PURPOSE: Crohn's disease is a chronic gastrointestinal inflammatory disease with possible extraintestinal symptoms. There are predisposing genetic factors and even monogenic variants of the disorder. One of the possible genetic factors are variants of the DUOX2 gene. The protein product of the DUOX2 gene is a dual oxidase enzyme producing H2O2 in the bowel. Reduced H2O2 levels impact mucosal homeostasis and contribute to the development of inflammatory bowel disease. Thus far, only 19 patients with IBD with the DUOX2 variants have been described. METHODS: Here we present a case report of an adolescent female diagnosed at eleven years of age with IBD that was subsequently reclassified as Crohn's disease. She was treated with immunosuppressants and biological therapy but experienced additional complications. Her peripheral blood lymphocyte DNA was studied using massive parallel sequencing. Detected variants were functionally studied. RESULTS: Whole exome sequencing found two novel DUOX2 gene variants: a de novo variant c.3646C>T; p.R1216W and a maternally inherited variant c.3391G>A; p.A1131T which were initially classified as variants of unknown significance. However, follow-up functional studies demonstrated that both DUOX2 variants led to impaired H2O2 generation, which led to their reclassification to the likely pathogenic class according to the ACMG.net. Therefore, we conclude that these variants are causative for the disease. CONCLUSIONS: Identifying novel variants in patients with Crohn's disease and their families is important for precision medicine approaches and understanding of the pathogenesis of likely "monogenic" rare forms of inflammatory bowel disease.

• Keywords
• Crohn’s Disease, DUOX2, Massive parallel sequencing, Whole exome sequencing,
• MeSH
• Crohn Disease * genetics   MeSH
• Dual Oxidases genetics   MeSH
• Inflammatory Bowel Diseases * genetics   MeSH
• Humans MeSH
• Adolescent MeSH
• Hydrogen Peroxide MeSH
• Check Tag
• Humans MeSH
• Adolescent MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Names of Substances
• Dual Oxidases MeSH
• DUOX2 protein, human MeSH Browser
• Hydrogen Peroxide MeSH

OBJECTIVE: By exposing mice carrying a deletion of NADPH oxidase isoform 4, NOX4, specifically in pancreatic β cells (βNOX4-/-) to nutrient excess stimulated by a high-fat diet (HFD), this study aimed to elucidate the role of β-cell redox status in the development of meta-inflammation within the diabetic phenotype. METHODS: The authors performed basic phenotyping of βNOX4-/- mice on HFD involving insulin and glycemic analyses, histochemistry of adipocytes, indirect calorimetry, and cytokine analyses. To characterize local inflammation, the study used caspase-1 activity assay, interleukin-1β immunochemistry, and real-time polymerase chain reaction during coculturing of β cells with macrophages. RESULTS: The phenotype of βNOX4-/- mice on HFD was not associated with hyperinsulinemia and hyperglycemia but showed accumulation of excessive lipids in epididymal fat and β cells. Surprisingly, mice showed significantly reduced systemic inflammation. Decreased interleukin-1β protein levels and downregulated NLRP3-inflammasome activity were observed on chronic glucose overload in βNOX4-/- isolated islets and NOX4-silenced INS1-E cells resulting in attenuated proinflammatory polarization of macrophages/monocytes in vitro and in situ and reduced local islet inflammation. CONCLUSIONS: Experimental evidence suggests that NOX4 pro-oxidant activity in β cells is involved in NLRP3-inflammasome activation during chronic nutrient overload and participates in local inflammatory signaling and perhaps toward peripheral tissues, contributing to a diabetic inflammatory phenotype.

