Pharmacological regulation of cytochrome P450 metabolites of arachidonic acid attenuates cardiac injury in diabetic rats.

Diabetic cardiomyopathy (DCM) is a well-established complication of type 1 and type 2 diabetes associated with a high rate of morbidity and mortality. DCM is diagnosed at advanced and irreversible stages. Therefore, it is of utmost need to identify novel mechanistic pathways involved at early stages to prevent or reverse the development of DCM. In vivo experiments were performed on type 1 diabetic rats (T1DM). Functional and structural studies of the heart were executed and correlated with mechanistic assessments exploring the role of cytochromes P450 metabolites, the 20-hydroxyeicosatetraenoic acids (20-HETEs) and epoxyeicosatrienoic acids (EETs), and their crosstalk with other homeostatic signaling molecules. Our data displays that hyperglycemia results in CYP4A upregulation and CYP2C11 downregulation in the left ventricles (LV) of T1DM rats, paralleled by a differential alteration in their metabolites 20-HETEs (increased) and EETs (decreased). These changes are concomitant with reductions in cardiac outputs, LV hypertrophy, fibrosis, and increased activation of cardiac fetal and hypertrophic genes. Besides, pro-fibrotic cytokine TGF-ß overexpression and NADPH (Nox4) dependent-ROS overproduction are also correlated with the observed cardiac functional and structural modifications. Of interest, these observations are attenuated when T1DM rats are treated with 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA), which blocks EETs metabolism, or N-hydroxy-N'-(4-butyl-2-methylphenol)Formamidine (HET0016), which inhibits 20-HETEs formation. Taken together, our findings confer pioneering evidence about a potential interplay between CYP450-derived metabolites and Nox4/TGF-ß axis leading to DCM. Pharmacologic interventions targeting the inhibition of 20-HETEs synthesis or the activation of EETs synthesis may offer novel therapeutic approaches to treat DCM.

Alamandine via MrgD receptor attenuates pulmonary fibrosis via NOX4 and autophagy pathway.

Alamandine (ALA) and its receptor MrgD were recently identified as components of the renin-angiotensin system, which confer protection against cardio-fibrosis and renal-fibrosis; however, the effects of ALA on pulmonary fibrosis are unknown. This study was designed to serve two goals: (i) to evaluate the ALA/MrgD axis ability in the prevention of angiotensin II (Ang II) - induced pulmonary fibrosis in fibroblasts, and (ii) to determine the effect of ALA in bleomycin (BLM) - treated C57B/6 mice. In vivo experiments revealed that the treatment of C57B/6 mice with ALA prevented BLM-induced fibrosis, and these findings were similar to those reported for pirfenidone. The antifibrosis actions of ALA were mediated via alleviation of oxidative injury and autophagy induction. In addition, in vitro studies revealed that ALA treatment attenuated Ang II-induced α-collagen I, CTGF, and α-SMA production in fibroblast which was blocked by D-Pro7-Ang-(1-7), a MrgD antagonist. This led to alleviation of oxidative injury and induction of autophagy similar to that reported for rapamycin. This study demonstrated that ALA via MrgD receptor reduced pulmonary fibrosis through attenuation of oxidative injury and induction of autophagy.

The Role of PI3K/AKT Pathway and NADPH Oxidase 4 in Host ROS Manipulation by Toxoplasma gondii.

Dendritic cell is one of the first innate immune cell to encounter T. gondii after the parasite crosses the host intestinal epithelium. T. gondii requires intact DC as a carrier to infiltrate into host central nervous system (CNS) without being detected or eliminated by host defense system. The mechanism by which T. gondii avoids innate immune defense of host cell, especially in the dendritic cell is unknown. Therefore, we examined the role of host PI3K/AKT signaling pathway activation by T. gondii in dendritic cell. T. gondii infection or T. gondii excretory/secretory antigen (TgESA) treatment to the murine dendritic cell line DC2.4 induced AKT phosphorylation, and treatment of PI3K inhibitors effectively suppressed the T. gondii proliferation but had no effect on infection rate or invasion rate. Furthermore, it is found that T. gondii or TgESA can reduce H2O2-induced intracellular reactive oxygen species (ROS) as well as host endogenous ROS via PI3K/AKT pathway activation. While searching for the main source of the ROS, we found that NADPH oxidase 4 (NOX4) expression was controlled by T. gondii infection or TgESA treatment, which is in correlation with previous observation of the ROS reduction by identical treatments. These findings suggest that the manipulation of the host PI3K/AKT signaling pathway and NOX4 expression is an essential mechanism for the down-regulation of ROS, and therefore, for the survival and the proliferation of T. gondii.

