Extracellular Hydrogen Peroxide Research Articles

Colorimetric Detection of Extracellular Hydrogen Peroxide Using an Integrated Microfluidic Device.

It is well known that hydrogen peroxide (H2O2) is a signaling molecule essential for vital physiological reactions in mammalian cells, such as cell survival, intercellular communication, and cancer metabolism. However, to fully understand the function of H2O2, it is critical to monitor its intracellular and/or extracellular concentrations. Current techniques implemented to address this need require large sample volumes, expensive instrumentation, and long sample preparation and analysis times, inapplicable to inline or online monitoring. In this paper, a new integrated microfluidic device capable of overcoming these limitations is demonstrated for the colorimetric detection of extracellular hydrogen peroxide H2O2. The device contains an optical waveguide to determine absorbance changes and micromixers to enable complete mixing of reagents using a passive approach. This novel H2O2-sensing device has allowed the detection of H2O2 in the range of 0.5-60 μM with a detection limit of 167 ± 5.8 nM and a sensitivity of 13.5 ± 0.1 AU/mM. Proof of concept of the device was demonstrated by quantifying H2O2 release from benign prostatic epithelial (BPH-1) cells upon stimulation with phorbol 12-myristate 13-acetate (PMA). Results show that this integrated device can be potentially utilized to continuously monitor cell-released metabolites autonomously without constant human supervision during the process. Furthermore, this can be achieved without interfering with the cell culture conditions, as only a very small volume of conditioned media (less than 0.4 μL), and not the cells, is required.

Development of an ATP-independent bioluminescent probe for detection of extracellular hydrogen peroxide.

This work reports a new ATP-independent bioluminescent probe (bor-DTZ) for detecting hydrogen peroxide that is compatible with the Nanoluciferase enzyme. The probe is designed with an arylboronate ester protecting group appended to a diphenylterazine core via a self-immolative phenolate linker. Reaction with hydrogen peroxide reveals diphenylterazine, which can then react with Nanoluciferase to produce a detectable bioluminescent signal. Bor-DTZ shows a dose-dependent response to hydrogen peroxide and selectivity over other biologically relevant reactive oxygen species and can be applied to detect either intra- or extracellular species. We further demonstrate the ability of this platform to monitor fluxes in extracellular hydrogen peroxide in a breast cancer cell line in response to the anticancer treatment, cisplatin.

Fe-Hemin-Metal Organic Frameworks/Three-Dimensional Graphene Composites with Efficient Peroxidase-Like Bioactivity for Real-Time Electrochemical Detection of Extracellular Hydrogen Peroxide

Real-time detection of extracellular hydrogen peroxide is important for dynamic monitoring of diseases and cytopathological research. Here, a novel composite of Fe-hemin-metal organic frameworks/three-dimensional graphene (Fe-hemin MOFs/3DG) was prepared by embedding hemin into amino-contained Fe-MOFs, then decorated with 3DG. The obtained Fe-hemin MOFs/3DG possessed efficient peroxidase-like bioactivity and could serve as an effective catalyst for construction of H2O2 electrochemical sensor. The electrochemical results show that the proposed sensor based on Fe-hemin MOFs/3DG has excellent catalytic activity for H2O2. With a linear range was 10−12080 μM and LOD was 0.34 μM, this sensor showed favorable selectivity, repeatability and stability, which could be used to detect H2O2 in real serum samples. Most importantly, this sensor realized the real-time determination of H2O2 released by A549 cells and possessed good biocompatibility. The outstanding electrochemical performance mainly benefited from the unique porous structure of MOFs, which could effectively protect the active center of hemin, and the introduction of 3DG greatly improved the conductivity of Fe-hemin MOFs. Therefore, the Fe-hemin MOFs/3DG could be a promising probe for real-time dynamic monitoring of H2O2.