• MeSH
• Diabetes Mellitus * MeSH
• Inflammasomes metabolism   MeSH
• Interleukin-1beta metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• NADPH Oxidase 4 genetics   MeSH
• NLR Family, Pyrin Domain-Containing 3 Protein * metabolism   MeSH
• Inflammation MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Inflammasomes MeSH
• Interleukin-1beta MeSH
• NADPH Oxidase 4 MeSH
• Nox4 protein, mouse MeSH Browser
• NLR Family, Pyrin Domain-Containing 3 Protein * MeSH

Oxidative stress closely related to the progression and severity of myocardial infarction (MI). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is one of the major enzymes that generate reactive oxygen species (ROS) in cardiovascular system. Here, we aim to elucidate the pathological role of NOX4 in MI. MI mouse model was created by the coronary artery ligation. NOX4 was specifically knocked down in heart through intramyocardial injection of siRNA. NOX4 expression and oxidative stress indicators were determined at different time points using qRT-PCR, Western blot, and ELISA, and then analyzed by Pearson's correlation. Cardiac function was evaluated by using echocardiographic technique. NOX4 was upregulated in myocardial tissues of MI mice, which positively correlated with the elevation of oxidative stress indicators. Knockdown of NOX4 in heart significantly reduced the production of ROS and the level of oxidative stress in left ventricle tissues, which was accompanied by significant improvement of cardiac function in MI mice. Selective knockdown of NOX4 in heart attenuates MI-induced oxidative stress and improves cardiac function, suggesting inhibition of NOX4/ROS axis in heart using siRNA is a potential therapeutic treatment for MI-induced cardiac dysfunction.

• MeSH
• Myocardial Infarction * genetics   MeSH
• Coronary Vessels MeSH
• Disease Models, Animal MeSH
• Mice MeSH
• NADPH Oxidase 4 * genetics   MeSH
• Oxidative Stress * MeSH
• Reactive Oxygen Species MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NADPH Oxidase 4 * MeSH
• Nox4 protein, mouse MeSH Browser
• Reactive Oxygen Species MeSH

Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca2+, phosphorylation, and protein-protein interactions on RBOH-dependent ROS production.

• Keywords
• Human embryonic kidney 293T (HEK293T), Luminol, NADPH oxidase, Respiratory oxidase homolog (RBOH),
• MeSH
• HEK293 Cells MeSH
• Kidney metabolism   MeSH
• Humans MeSH
• NADPH Oxidases * metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Gene Expression Regulation, Plant * MeSH
• Plants metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NADPH Oxidases * MeSH
• Reactive Oxygen Species MeSH

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

• Keywords
• HL-60 cells, MPO, N-acetylcysteine, NOX, SOD, U937 cells, oxidative stress,
• MeSH
• Acetylcysteine pharmacology   MeSH
• HL-60 Cells MeSH
• Catalase genetics   MeSH
• Leukemia drug therapy   genetics   metabolism   MeSH
• Humans MeSH
• NADPH Oxidase 2 genetics   MeSH
• Oxidative Stress drug effects   MeSH
• Peroxidase genetics   MeSH
• Cell Proliferation drug effects   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Gene Expression Profiling MeSH
• Superoxide Dismutase genetics   MeSH
• U937 Cells MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcysteine MeSH
• CYBB protein, human MeSH Browser
• Catalase MeSH
• MPO protein, human MeSH Browser
• NADPH Oxidase 2 MeSH
• Peroxidase MeSH
• Reactive Oxygen Species MeSH
• Superoxide Dismutase MeSH