NOX4/H2O2/mTORC1 Pathway in Salt-Induced Hypertension and Kidney Injury.

We have reported that a high-salt (4.0% NaCl) dietary intake activates mTORC1 and inhibition of this pathway with rapamycin blunts the chronic phase of salt-induced hypertension and renal injury in Dahl salt-sensitive (SS) rats. In SS rats, high-salt intake is known to increase the renal production of H2O2 by NOX4, the most abundant NOX isoform in the kidney, and the global knockout of NOX4 blunts salt-sensitivity in these rats. Here, we explored the hypothesis that elevations of H2O2 by NOX4 in high-salt fed SS rat stimulate mTORC1 for the full development of salt-induced hypertension and renal injury. Our in vitro studies found that H2O2 activates mTORC1 independent of PI3K/AKT and AMPK pathways. To determine the in vivo relevance of NOX4/H2O2/mTORC1 in the salt-induced hypertension, SS-Nox4 knockout (SSNox4-/-) rats were daily administrated with vehicle/rapamycin fed a high-salt diet for 21 days. Rapamycin treatment of SSNox4-/- rats had shown no augmented effect on the salt-induced hypertension nor upon indices of renal injury. Significant reductions of renal T lymphocyte and macrophage together with inhibition of cell proliferation were observed in rapamycin treated rats suggesting a role of mTORC1 independent of NOX4 in the proliferation of immune cell. Given the direct activation of mTORC1 by H2O2 and absence of any further protection from salt-induced hypertension in rapamycin-treated SSNox4-/- rats, we conclude that NOX4-H2O2 is a major upstream activator of mTORC1 that contributes importantly to salt-induced hypertension and renal injury in the SS rat model.

Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4.

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of ß-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, ß-cell-specific knockout mice (NOX4ßKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4ßKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4ßKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause ß-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

Nox4-mediated ROS production is involved, but not essential for TGFß-induced lens EMT leading to cataract.

The reactive oxygen species (ROS) producing enzyme, NADPH oxidase 4 (Nox4), is upregulated in response to TGFß in lens epithelial cells in vitro, and its selective inhibition was shown to block aspects of TGFß-induced epithelial-mesenchymal transition (EMT). In the present in situ study we validate the role(s) of Nox4 in TGFß-induced lens EMT leading to anterior subcapsular cataract (ASC) formation. Mice overexpressing TGFß in the lens, that develop ASC, were crossed to Nox4-deficient mice. When comparing mice overexpressing TGFß in lens, to mice that were also deficient for Nox4, we see the delayed onset of cataract, along with a delay in EMT protein markers normally associated with TGFß-induced fibrotic cataracts. In the absence of Nox4, we also see elevated levels of ERK1/2 activity that was shown to be required for TGFß/Smad2/3-signaling. qRT-PCR revealed upregulation of Nox2 and its regulatory subunit in TGFß-overexpressing lens epithelial cells devoid of Nox4. Taken together, these findings provide an improved platform to delineate putative Nox4 (and ROS) interactions with Smad2/3 and/or ERK1/2, in particular in the development of fibrotic diseases, such as specific forms of cataract.

NADPH oxidase 4 expression in the normal endometrium and in endometrial cancer.

The aim of this study was to explore the role of NOX4 in the biology of the normal endometrium and endometrial cancer. NOX4 plays a key role in other adenocarcinomas and has been implicated in the pathogenesis of diabetes and obesity, which are important risk factors for endometrial cancer. NOX4 expression was assessed in 239 endometrial cancer and 25 normal endometrium samples by quantitative real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry. DNA methylation of the NOX4 promoter was determined by means of MethyLight PCR. Data were correlated with clinicopathological parameters and analyzed in the context of diabetes and body mass index. In the normal endometrium, NOX4 microRNA expression was significantly higher in the secretory transformed compared with proliferative endometrium ( p = 0.008). In endometrial cancer specimens, NOX4 expression did not differ between diabetic and non-diabetic patients, but was the highest in patients with a body mass index ≤ 26 ( p = 0.037). The lowest NOX4 expression was found in carcinosarcomas ( p = 0.007). High NOX4 expression predicted poorer clinical outcome with regard to overall survival, especially in non-diabetic patients and those with a body mass index > 20. Independent prognostic significance of NOX4 transcripts was retained in type I endometrial cancer and was the most meaningful in patients with a body mass index > 20. No prognostic impact was shown for NOX4 promoter methylation in endometrial cancer. For the first time, we demonstrate that NOX4 plays a considerable role in the cycle-dependent changes in the normal endometrium and in the biology of endometrial cancer.