Co–Doped Fe–Co@C–Nx Nano-Arrays Grown on Carbon Cloth as a Novel Flexible Electrode for In Situ Detection of Extracellular Hydrogen Peroxide

Flexible electrodes play an increasingly important role in the development of portable and wearable electronics. Herein, a novel flexible and self-supporting electrode supported on active electrocatalyst (Au/Fe–Co@C–Nx/GO/CC) has been prepared for in situ detection of extracellular H2O2. The Fe, N co-doping of Fe–Co@C–Nx with abundant active sites promotes electron transfer efficiency, and Fe–Co@C–Nx nano-arrays with a unique spatial structure are beneficial for the deposition of Au nanoparticles (AuNPs). Graphene oxide (GO) serves as a chemical bridge to ensure that the nanomaterials grow more uniformly and firmly on the carbon cloth. Meanwhile, the flexible carbon cloth possesses stable mechanical and electrochemical properties. Based on these unique superiorities, this flexible electrode exhibits excellent sensing performance toward H2O2 with high selectivity and sensitivity, low LOD (0.56 μM), and wide linear range (from 5 to 21195 μM). Most importantly, the prepared electrode can adhere to cells for in situ and sensitive determination of H2O2 released from cells. Compared to detecting H2O2 in cell suspensions, in situ detection can capture the signal molecules more sensitively and effectively, avoiding the diffusion of H2O2 from the solution to the electrode surface. This flexible electrode has great potential in development of molecular detection and portable electronics.

Plasma‐activated Ringer's lactate solution inhibits the cellular respiratory system in HeLa cells

AbstractNonequilibrium atmospheric pressure plasma has enabled a variety of new applications in medicine, agriculture, and other industries. It is particularly noteworthy that plasma itself and/or plasma‐activated culture medium have been shown to preferentially kill various cancer cells. We have previously developed a plasma‐activated Ringer's lactate solution (PAL) for use as a new cancer treatment. In this study, behaviors of extracellular and intracellular reactive oxygen and nitrogen species in the cellular respiratory system of PAL‐treated HeLa cells were investigated using an extracellular flux analyzer and a probe to measure mitochondrial membrane potential. In PAL‐treated HeLa cells, extracellular hydrogen peroxide in PAL was found to be responsible for the induction of intracellular hydrogen peroxide and apoptosis, while other components in PAL are responsible for the induction of non‐H2O2 intracellular ROS and non‐apoptotic cell death, which should be clarified by further experiments. We believe that these are long‐lived species derived from plasma‐activated lactates. Furthermore, we found that the plasma‐activated lactates inhibited glycolysis and the tricarboxylic acid (TCA) cycle, but not the electron transport chain in HeLa cells. These results suggest that PAL induces multiple modes of cell death, including apoptosis through hydrogen peroxide, and non‐apoptotic cell death associated with the impairment of mitochondrial functions (glycolysis and TCA cycle). These findings shed light on the novel mechanism underlying plasma‐activated lactate‐induced cell death.

Abstract 1755: E. coli genomic DNA fragments induce cGAS-STING-mediated DUOX2 expression in human pancreatic cancer cells that is associated with H2O2-related DNA damage

