Cellular Peroxidation Research Articles

In vitro silicon supplementation enhanced acclimatisation and growth of sugarcane (Saccharum officinarum) via improved antioxidant and nutrient acquisition patterns in saline soil.

Salinity affects crop growth by modulating cellular ionic concentrations and generation of reactive oxygen species. Application of silicon (Si) has proved beneficial in ameliorating salinity-triggered plant growth and yield retardations. Leaf roll explants of three sugarcane (Saccharum officinarum ) genotypes (HSF-240, CPF-246, CPF-250) were cultured in Murashige and Skoog (MS) medium supplemented with K2 SiO3 . In vitro regenerated plantlets were acclimatised and grown in natural saline soil. In absence of Si, cv. CPF-246 exhibited better salt tolerance as indicted by maximum chlorophyll a and chlorophyll b contents, rate of photosynthesis and root K+ uptake along with less cellular hydrogen peroxide content. Silicon restricted root Na+ uptake but assisted in K+ , Ca2+ , Mg2+ and Fe2+ accretion in roots and their translocation towards shoots. Cv. HSF-240 and cv. CPF-250 exhibited more increase in photosynthetic pigment content, stomatal conductance and photosynthetic rate after addition of 25 or 50mgL-1 Si than control group. Optimum phenolic content and antioxidant enzyme activity along with decreased lipid peroxidation and hydrogen peroxide content were recorded in all three sugarcane genotypes raised in presence of 25 or 50mgL-1 Si. These findings signify Si supplementation (50mgL-1 ) in tissue culture medium and plant adaptation in saline soil. Further in vitro studies involving Si-mediated gene expression modulations in sugarcane protoplasts shall assist in deciphering cross-talk between Si uptake and cellular responses. The application of Si can further be tested for other plant species to devise strategies for improved crop growth and utilisation of saline areas for crop cultivation.

Maternal catalase supplementation regulates fatty acid metabolism and antioxidant ability of lactating sows and their offspring.

As a crucial antioxidant enzyme, catalase (CAT) could destroy the cellular hydrogen peroxide to mitigate oxidative stress. The current study aimed to investigate the effects of maternal CAT supplementation from late gestation to day 14 of lactation on antioxidant ability and fatty acids metabolism with regard to the sow-piglet-axis. On day 95 of gestation, forty sows were divided into control (CON) group (fed a basal diet) and CAT group (fed a basal diet supplemented with 660 mg/kg CAT), the feeding experiment ended on day 14 of lactation. The lactating sows in the CAT group produced more milk, and had higher antioxidant enzymes activity including POD and GSH-Px (P < 0.05), lower content of serum LDL as well as plasmic C18:3n3 content (P < 0.05). Additionally, maternal CAT supplementation improved offspring's body weight at day 14 of nursing period and ADG (P < 0.05), and regulated the antioxidant ability as evidenced by decreased related enzymes activity such as T-AOC and CAT and changed genes expression level. It significantly affected lipid metabolism of suckling piglets manifested by increasing the serum ALT, CHOL, and LDL (P < 0.05) level and modulating plasma medium- and long-chain fatty acids (MCFAs and LCFAs), as well as regulating the genes expression involved in lipid metabolism. Maternal CAT supplementation could regulate the fatty acid composition and enhance the antioxidant ability of sows and offspring during the lactating period and further promote the growth of suckling piglets. These findings might provide a reference value for the utilization of CAT as supplement for mother from late pregnancy to lactation period to promote the fatty acid metabolism of offspring.

DDDR-11. TREATING DIPG WITH A MITOCHONDRIALLY TARGETED PLATINUM(IV)-BASED PRODRUG

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal childhood cancer of the brain with 5-year survival rates of &amp;lt; 2%. Despite the introduction of conventional chemotherapy and radiotherapy, improvements in survival have been marginal and long-term survivorship is rare. We previously developed MP-Pt(IV), monoamine oxidase B (MAOB) sensitive, mitochondrial targeting platinum-(IV) based prodrug derived from the chemotherapeutic cisplatin. MP-Pt(IV) is well tolerated and is highly effective in a mice xenograft GBM brain tumour model. Unlike in GBM, the expression levels of MAOB in DIPG are unknown. We tested MP-Pt(IV) against DIPG to test whether DIPG exhibit a similar sensitivity to mitochondrially targeted Pt(IV)-based prodrugs. METHODS In-vitro cell growth inhibition studies were conducted on DIPG cells using MP-Pt(IV). Labeling studies were carried out with MitoSox, MitoTracker, and H2DCFAM. Oxygen consumption studies were performed to show mitochondrial targeting. Clonogenic survival assay and scratch assay were performed. RESULTS MP-Pt(IV) shows potent growth inhibition of DIPG in cell culture studies. Mechanistic studies indicate mitochondrial targeting of the prodrug. Significant increases in cellular super-oxide and hydrogen peroxide levels are observed upon MP-Pt(IV) treatment. Oxygen consumption studies confirm mitochondrial targeting of MP-Pt(IV). MP-Pt(IV) strongly inhibits clonogenic survival and cell migration in treated cells. CONCLUSIONS We demonstrate that MP-Pt(IV) is highly effective in DIPG in vitro and is a good candidate for testing in a DIPG xenograft animal model and further development.