Reactive oxygen species (ROS) such as superoxide (O2-) generated by NAD(P)H oxidases have emerged as important molecules in blood pressure regulation. This study investigated the effect of apocynin and catalase on blood pressure and renal haemodynamic and excretory function in an L-NAME induced hypertension model. Forty Male Wistar-Kyoto (WKY) rats (n=8 per group) were treated with either: vehicle (WKY-C); L-NAME (WKY-L, 15 mg/kg/day in drinking fluid); WKY-L given apocynin to block NAD(P)H oxidase (WKY-LApo, 73 mg/kg/day in drinking water.); WKY-L given catalase to enhance ROS scavenging (WKY-LCat, 10000 U/kg/day i.p.); and WKY-L receiving apocynin plus catalase (WKY-LApoCat) daily for 14 days. L-NAME elevated systolic blood pressure (SBP), 116+/-1 to 181±4 mmHg, reduced creatinine clearance, 1.69+/-0.26 to 0.97+/-0.05 ml/min/kg and fractional sodium excretion, 0.84+/-0.09 to 0.55+/-0.09 % at day 14. Concomitantly, plasma malondialdehyde (MDA) increased six fold, while plasma total superoxide dismutase (T-SOD), plasma nitric oxide (NO) and plasma total antioxidant capacity (T-AOC) were decreased by 60-70 % and Nox 4 mRNA expression was increased 2-fold. Treatment with apocynin and catalase attenuated the increase in SBP and improved renal function, enhanced antioxidative stress capacity and reduced the magnitude of Nox4 mRNAs expression in the L-NAME treated rats. This study demonstrated that apocynin and catalase offset the development of L-NAME induced hypertension, renal dysfunction and reduced oxidative stress status, possibly contributed by a reduction in Nox4 expression during NOS inhibition. These findings would suggest that antioxidant compounds such as apocynin and catalase have potential in treating cardiovascular diseases.

• MeSH
• Acetophenones pharmacology   MeSH
• Antioxidants pharmacology   MeSH
• Hemodynamics MeSH
• Hypertension chemically induced   drug therapy   physiopathology   MeSH
• Enzyme Inhibitors toxicity   MeSH
• Catalase pharmacology   MeSH
• Drug Therapy, Combination MeSH
• Rats MeSH
• Disease Models, Animal MeSH
• NADPH Oxidase 4 metabolism   MeSH
• NG-Nitroarginine Methyl Ester toxicity   MeSH
• Rats, Inbred WKY MeSH
• Reactive Oxygen Species metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetophenones MeSH
• acetovanillone MeSH Browser
• Antioxidants MeSH
• Enzyme Inhibitors MeSH
• Catalase MeSH
• NADPH Oxidase 4 MeSH
• NG-Nitroarginine Methyl Ester MeSH
• Nox4 protein, rat MeSH Browser
• Reactive Oxygen Species MeSH

Numerous pathological changes of subcellular structures are characteristic hallmarks of neurodegeneration. The main research has focused to mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomal networks as well as microtubular system of the cell. The sequence of specific organelle damage during pathogenesis has not been answered yet. Exposition to rotenone is used for simulation of neurodegenerative changes in SH-SY5Y cells, which are widely used for in vitro modelling of Parkinson´s disease pathogenesis. Intracellular effects were investigated in time points from 0 to 24 h by confocal microscopy and biochemical analyses. Analysis of fluorescent images identified the sensitivity of organelles towards rotenone in this order: microtubular cytoskeleton, mitochondrial network, endoplasmic reticulum, Golgi apparatus and lysosomal network. All observed morphological changes of intracellular compartments were identified before alphaS protein accumulation. Therefore, their potential as an early diagnostic marker is of interest. Understanding of subcellular sensitivity in initial stages of neurodegeneration is crucial for designing new approaches and a management of neurodegenerative disorders.

• MeSH
• Apoptosis MeSH
• Insecticides toxicity   MeSH
• Humans MeSH
• Microtubules drug effects   metabolism   pathology   MeSH
• Mitochondria drug effects   metabolism   pathology   MeSH
• Cell Line, Tumor MeSH
• NADPH Oxidases metabolism   MeSH
• Neuroblastoma complications   MeSH
• Neurodegenerative Diseases etiology   metabolism   pathology   MeSH
• Rotenone toxicity   MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insecticides MeSH
• NADPH Oxidases MeSH
• Rotenone MeSH