Abstract There is a growing appreciation that pancreatic cancers harbor higher numbers of bacteria & fungi compared to normal pancreatic tissue in both humans & mice. These microbes have been suggested to contribute to pancreatic carcinogenesis, & impact therapeutic sensitivity & resistance. Dual oxidase 2 (DUOX2), one of the seven NADPH oxidases, is expressed at the pancreatic cancer plasma membrane & generates extracellular hydrogen peroxide (H2O2). We previously demonstrated that DUOX protein was significantly upregulated in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), & some early stage pancreatic ductal adenocarcinomas (PDACs). Moreover, DUOX expression in these tissues was associated with H2O2-mediated DNA double strand breaks & upregulation of hypoxia markers VEGF & HIF-1α. Incidentally, we also discovered that mycoplasma-contaminated BxPC-3 human pancreatic cancer cells upregulated known oncogenes such as EGFR, MET, & VEGF-A, in concert with ~20-fold upregulation of DUOX2 mRNA. Recently, the cytosolic DNA-sensing cGAS-STING pathway has been implicated as a critical mediator of tumor-targeting immune responses. Using a panel of human colon & pancreatic cancer cell lines coupled with Western blot analysis & qPCR methods, we have demonstrated that the introduction of purified E. coli genomic DNA fragments, as well as several kinds of plasmid DNA, into the pancreatic cancer cell cytosol significantly enhanced DUOX2 RNA & protein expression, leading to an enhanced DNA damage response. For BxPC-3, CFPAC-1, & HTB134 human pancreatic cancer cells, enhanced DUOX2 expression following introduction of exogenous DNA was associated with the activation of the cGAS-STING pathway, as indicated by enhanced IRF-3 and TBK1 phosphorylation. Treatment of these cell lines with exogenous cGAMP for 24h also activated the cGAS-STING pathway and significantly increased DUOX2 expression. Correspondingly, in BxPC-3 cells treated with human cGAS-specific small interfering RNAs, we observed the significant suppression of cGAS RNA and protein expression & related downstream signaling, along with the significant attenuation of DNA-induced DUOX2 RNA & protein. In contrast, exogenous DNA did not upregulate DUOX2 in colon cancer cell lines. In summary, our data suggest that, in pancreatic cancers, a bacteria-containing microenvironment is likely to promote DUOX2-related H2O2 production as a consequence of the bacterial DNA-induced activation of the cGAS-STING innate immunity pathway, which, in turn, could enhance the oxidative milieu, augment leukocyte recruitment, & increase genetic instability, thus contributing to the molecular evolution of pancreatic tumors. Citation Format: Yongzhong Wu, Stephen L. Wang, Smitha Antony, Jennifer L. Meitzler, Jiamo Lu, Guojian Jiang, Becky Diebold, Mallick David, Mariam M. Konate, Roy Krishnendu, James H. Doroshow. E. coli genomic DNA fragments induce cGAS-STING-mediated DUOX2 expression in human pancreatic cancer cells that is associated with H2O2-related DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1755.

Assessing Bioenergetic Function in Response to Reactive Oxygen Species in Neural Cells

In this study, we characterized the bioenergetic response of the Lund human mesencephalic (LUHMES) cell line and a mouse astrocyte cell line to oxidative stress. Extracellular hydrogen peroxide (H2O2) levels and bioenergetic response were investigated in these cell lines after exposure to paraquat (PQ), a redox cycling compound that causes oxidative stress in cells. We used extracellular flux analysis to measure mitochondrial function in adherent astrocytes and LUHMES cells. Extracellular H2O2 was measured fluorometrically. H2O2 levels increased in both cell lines after exposure to 5 µM PQ for 18 h; however, the extent of H2O2 increase with astrocytes was significantly lower than that with LUHMES cells (33% vs. 67%). Measurements of basal mitochondrial respiration showed that PQ almost completely eliminated oxygen consumption rate (OCR) in astrocytes and significantly reduced it in LUHMES cells. Notably, OCR in LUHMES cells was higher than that in astrocytes, indicating that neuronal cells maintain higher oxidative metabolism than glial cells, which is also consistent with higher energy demands of the neuronal cells. Moreover, LUHMES cells exhibited a higher amount of adenosine triphosphate (ATP) being produced by oxidative phosphorylation than by glycolysis. In contrast, astrocytes demonstrated a higher glycolytic capacity and glycolytic reserve than LUHMES cells and higher ATP production rate by glycolysis than its production by mitochondrial oxidative metabolism. Collectively, this study showed the differential bioenergetic responses between astrocytes and LUHMES cells in responding to oxidative stress and the findings may provide insights into the mitochondrial reserve capacity in neurons and astrocytes in responding to oxidative stress. (First online: Mar 30, 2021)

Electrochemical sensor based on the Mn3O4/CeO2 nanocomposite with abundant oxygen vacancies for highly sensitive detection of hydrogen peroxide released from living cells.

