO attenuated DFP-induced oxidative stress and proinflammatory cytokines in peripheral circulation but only minimally reduced reactive microgliosis and astrogliosis in the brain. Considering the known pharmacokinetic challenges of apocynin and DPO, MPO also revealed bioavailability concerns, which are under further investigation. Our study provides evidence that at the dose of 10 mg/kg, MPO is not effective in the brain despite its ability to cross the blood-brain-barrier. Optimization of the dosing regimen and drug formulation is required to determine the efficacy of MPO in the DFP model.

The original contributions presented in this study are included in this article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Iowa State University IACUC committee.

TT conceptualized, designed and supervised the experiments, secured the funding, and edited the manuscript. CM conducted the experiments, acquired and analyzed the data, and drafted the manuscript. NR, SV, MG, and MP assisted the experiments, compiled and cross-verified the data, and proofread the manuscript. BP conducted the IHC and image analysis. CH and JH contributed to the cell quantification. All authors contributed to the article and approved the submitted version.