A novel Gallic acid derivative attenuates BLM-induced pulmonary fibrosis in mice.

Idiopathic Pulmonary fibrosis is a disease with high morbidity and mortality. Therefore, the development of new drugs is imperative. Gallic acid derivative is a derivative of Gallic acid that can be extracted from Chinese herbal medicine. In previous experiments, we found that Gallic acid derivative played dual roles in inflammatory and antioxidant activities. Meanwhile, Gallic acid derivative could inhibit the proliferation of lung fibroblast. In the present study, we investigated the function of Gallic acid derivative in inhibiting lung fibrosis. 5 mg/kg of bleomycin was administered to mice by a single intratracheal instillation. Three dosages of Gallic acid derivative (75 mg/kg, 150 mg/kg, 300 mg/kg) and Pirfenidone (80 mg/kg) were given to mice for 21 day. Gallic acid derivative treatment significantly reduced lung histological changes and decreased inflammatory cell infiltration. The content of collagen decreased with the decrease of hydroxyproline level. Analogously, the expression of alpha smooth muscle actin was reduced. Gallic acid derivative enhanced the antioxidant status, but reduced the expression of interleukin 6, NADPH oxidase-4. Our study proved that Gallic acid derivative reduced inflammation activation to some extent and could exert its effects through transforming growth factor ß1/Smad2 signaling pathway and balancing NOX4/Nrf2.

Genome-Wide Methylation Analysis Identifies NOX4 and KDM5A as Key Regulators in Inhibiting Breast Cancer Cell Proliferation by Ginsenoside Rg3.

Ginsenoside Rg3 is a key metabolite of ginseng and is known to inhibit cancer cell growth. However, the epigenetics of CpG methylation and its regulatory mechanism have yet to be determined. Genome-wide methylation analysis of MCF-7 breast cancer cells treated with Rg3 was performed to identify epigenetically regulated genes and pathways. The effect of Rg3 on apoptosis and cell proliferation was examined by a colony formation assay and a dye-based cell proliferation assay. The association between methylation and gene expression was monitored by RT-PCR and Western blot analysis. Genome-wide methylation analysis identified the "cell morphology"-related pathway as the top network. Rg3 induced late stage apoptosis but inhibited cell proliferation up to 60%. Hypermethylated TRMT1L, PSMC6 and NOX4 were downregulated by Rg3, while hypomethylated ST3GAL4, RNLS and KDM5A were upregulated. In accordance, downregulation of NOX4 by siRNA abrogated the cell growth effect of Rg3, while the effect was opposite for KDM5A. Notably, breast cancer patients with a higher expression of NOX4 and KDM5A showed poor and good prognosis of survival, respectively. In conclusion, Rg3 deregulated tumor-related genes through alteration of the epigenetic methylation level leading to growth inhibition of cancer cells.

Canonical transient receptor potential 3 channels in atrial fibrillation.

The pathogenesis of atrial fibrillation (AF) is largely dependent on structural remodeling and electrical reconfiguration, which in turn drive localized fibrosis. Canonical transient receptor potential 3 (TRPC3) channel is indispensable regulator of fibrosis development, promoting fibroblasts to transition into myofibroblasts via intracellular Ca2+ overload. TRPC3 is a non-voltage gated, non-selective cation channel that regulates the permeability of the cell to Ca2+. When subjected to various external physical and chemical stimuli, such as angiotensin II (AngII), mechanical stretch, hypoxia, or oxidative stress, TRPC3 coordinates with downstream signal transduction pathways to alter gene expression and thereby regulate a number of distinct pathological patterns and mechanisms. This review will focus on how TRPC3 affects AF pathogenesis by exploring the underlying mechanisms governing fibrosis associated with particular signaling proteins, ultimately highlighting the characteristics of TPRC3 that mark it as a novel therapeutic target for AF alleviation.

Reactive oxygen species and NADPH oxidase 4 involvement in osteoarthritis.