Based on the strategy of increasing the number of oxygen vacancies to improve the catalytic performance, we have developed a novel electrochemical sensor based on the multivalent metal oxides cerium dioxide and manganous oxide (Mn3O4/CeO2) for reliable determination of extracellular hydrogen peroxide (H2O2) released from living cells. The Mn3O4/CeO2 nanocomposite was characterized by high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The electrochemical performance of the Mn3O4/CeO2 nanocomposite modified glassy carbon electrode (Mn3O4/CeO2/GCE) was investigated. Owing to the abundant oxygen vacancies and strong synergistic effect between the multivalent Ce and Mn, the sensor exhibited excellent catalytic activity and selectivity for the electrochemical detection of H2O2 with a low quantitation limit of 2 nM. Moreover, Mn3O4/CeO2/GCE exhibited excellent reproducibility, repeatability, and long-term storage stability. Because of these remarkable analytical advantages, the constructed sensor was able to determine H2O2 released from living cells with satisfactory results. The results showed that the Mn3O4/CeO2 sensor is a promising candidate for a nanoenzymatic H2O2 sensor with the possibility of applications in physiology and diagnosis.

Effect of Anticancer Quinones on Reactive Oxygen Production by Adult Rat Heart Myocytes.

This study investigated the effect of anthracycline antibiotics, mitomycin C, and menadione on oxygen consumption and hydrogen peroxide production by intact, beating, rat heart myocytes. Doxorubicin produced a dose-dependent increase in the rate of cyanide-resistant respiration by beating myocytes. The anthracycline analogs 4-demethoxydaunorubicin, 4′-epidoxorubicin, 4′-deoxydoxorubicin, and menogaril, as well as the anticancer quinones mitomycin C and menadione, also significantly increased oxygen consumption by cardiac myocytes. However, 5-iminodaunorubicin (which has a substituted quinone group) and mitoxantrone (which is not easily reduced by flavin dehydrogenases) had no effect on cardiac respiration. Both catalase (43%) and acetylated cytochrome c (19%) significantly decreased oxygen consumption that had been stimulated by doxorubicin; furthermore, extracellular hydrogen peroxide production was increased from undetectable control levels to 1.30 ± 0.02 nmol/min/107 myocytes (n = 4, P < 0.01) in the presence of 400 μM doxorubicin. These experiments suggest that the anthracycline antibiotics and other anticancer quinones stimulate cardiac oxygen radical production in intact heart myocytes; such a free radical cascade could contribute to the cardiac toxicity of these drugs.

Production of superoxide and hydrogen peroxide in the mitochondrial matrix is dominated by site IQ of complex I in diverse cell lines

Understanding how mitochondria contribute to cellular oxidative stress and drive signaling and disease is critical, but quantitative assessment is difficult. Our previous studies of cultured C2C12 cells used inhibitors of specific sites of superoxide and hydrogen peroxide production to show that mitochondria generate about half of the hydrogen peroxide released by the cells, and site IQ of respiratory complex I produces up to two thirds of the superoxide and hydrogen peroxide generated in the mitochondrial matrix. Here, we used the same approach to measure the engagement of these sites in seven diverse cell lines to determine whether this pattern is specific to C2C12 cells, or more general. These diverse cell lines covered primary, immortalized, and cancerous cells, from seven tissues (liver, cervix, lung, skin, neuron, heart, bone) of three species (human, rat, mouse). The rate of appearance of hydrogen peroxide in the extracellular medium spanned a 30-fold range from HeLa cancer cells (3 pmol/min/mg protein) to AML12 liver cells (84 pmol/min/mg protein). The mean contribution of identified mitochondrial sites to this extracellular hydrogen peroxide signal was 30 ± 7% SD; the mean contribution of NADPH oxidases was 60 ± 14%. The relative contributions of different sites in the mitochondrial electron transport chain were broadly similar in all seven cell types (and similar to published results for C2C12 cells). 70 ± 4% of identified superoxide/hydrogen peroxide generation in the mitochondrial matrix was from site IQ; 30 ± 4% was from site IIIQo. We conclude that although absolute rates vary considerably, the relative contributions of different sources of hydrogen peroxide production are similar in nine diverse cell types under unstressed conditions in vitro. Identified mitochondrial sites account for one third of total cellular hydrogen peroxide production (half each from sites IQ and IIIQo); in the mitochondrial matrix the majority (two thirds) of superoxide/hydrogen peroxide is from site IQ.