INTRODUCTION: Against the background of significant progress in anticancer therapy, which makes it possible to improve the quality of life and life expectancy of patients with cancer, anthracycline antibio-tics retain their relevance, both for scientific research and for clinical practice. However, the therapeutic efficacy of anthracyclines is associated with the development of various complications, among which the most common is cardiotoxicity. Our study was dedicated to searching for possible associations between rs4673 and rs28714259 single nucleotide polymorphisms (SNPs) and the risk of cardiotoxicity in breast cancer patients who underwent anthracycline-containing chemotherapy. MATERIALS AND METHODS: The study included 256 patients with a dia-gnosis of breast cancer without dia-gnosed cardiovascular changes who were treated at the National Medical Research Center of Oncology in Rostov-on-Don in 2019-2020. For SNP genotyping, DNA was extracted from blood and high resolution melting analysis was performed. The presence of SNPs was confirmed by Sanger sequencing. RESULTS: The presence of the T-allele rs4673 increased the risk of cardiotoxicity in breast cancer patients 6.49x (95% CI 1.48-28.53; P = 0.002), and the presence of the A-allele rs28714259 increased the risk 3.27x (95% CI 1.23-8.75; P = 0.026). For tests based on genotyping rs4673 and rs28714259 SNPs, the areas under the receiver operating characteristic (ROC) curves were equal to 71.9% and 76.3%, respectively. The two-locus SNP-SNP model turned out to be statistically significant: the training balanced accuracy was 0.77; similarly, the testing balanced accuracy, and the cross-validation consistency was 10/10. CONCLUSION: Our study confirmed the predictive value of genetic tests based on the determination of the rs4673 and rs28714259 SNPs. Genotyping of both SNPs will significantly improve the accuracy of predicting the development of cardiotoxicity against the background of anthracycline-containing therapy and timely identify the risk group of breast cancer patients for whom it is necessary to adjust the therapeutic strategy.

• Keywords
• SNP-SNP interactions, anthracycline-mediated cardiotoxicity– rs4673, breast cancer, rs28714259,
• MeSH
• Anthracyclines adverse effects   MeSH
• Adult MeSH
• Polymorphism, Single Nucleotide MeSH
• Cardiotoxicity etiology   genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Breast Neoplasms drug therapy   genetics   MeSH
• NADPH Oxidases genetics   MeSH
• Antibiotics, Antineoplastic adverse effects   MeSH
• Risk MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anthracyclines MeSH
• CYBA protein, human MeSH Browser
• NADPH Oxidases MeSH
• Antibiotics, Antineoplastic MeSH

L-arginine is a substrate for nitric oxide synthase (NOS) responsible for the production of NO. This investigation studied the effect of apocynin, an NADPH oxidase inhibitor and catalase, an H2O2 scavenger on L-arginine induced oxidative stress and hypotension. Forty Wistar-Kyoto rats were treated for 14 days with vehicle, L-arginine (12.5mg/ml p.o.), L-arginine+apocynin (2.5mmol/L p.o.), L-arginine+catalase (10000U/kg/day i.p.) and L-arginine plus apocynin+catalase respectively. Weekly renal functional and hemodynamic parameters were measured and kidneys harvested at the end of the study for histopathological and renal NADPH oxidase 4 (Nox4) assessments. L-arginine administration in normotensive rats decreased systolic blood pressure (120±2 vs 91±2mmHg) and heart rate (298±21 vs 254±15b/min), enhanced urinary output (21.5±4.2 vs 32±1.9ml/24h , increased creatinine clearance (1.72±0.56 vs 2.62±0.40ml/min/kg), and fractional sodium excretion (0.88±0.16 vs 1.18±0.16 %), caused proteinuria (28.10±1.93 vs 35.26±1.69mg/kg/day) and a significant decrease in renal cortical blood perfusion (292±3 vs 258±5bpu) and pulse wave velocity (3.72±0.20 vs 2.84±0.13m/s) (all P<0.05). L-arginine increased plasma malondialdehyde (by ~206 % P<0.05) and NO (by~51 %, P<0.05) but decreased superoxide dismutase (by~31 %, P<0.05) and total antioxidant capacity (by~35 %, P<0.05) compared to control. Renal Nox4 mRNA activity was approximately 2.1 fold higher (P<0.05) in the L-arginine treated rats but was normalized by apocynin and apocynin plus catalase treatment. Administration of apocynin and catalase, but not catalase alone to rats fed L-arginine, restored the deranged renal function and structure, prevented hypotension and enhanced the antioxidant capacity and suppressed Nox4 expression. These findings suggest that apocynin and catalase might be used prophylactically in states of oxidative stress.