Osteoarthritis (OA) is a degenerative chronic disease affecting >300,000 million people around the world as of 2016. Symptomatic measures exist, but there are hardly any curative treatments available. Disruption of the cartilage homeostasis in favor of catabolism leads to cartilage destruction. ROS-macromolecular-induced damage is significantly greater in OA cartilage and OA is described as low-grade chronic systemic inflammation. This review aimed to assess the critical role of cartilage ageing and oxidative stress in the OA process, focusing in particular on NADPH oxidase and especially Nox4 involvement. With age, hypertrophic senescent cells with an altered redox cell profile accumulated. Chondrocytes are more sensitive to oxidant-mediators and the serum level of pro-inflammatory mediators increases. Age-related advanced glycation end products impact on extra cellular matrix (ECM) properties leading to the apoptosis of chondrocytes. A focus on NADPH oxidase-mediated-ROS signaling highlighted the very specific Nox4 isoform, which plays a role on the final common pathway targeting chondrocyte cells. IL-1ß-mediated Nox4 stimulation induced an increase in the levels released by the chondrocyte of MMP-1 and MMP-13 proteins, which are involved in ECM degradation. In comparison with the other Nox isoforms, Nox4 remains unusual, since it is constitutively active, does not depend on cytosolic activator proteins and seems to generate H2O2 thanks to the specific conformation of the Nox4 E-loop. Nox4-induced ROS production appears an essential actor in the OA process and it could be relevant to focus on this target in the aim of discovering and developing new therapeutic strategies.

NOX4, a new genetic target for anti-cancer therapy in digestive system cancer.

Oxidative stress has been implicated as an important factor in tumorigenesis and tumor progression. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit 4 (NOX4), a substrate of NADPH that can generate H2 O2 reactive oxygen species, has been reported to be highly expressed in gastrointestinal tumors. In this review we summarize the available evidence on the biological function of NOX4 in digestive system tumors by focusing on its correlation with classical cell signaling pathways, including VEGF, MAPK and PI3K/AKT, and with biochemical mediators, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP)-1 and transforming growth factor (TGF)-ß. According to the clinical and database studies on tumors of the digestive system, such as colorectal, gastric and pancreatic cancer, there are significant associations between NOX4 expression and tumor prognosis as well as patient's survival. Animal studies using NOX4 inhibitors such as diphenylene iodonium and GKT137831, which selectively block NOX4, indicate their potential as therapeutic agents for targeting cancer cells.

NOX4-driven ROS formation regulates proliferation and apoptosis of gastric cancer cells through the GLI1 pathway.

NADPH Oxidase 4 (NOX4), a member of the NOX family, has emerged as a significant source of reactive oxygen species, playing an important role in tumor cell proliferation, apoptosis, and other physiological processes. However, the potential function of NOX4 in gastric cancer (GC) cell proliferation is yet unknown. The aim of this study was to illustrate whether NOX4 plays a role in regulating gastric cancer cell growth. First, the clinical information from 90 patients was utilized to explore the clinical value of NOX4 as a predictive tool for tumor size and prognosis. Results showed that NOX4 expression was correlated with tumor size and prognosis. In vitro assays confirmed that knockdown of NOX4 expression blocked cell proliferation and the expression of Cyclin D1, BAX, and so on. Interestingly, NOX4 promoted cell proliferation via activation of the GLI1 pathway. GLI1, a well-known transcription factor in the Hedgehog signaling pathway, was overexpressed to test whether NOX4 activates downstream signaling via GLI1. Overexpression of GLI1 reversed the inhibition of proliferation induced by NOX4 knockdown. In addition, overexpression of NOX4 increased GLI1 expression, and depletion of GLI1 expression decreased the effects induced by NOX4 overexpression. Further, ROS generated by NOX4 was required for GLI1 expression, as shown by use of the ROS inhibitor, diphenylene iodonium (DPI). In summary, the findings indicate that NOX4 plays an important role in gastric cancer cell growth and apoptosis through the generation of ROS and subsequent activation of GLI1 signaling. Hence, the targeting of NOX4 may be an attractive therapeutic strategy for blocking gastric cancer cell proliferation.

The involvement of Nox4 in fine particulate matter exposure-induced cardiac injury in mice.