A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells.

Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H2O2-induced cell death: POR (encoding cytochrome P450 oxidoreductase), RETSAT (retinol saturase), KEAP1 (Kelch-like ECH-associated protein-1), and SLC52A2 (riboflavin transporter). Among these genes, only POR also mediated methyl viologen dichloride hydrate (paraquat)-induced cell death. Because the identification of SLC52A2 as a mediator of H2O2 was both novel and unexpected, we performed additional experiments to characterize the specificity and mechanism of its effect. These experiments showed that paralogs of SLC52A2 with lower riboflavin affinities could not mediate H2O2-induced cell death and that riboflavin depletion protected HAP1 cells from H2O2 toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H2O2 entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H2O2-induced cell death and also show for the first time that a vitamin can regulate membrane transport.IMPORTANCE Using a genetic screen, we discovered that riboflavin controls the entry of hydrogen peroxide into a white blood cell line. To our knowledge, this is the first report of a vitamin playing a role in controlling transport of a small molecule across the cell membrane.

NADPH oxidase DUOX1 sustains TGF-β1 signalling and promotes lung fibrosis.

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF)-β1/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular hydrogen peroxide (H2O2--)-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis.Our in vivo data (idiopathic pulmonary fibrosis patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1+/- and DUOX1-/-) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H2O2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation.These findings highlight a role for DUOX1-derived H2O2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis.

Bimetallic AgAu decorated MWCNTs enable robust nonenzyme electrochemical sensors for in-situ quantification of dopamine and H2O2 biomarkers expelled from PC-12 cells

Abstract Construction of efficient and reliable biosensor for real-time quantification of neurochemicals and reactive oxygen species is highly essential for clinical analysis because of their close connection to the health of living beings, however, remains challenging. Here, we demonstrated a novel amperometric sensor based on tiny AgAu nanoparticles (~2.15 nm) decorated multiwalled carbon nanotubes (MWCNTs) with boosted sensitivity and specificity for in-vivo monitoring of extracellular dopamine (DA) and hydrogen peroxide (H2O2). According to basic analysis, AgAu-MWCNTs nanohybrid electrode showed significantly higher electroactive surface area (0.259 cm2), and superior heterogeneous electron transfer rate constant (9.25 × 102 cm s−1). Profiting from the synergism and high conductivity of nanoparticular AgAu and MWCNTs, fabricated sensor delivered excellent electrochemical activity, can quantify DA and H2O2 with a broad dynamic response in the range of 3 nM-2.3 mM and 7 nM-7.8 mM, respectively. Also, the estimated limit of detection of DA and H2O2 to be 0.23 nM and 0.59 nM, correspondingly, which is well inside the desired level for in-situ monitoring. It was successfully applied to measure DA and H2O2 concentration expelled from the PC-12 cells upon the external stimulation, signifying a potential value of such biosensor in cell biology and clinical disease and cancer analysis.

Response of Human Neutrophil Granulocytes to the Hyphae of the Emerging Fungal Pathogen Curvularia lunata.