SERS and dark-field scattering dual-mode detection of intracellular hydrogen peroxide using biocompatible Au@COF nanosensor

Hydrogen peroxide is widely involved in intracellular physiological activities, playing vital roles in regulating various intracellular metabolite and physiological events. Surface-enhanced Raman scattering (SERS) may serve as a promising technique to efficiently sensing metabolites in living cells owing to its non-invasiveness, low autofluorescence, and rapid detection capability. Herein, a size-controllable core-shell structure was constructed by covering 2D covalent organic frameworks (COFs) on Au nanoparticles (Au@COF), and the cellular hydrogen peroxide were detected by Raman spectroscopy/Dark-field scattering imaging dual-mode technique. The introduction of COFs endows Au@COF with perfect enrichment capacity as well as high stability and good biocompatibility. Combining the superior fingerprint identification of SERS and the imaging capability of dark-field microscopy, the present method exhibits high applicability for hydrogen peroxide detection in living cells. Results show that when the living MCF-7 cells were treated by different drugs, the level of hydrogen peroxide metabolite can be monitored in real-time. This study provides a simple but sensitive and facile platform for biosensing hydrogen peroxide and cellular metabolites.

Moisture-resistant and green cyclodextrin metal-organic framework nanozyme based on cross-linkage for visible detection of cellular hydrogen peroxide.

Amoisture-resistant and green cyclodextrin metal-organic framework (CD-MOF) nanosheet has been preparedvia anone-pot antisolvent synthesis procedure. After the treatment of in situ chemical cross-linkage, the two-dimensional (2D) cross-linked CD-MOF exhibited both peroxidase (POD) and oxidase (OXD) enzymatic activities, as well as hydrolytic stability. On the basis of its POD mimics function, the proof-of-concept biosensors were constructed to realize the colorimetric detection for H2O2 and glucose, respectively. In vitro cytotoxicity experiments showed that the 2D cross-linked CD-MOF nanozymes still maintained excellent biocompatibility even at a concentration reaching up to several mg/mL. The in situ colorimetric detection of H2O2 secreted by HepG2 cells further confirmed its promising biocompatibility, showing its great promises as label-free colorimetric probe in early cancer detection and pathological process monitoring.

Obesity and Fatty Acids Promote Mitochondrial Translocation of STAT3 Through ROS-Dependent Mechanisms.

Obesity promotes the onset and progression of metabolic and inflammatory diseases such as type 2 diabetes. The chronic low-grade inflammation that occurs during obesity triggers multiple signaling mechanisms that negatively affect organismal health. One such mechanism is the persistent activation and mitochondrial translocation of STAT3, which is implicated in inflammatory pathologies and many types of cancers. STAT3 in the mitochondria (mitoSTAT3) alters electron transport chain activity, thereby influencing nutrient metabolism and immune response. PBMCs and CD4+ T cells from obese but normal glucose-tolerant (NGT) middle-aged subjects had higher phosphorylation of STAT3 on residue serine 727 and more mitochondrial accumulation of STAT3 than cells from lean subjects. To evaluate if circulating lipid overabundance in obesity is responsible for age- and sex-matched mitoSTAT3, cells from lean subjects were challenged with physiologically relevant doses of the saturated and monounsaturated fatty acids, palmitate and oleate, respectively. Fatty acid treatment caused robust accumulation of mitoSTAT3 in all cell types, which was independent of palmitate-induced impairments in autophagy. Co-treatment of cells with fatty acid and trehalose prevented STAT3 phosphorylation and mitochondrial accumulation in an autophagy-independent but cellular peroxide–dependent mechanism. Pharmacological blockade of mitoSTAT3 either by a mitochondria-targeted STAT3 inhibitor or ROS scavenging prevented obesity and fatty acid–induced production of proinflammatory cytokines IL-17A and IL-6, thus establishing a mechanistic link between mitoSTAT3 and inflammatory cytokine production.