• MeSH
• Acetophenones pharmacology   MeSH
• Pulse Wave Analysis methods   MeSH
• Antioxidants pharmacology   MeSH
• Arginine pharmacology   MeSH
• Hypotension chemically induced   drug therapy   metabolism   pathology   MeSH
• Catalase pharmacology   MeSH
• Blood Pressure drug effects   MeSH
• Rats MeSH
• Kidney drug effects   metabolism   pathology   MeSH
• Disease Models, Animal MeSH
• NADPH Oxidase 4 metabolism   MeSH
• Oxidative Stress drug effects   MeSH
• Rats, Inbred WKY MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetophenones MeSH
• acetovanillone MeSH Browser
• Antioxidants MeSH
• Arginine MeSH
• Catalase MeSH
• NADPH Oxidase 4 MeSH
• NOX4 protein, human MeSH Browser

Hypersensitive pain response is observed in patients with Parkinson's disease (PD). However, the signal pathways leading to hyperalgesia still need to be clarified. Chronic oxidative stress is one of the hallmarks of PD pathophysiology. Since the midbrain periaqueductal gray (PAG) is an important component of the descending inhibitory pathway controlling on central pain transmission, we examined the role NADPH oxidase (NOX) of the PAG in regulating exaggerated pain evoked by PD. PD was induced by central microinjection of 6-hydroxydopamine to lesion the left medial forebrain bundle of rats. Then, Western Blot analysis and ELISA were used to determine NOXs and products of oxidative stress (i.e., 8-isoprostaglandin F2alpha and 8-hydroxy-2'-deoxyguanosine). Pain responses to mechanical and thermal stimulation were further examined in control rats and PD rats. In results, among the NOXs, protein expression of NOX4 in the PAG of PD rats was significantly upregulated, thereby the products of oxidative stress were increased. Blocking NOX4 pathway in the PAG attenuated mechanical and thermal pain responses in PD rats and this was accompanied with decreasing production of oxidative stress. In addition, inhibition of NOX4 largely restored the impaired GABA within the PAG. Stimulation of GABA receptors in the PAG of PD rats also blunted pain responses. In conclusions, NOX4 activation of oxidative stress in the PAG of PD rats is likely to impair the descending inhibitory GABAergic pathways in regulating pain transmission and thereby plays a role in the development of pain hypersensitivity in PD. Inhibition of NOX4 has beneficial effects on the exaggerated pain evoked by PD.

• MeSH
• Pain drug therapy   etiology   metabolism   pathology   MeSH
• Medial Forebrain Bundle drug effects   metabolism   MeSH
• gamma-Aminobutyric Acid metabolism   MeSH
• Rats MeSH
• Disease Models, Animal MeSH
• NADPH Oxidase 4 antagonists & inhibitors   MeSH
• Parkinson Disease enzymology   metabolism   pathology   MeSH
• Rats, Sprague-Dawley MeSH
• Pain Threshold drug effects   physiology   MeSH
• Pyrazolones pharmacology   MeSH
• Pyridones pharmacology   MeSH
• Signal Transduction drug effects   MeSH
• Periaqueductal Gray drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• gamma-Aminobutyric Acid MeSH
• NADPH Oxidase 4 MeSH
• Nox4 protein, rat MeSH Browser
• Pyrazolones MeSH
• Pyridones MeSH
• setanaxib MeSH Browser