Curvularia lunata is an ascomycete filamentous fungus causing local and invasive phaeohyphomycoses in both immunocompromised and immunocompetent patients. Neutrophils are crucial participants of the first line host defense against fungal infections. They migrate to the infected site and eliminate the infectious agents by various mechanisms including phagocytoses, oxidative damage, or formation of neutrophil extracellular trap (NET). Neutropenia may be a risk factor for phaeohyphomycoses, and restoration of the neutrophil function can improve the outcome of the infection. In the present study, interaction of primary human neutrophil granulocytes with the hyphae C. lunata was examined and compared to that with the well characterized filamentous fungal pathogen Aspergillus fumigatus. Neutrophils could recognize the serum opsonized hyphae of C. lunata and attach to them. Myeloperoxidase release was also activated by a soluble factor present in the culture supernatant of the fungus. Induction of the oxidative burst was found to depend on serum opsonization of the hyphae. Although extracellular hydrogen peroxide production was induced, the fungus efficiently blocked the oxidative burst by acidifying the reaction environment. This blockage also affected the NET forming ability of the neutrophils.

Scavenging reactive oxygen species selectively inhibits M2 macrophage polarization and their pro-tumorigenic function in part, via Stat3 suppression

Tumor associated macrophages (TAM) enhance the aggressiveness of breast cancer via promoting cancer cell growth, metastasis, and suppression of the patient's immune system. These TAMs are polarized in breast cancer with features more closely resembling the pro-tumorigenic and immunosuppressive M2 type rather than the anti-tumor and pro-inflammatory M1 type. The goal of our study was to examine primary human monocyte-derived M1 and M2 macrophages for key redox differences and determine sensitivities of these macrophages to the redox-active drug, MnTE-2-PyP5+. This compound reduced levels of M2 markers and inhibited their ability to promote cancer cell growth and suppress T cell activation. The surface levels of the T cell suppressing molecule, PD-L2, were reduced by MnTE-2-PyP5+ in a dose-dependent manner. This study also examined key differences in ROS generation and scavenging between M1 and M2 macrophages. Our results indicate that M2 macrophages have lower levels of reactive oxygen species (ROS) and lower production of extracellular hydrogen peroxide compared to the M1 macrophages. These differences are due in part to reduced expression levels of pro-oxidants, Nox2, Nox5, and the non-enzymatic members of the Nox complex, p22phox and p47phox, as well as higher levels of antioxidant enzymes, Cu/ZnSOD, Gpx1, and catalase. More importantly, we found that despite having lower ROS levels, M2 macrophages require ROS for proper polarization, as addition of hydrogen peroxide increased M2 markers. These TAM-like macrophages are also more sensitive to the ROS modulator and a pan-Nox inhibitor. Both MnTE-2-PyP5+ and DPI inhibited expression levels of M2 marker genes. We have further shown that this inhibition was partly mediated through a decrease in Stat3 activation during IL4-induced M2 polarization. Overall, this study reveals key redox differences between M1 and M2 primary human macrophages and that redox-active drugs can be used to inhibit the pro-tumor and immunosuppressive phenotype of TAM-like M2 macrophages. This study also provides rationale for combining MnTE-2-PyP5+ with immunotherapies.

Inhibition of the thyroid hormonogenic H2O2 production by Duox/DuoxA in zebrafish reveals VAS2870 as a new goitrogenic compound

Thyroid hormone (TH) synthesis requires extracellular hydrogen peroxide generated by the NADPH oxidases, DUOX1 and DUOX2, with maturation factors, DUOXA1 and DUOXA2. In zebrafish, only one duox and one duoxa gene are present. Using a thyroid-specific reporter line, we investigated the role of Duox and Duoxa for TH biosynthesis in zebrafish larvae. Analysis of several zebrafish duox and duoxa mutant models consistently recovered hypothyroid phenotypes with hyperplastic goiter caused by impaired TH synthesis. Mutant larvae developed enlarged thyroids and showed increased expression of the EGFP reporter and thyroid functional markers including wild-type and mutated duox and duoxa transcripts. Treatment of zebrafish larvae with the NADPH oxidase inhibitor VAS2870 phenocopied the thyroid effects observed in duox or duoxa mutants. Additional functional in vitro assays corroborated the pharmacological inhibition of Duox activity by VAS2870. These data support the utility of this new experimental model to characterize endocrine disruptors of the thyroid function.