Effects of sugars, fatty acids and amino acids on cytosolic and mitochondrial hydrogen peroxide release from liver cells

The rates of formation of superoxide and hydrogen peroxide at different electron-donating sites in isolated mitochondria are critically dependent on the substrates that are added, through their effects on the reduction level of each site and the components of the protonmotive force. However, in intact cells the acute effects of added substrates on different sites of cytosolic and mitochondrial hydrogen peroxide production are unclear. Here we tested the effects of substrate addition on cytosolic and mitochondrial hydrogen peroxide release from intact AML12 liver cells. In 30-min starved cells replete with endogenous substrates, addition of glucose, fructose, palmitate, alanine, leucine or glutamine had no effect on the rate or origin of cellular hydrogen peroxide release. However, following 150-min starvation of the cells to deplete endogenous glycogen (and other substrates), cellular hydrogen peroxide production, particularly from NADPH oxidases (NOXs), was decreased, GSH/GSSH ratio increased, and antioxidant gene expression was unchanged. Addition of glucose or glutamine (but not the other substrates) increased hydrogen peroxide release. There were similar relative increases from each of the three major sites of production: mitochondrial sites IQ and IIIQo, and cytosolic NOXs. Glucose supplementation also restored ATP production and mitochondrial NAD reduction level, suggesting that the increased rates of hydrogen peroxide release from the mitochondrial sites were driven by increases in the protonmotive force and the degree of reduction of the electron transport chain. Long-term (24 h) glucose or glutamine deprivation also diminished hydrogen peroxide release rate, ATP production rate and (for glucose deprivation) NAD reduction level. We conclude that the rates of superoxide and hydrogen peroxide production from mitochondrial sites in liver cells are insensitive to extra added substrates when endogenous substrates are not depleted, but these rates are decreased when endogenous substrates are lowered by 150 min of starvation, and can be enhanced by restoring glucose or glutamine supply through improvements in mitochondrial energetic state.

Peroxisome-Derived Hydrogen Peroxide Modulates the Sulfenylation Profiles of Key Redox Signaling Proteins in Flp-In T-REx 293 Cells.

The involvement of peroxisomes in cellular hydrogen peroxide (H2O2) metabolism has been a central theme since their first biochemical characterization by Christian de Duve in 1965. While the role of H2O2 substantially changed from an exclusively toxic molecule to a signaling messenger, the regulatory role of peroxisomes in these signaling events is still largely underappreciated. This is mainly because the number of known protein targets of peroxisome-derived H2O2 is rather limited and testing of specific targets is predominantly based on knowledge previously gathered in related fields of research. To gain a broader and more systematic insight into the role of peroxisomes in redox signaling, new approaches are urgently needed. In this study, we have combined a previously developed Flp-In T-REx 293 cell system in which peroxisomal H2O2 production can be modulated with a yeast AP-1-like-based sulfenome mining strategy to inventory protein thiol targets of peroxisome-derived H2O2 in different subcellular compartments. By using this approach, we identified more than 400 targets of peroxisome-derived H2O2 in peroxisomes, the cytosol, and mitochondria. We also observed that the sulfenylation kinetics profiles of key targets belonging to different protein families (e.g., peroxiredoxins, annexins, and tubulins) can vary considerably. In addition, we obtained compelling but indirect evidence that peroxisome-derived H2O2 may oxidize at least some of its targets (e.g., transcription factors) through a redox relay mechanism. In conclusion, given that sulfenic acids function as key intermediates in H2O2 signaling, the findings presented in this study provide valuable insight into how peroxisomes may be integrated into the cellular H2O2 signaling network.

Data Processing to Probe the Cellular Hydrogen Peroxide Landscape.

Hydrogen peroxide (H2O2) regulates signaling pathways by modulating the activity of redox-sensitive proteins denominated redox switches. The magnitude of the transient variations in localized H2O2 pools during signaling events and how these variations impact redox switches present in the cell remain elusive. A canonical model with two chemical reactions comprising the oxidation/reduction cycle of a redox switch is described. The model is dimensionless with respect to the redox switch concentration. Thus, the time-series data required to apply the equations deduced is the percentage of oxidation of a redox switch, avoiding the application of absolute concentrations that are often difficult to measure experimentally. Here, we describe detailed protocols for the processing of experimental data with the canonical model to probe the absolute concentrations of H2O2 found in the vicinity of redox switches and probes, as well as the kinetic parameters that describe the reduction and oxidation of redox switches. The protocols are an analytical tool that helps to depict the cellular hydrogen peroxide signaling landscape, giving new insights on H2O2 signaling mechanisms, and hold the potential to be a framework for a future redox kinetomics analytical platform.

Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli

Previous studies demonstrated that superoxide could initiate and amplify LDH-catalyzed hydrogen peroxide production in aqueous phase, but its physiological relevance is unknown. Here we showed that LDHA and LDHB both exhibited hydrogen peroxide-producing activity, which was significantly enhanced by the superoxide generated from the isolated mitochondria from HeLa cells and patients’ cholangiocarcinoma specimen. After LDHA or LDHB were knocked out, hydrogen peroxide produced by Hela or 4T1 cancer cells were significantly reduced. Re-expression of LDHA in LDHA-knockout HeLa cells partially restored hydrogen peroxide production. In HeLa and 4T1 cells, LDHA or LDHB knockout or LDH inhibitor FX11 significantly decreased ROS induction by modulators of the mitochondrial electron transfer chain (antimycin, oligomycin, rotenone), hypoxia, and pharmacological ROS inducers piperlogumine (PL) and phenethyl isothiocyanate (PEITC). Moreover, the tumors formed by LDHA or LDHB knockout HeLa or 4T1 cells exhibited a significantly less oxidative state than those formed by control cells. Collectively, we provide a mechanistic understanding of a link between LDH and cellular hydrogen peroxide production or oxidative stress in cancer cells in vitro and in vivo.

Chemoresistive Nanostructured Metal-Oxide Sensors for Human Tumor Tissue and Blood Exhalations Analysis

Early-stage tumor detection is one of the most coveted goals for the world research community. An effective prevention, associated to reliable screening protocols, is crucial to favor a early diagnosis, allowing doctors and surgeons to intervene with high probabilities of success on tumor affected patients. The main purpose of this study is cancer detection, exploiting the volatile organic compounds (VOCs) produced by cancer cells as tumor biomarkers [1][2], by investigating tumor tissue and blood samples. Tumor biomarkers exhalation is attributed to two main cell involving biological mechanisms: altered metabolism and cellular membrane peroxidation [3]. This experimental work was carried on using an innovative, fast-responding and reliable patented device, named SCENT B1 [4], entirely designed and assembled in the Sensor Laboratory of the University of Ferrara. It hosts an array of four specific metal-oxide (MOX) sensors able to detect gases in low concentration (up to 10 ppm) with high stability and repeatability, chosen after many tests on different types of biological samples (feces, blood, cell cultures, etc.) [5,6]; the chosen sensors are based on different mixtures of tin, titanium, tungsten, niobium, tantalum and vanadium oxides (ST25Au, W11, STN, TiTaV). Moreover, SCENT B1 is gifted of a power supply and sensor signal transduction electronic boards, a pneumatic air system (necessary to direct the VOCs contaminated air from the sample chamber to the sensors) and an ad hoc management and data acquisition software (LSS4) [6]. The sensor output signal is a voltage directly proportional to the sensor conductance, depending on the chemical redox reactions taking place on the surface of the sensor sensing material. The sensor response is: R=Vgas/Vair , where Vgas and Vair are the sensor voltage in gas presence and in dry air after the steady state achievement respectively [6].Sensor responses were further elaborated, using different statistical approaches as principal component analysis and receiver operating characteristics methods.Measurements have been performed on colorectal cancer and healthy tissues surgically removed from the same operatory piece (directly and far enough from tumor mass respectively) (Figure 1a), and on blood samples collected from tumor affected subjects and healthy ones as controls (Figure 1b). with the future aim of extending the study to other tumor types. All four sensors gave larger responses (although with different amplitudes) to the tumor tissue with respect to the healthy one. Smaller and reproducible responses were given by the breeding ground (DMEM) only, confirming that it does not alter the measurements. The results are consistent with the stronger and altered metabolism of tumor cells, leading them to emit a larger amount of volatile biomarkers with respect to the healthy ones;.All the four sensors hosted by SCENT B1 proved to be capable of distinguishing between healthy and tumor tissue and blood samples, although with different discrimination power. The encouraging results of this feasibility study are the basis of a new in-depth study, which includes also a follow-up protocol based on post surgery blood monitoring of patients. Our future aims foresee the extension of this study to other tumor types and the obtainment of the SCENT B1 clinical validation as oncologic screening device. Figure 1