Epidemiological studies have confirmed that ambient fine particulate matter (PM2.5) exposure is associated with cardiovascular disease (CVD). However, the underlying mechanisms in PM2.5 exposure-induced heart injury are largely unknown. It has been acknowledged that NADPH oxidase (Nox) 4 plays a critical role in CVD development. To investigate the acute effects of PM2.5 on the mouse heart and the role of Nox4 in PM2.5 exposure-induced cardiac injury, C57BL/6J mice were instilled with saline or 1.5, 3.0, 6.0 mg/kg BW PM2.5 suspension for two weeks (five days per week). The levels of malondialdehyde (MDA), super oxide dismutase (SOD), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß in heart supernatants were determined using related kits. The expression of Nox4, p67phox, p47phox and p22phox in heart tissue was evaluated by immunofluorescence staining or Western blotting, respectively. Protein levels of p53, Bax, Bcl-2 and Caspase-3 in the heart were examined using immunohistochemical staining and Western blotting. TUNEL assay was used to measure myocardial apoptosis. PM2.5 exposure leads to obvious cardiac injury. PM2.5 exposure increases MDA level and iNOS activity, and decreases activity of SOD in heart supernatants of mice. High levels of TNF-α and IL-1ß in heart supernatants of mice with PM2.5 instillation were determined. Nox4 and Nox-associated subunits such as p67phox, p47phox and p22phox expression levels were increased in heart tissue of mice after PM2.5 exposure. Additionally, PM2.5 exposure causes myocardial apoptosis in the mouse heart. This study suggested that Nox4 is involved in PM2.5 exposure-induced cardiac injury in mice.

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.

NADPH oxidase 4 modulates hepatic responses to lipopolysaccharide mediated by Toll-like receptor-4.

Chronic inflammation plays a key role in development of many liver diseases. Stimulation of Toll-like receptor 4 (TLR4) by bacterial lipopolysaccharide (LPS) initiates inflammation and promotes development of hepatocellular carcinoma and other liver diseases. NADPH oxidases contribute to LPS-induced reactive oxygen species (ROS) production and modulate TLR responses, but whether these enzymes function in TLR4 responses of hepatocytes is unknown. In the present work, we examined the role of NADPH oxidase 4 (Nox4) in LPS-induced TLR4 responses in human hepatoma cells and wildtype and Nox4-deficient mice. We found that LPS increased expression of Nox4, TNF-α, and proliferating cell nuclear antigen (PCNA). Nox4 silencing suppressed LPS-induced TNF-α and PCNA increases in human cells. The LPS-induced TNF-α increases were MyD88-dependent, and were attenuated in primary hepatocytes isolated from Nox4-deficient mice. We found that Nox4 mediated LPS-TLR4 signaling in hepatocytes via NF-ĸB and AP-1 pathways. Moreover, the effect of Nox4 depletion was time-dependent; following six weeks of repeated LPS stimulation in vivo, hepatic TNF-α and PCNA responses subsided in Nox4-deficient mice compared with wildtype mice. Therefore, our data suggest that Nox4 mediates LPS-TLR4 signaling in human hepatoma cells and murine hepatocytes and may contribute to the ability of LPS to stimulate liver pathology.

NADPH oxidase 4 is involved in the triethylene glycol dimethacrylate-induced reactive oxygen species and apoptosis in human embryonic palatal mesenchymal and dental pulp cells.

OBJECTIVES: Triethylene glycol dimethacrylate (TEGDMA) is a common component of resin-based dental composites and endodontic sealers. TEGDMA induces apoptosis in several types of cells. However, the mechanisms are not completely understood. The aim of this study was to investigate the mechanisms underlying TEGDMA-induced apoptosis in human embryonic palatal mesenchymal (HEPM) pre-osteoblasts and primary human dental pulp (HDP) cells. MATERIAL AND METHODS: Cell viability was examined after TEGDMA treatment. Cell cycle progression was checked by flow cytometry. Apoptotic cells were evaluated using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling assay and visualized by fluorescence microscopy. Western blot analyses were performed to determine expressions of apoptosis-related proteins. The production of reactive oxygen species (ROS) was detected using flow cytometry. NADPH oxidase 4 (NOX4) expression levels were investigated using real-time quantitative polymerase chain reaction and Western blot analyses. RESULTS: TEGDMA increased cytosol cytochrome c levels and activated caspase-9 in HEPM and HDP cells. TEGDMA decreased the expression of anti-apoptotic protein Bcl-XL. TEGDMA-induced apoptosis was inhibited by caspase-9-specific inhibitor, anti-oxidants, NOX inhibitor, NOX4 inhibitor, and NOX4 small interfering RNA (siRNA). TEGDMA increased ROS production and upregulated NOX4 mRNA and protein expression. TEGDMA-induced intracellular ROS production was inhibited by NOX inhibitor and NOX4 inhibitor. CONCLUSIONS: We demonstrate significant involvement of NOX4 in the TEGDMA-induced ROS. NOX4-derived ROS subsequently induces mitochondrial cytochrome c release leading to apoptosis through activation of the intrinsic apoptotic pathway. CLINICAL RELEVANCE: NOX4 may be a potential target for strategies to prevent or ameliorate the TEGDMA-induced toxicity in HEPM and HDP cells.