3-Amidinophenylalanine-derived matriptase inhibitors can modulate hepcidin production in vitro

Matriptase-2 (MT-2) is a type II transmembrane serine protease and predominantly attached to the surface of hepatocytes. MT-2 decreases the production of hepcidin, a key regulator of iron homeostasis. In this study, the effects of four 3-amidinophenylalanine-derived combined matriptase-1/matriptase-2 (MT-1/2) inhibitors (MI-432, MI-441, MI-460, and MI-461) on hepcidin production were investigated in hepatocyte mono- and hepatocyte-Kupffer cell co-cultures. In MI-461-treated cell cultures, the extracellular hydrogen peroxide contents and the interleukin-6 and -8 (IL-6 and IL-8) levels were determined and compared to controls. Hepcidin overproduction was observed in hepatocytes upon treatment with MI-432, MI-441 and MI-461 at 50 μM. In contrast, extracellular hydrogen peroxide levels were not elevated significantly after matriptase inhibition with MI-461. Furthermore, MI-461 did not induce increases in IL-6 and IL-8 levels in these hepatic models. A model of the binding mode of inhibitor MI-461 in complex with MT-2 revealed numerous polar contacts contributing to the nanomolar potency of this compound. Based on the in vitro data on hepcidin regulation, treatment with MI-461 might be valuable in pathological states of iron metabolism without causing excessive oxidative stress.

Monitoring cytosolic H2O2 fluctuations arising from altered plasma membrane gradients or from mitochondrial activity

Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.

Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress

IntroductionThe etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic. AimTo focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells. MethodsBovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10–15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H2O2) was measured using the Amplex Red assay and intracellular H2O2 using a fluorescent sensor. In addition, ROS superoxide (O2•-) was measured using a O2•- chemiluminescence enhancer. Both H2O2 and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO−) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C16-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells’ dysfunction. Main Outcome MeasuresSingle high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway. ResultsHere, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O2•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis. Strengths and LimitationsThis is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O2•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED. ConclusionsThis study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED.Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721–1733.

The peroxidase PRDX1 inhibits the activated phenotype in mammary fibroblasts through regulating c-Jun N-terminal kinases

BackgroundReactive oxygen species (ROS), including hydrogen peroxide, drive differentiation of normal fibroblasts into activated fibroblasts, which can generate high amounts of hydrogen peroxide themselves, thereby increasing oxidative stress in the microenvironment. This way, activated fibroblasts can transition into cancer-associated fibroblasts (CAFs).MethodsMammary fibroblasts from either female 8 weeks old PRDX1 knockout and wildtype mice or Balb/c mice were studied for characteristic protein expression using immunofluorescence and immunoblotting. Cancer-associated fibroblasts was examined by transwell migration and invasion assays. The binding of PRDX1 to JNK1 was assessed by co-immuneprecipitation and JNK regulation of CAF phenotypes was examined using the JNK inhibitor SP600125. Extracellular hydrogen peroxide levels were measured by chemiluminescence via the reaction between hypochlorite and luminol. Statistical analyses were done using Students t-test.ResultsWe show here PRDX1 activity as an essential switch in regulating the activated phenotype as loss of PRDX1 results in the development of a CAF-like phenotype in mammary fibroblasts. We also show that PRDX1 regulates JNK kinase signaling thereby inhibiting CAF-like markers and CAF invasion. Inhibition of JNK activity reduced these behaviors.ConclusionsThese data suggest that PRDX1 repressed the activated phenotype of fibroblasts in part through JNK inhibition which may present a novel therapeutic option for CAF-enriched cancers such as breast cancer.