Modeling of cellular hydrogen peroxide landscape

Modeling of cellular hydrogen peroxide landscape

Redox Regulation of PTEN by Peroxiredoxins.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known as a tumor suppressor gene that is frequently mutated in numerous human cancers and inherited syndromes. PTEN functions as a negative regulator of PI3K/Akt signaling pathway by dephosphorylating phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) to phosphatidylinositol (4, 5)-bisphosphate (PIP2), which leads to the inhibition of cell growth, proliferation, cell survival, and protein synthesis. PTEN contains a cysteine residue in the active site that can be oxidized by peroxides, forming an intramolecular disulfide bond between Cys124 and Cys71. Redox regulation of PTEN by reactive oxygen species (ROS) plays a crucial role in cellular signaling. Peroxiredoxins (Prxs) are a superfamily of peroxidase that catalyzes reduction of peroxides and maintains redox homeostasis. Mammalian Prxs have 6 isoforms (I-VI) and can scavenge cellular peroxides. It has been demonstrated that Prx I can preserve and promote the tumor-suppressive function of PTEN by preventing oxidation of PTEN under benign oxidative stress via direct interaction. Also, Prx II-deficient cells increased PTEN oxidation and insulin sensitivity. Furthermore, Prx III has been shown to protect PTEN from oxidation induced by 15s-HpETE and 12s-HpETE, these are potent inflammatory and pro-oxidant mediators. Understanding the tight connection between PTEN and Prxs is important for providing novel therapies. Herein, we summarized recent studies focusing on the relationship of Prxs and the redox regulation of PTEN.

Effect of Engineered Nickel Oxide Nanoparticle on Reactive Oxygen Species-Nitric Oxide Interplay in the Roots of Allium cepa L.

Scientists anxiously follow instances of heavy metals augmenting in the environment and undergoing bioaccumulation and trace their biomagnification across food webs, wary of their potent toxicity on biological entities. Engineered nanoparticles supplement natural pools of respective heavy metals and can mimic their effects, exerting toxicity at higher concentrations. Thus, a thorough understanding of the underlying mechanism of this precarious interaction is mandatory. Most urban and industrial environments contain considerable quantities of nickel oxide nanoparticles. These in excess can cause considerable damage to plant metabolism through a significant increase in cellular reactive oxygen species and perturbation of its cross-talk with the reactive nitrogen species. In the present work, the authors have demonstrated how the intrusion of nickel oxide nanoparticles (NiO-NP) affected the exposed roots of Allium cepa: starting with disruption of cell membranes, before being interiorized within cell organelles, effectively disrupting cellular homeostasis and survival. A major shift in the reactive oxygen species (ROS) and nitric oxide (NO) equanimity was also observed, unleashing major altercations in several crucial biochemical profiles. Altered antioxidant contents and upregulation of stress-responsive genes, namely, Catalase, Ascorbate peroxidase, Superoxide dismutase, and Rubisco activase, showing on average 50–250% rise across NiO-NP concentrations tested, also entailed increased cellular hydrogen peroxide contents, with tandem rise in cellular NO. Increased NO content was evinced from altered concentrations of nitric oxide synthase and nitrate reductase, along with NADPH oxidase, when compared with the negative control. Though initially showing a dose-dependent concomitant rise, a significant decrease of NO was observed at higher concentrations of NiO-NP, while cellular ROS continued to increase. Modified K/Na ratios, with increased proline concentrations and GABA contents, all hallmarks of cellular stress, correlated with ROS–NO perturbations. Detailed studies showed that NiO-NP concentration had a significant role in inducing toxicity, perturbing the fine balance of ROS–NO, which turned lethal for the cell at higher dosages of the ENP precipitating in the accumulation of stress markers and an inevitable shutdown of cellular mechanisms.

Environment-insensitive two-photon ratiometric probe for in cellulo quantitative measurement of hydrogen peroxide

The quantitative analysis of a biological analyte directly through fluorescence imaging is essential in biomedical sciences but remains as a challenge owing to the environment-sensitive nature of fluorescence intensity. We show that a fluorescent hydrogen peroxide probe based on an in cellulo super-bright benzothiazolyl-benzocoumarin dye of which emission properties are insensitive to environmental changes offers a reliable and practical means for the quantitative analysis of hydrogen peroxide in cells and tissues directly through ratiometric imaging both under one-photon and two-photon excitation conditions. Different cellular hydrogen peroxide concentrations were determined for several cell lines and also for tumour and normal tissues.

Development of Au-Pd@UiO-66-on-ZIF-L/CC as a self-supported electrochemical sensor for in situ monitoring of cellular hydrogen peroxide.

Integrating metal-organic frameworks (MOFs) of different components or structures together and exploiting them as electrochemical sensors for electrochemical sensing has aroused great interest. Furthermore, the incorporation of noble metals with MOFs is conducive to the improvement of catalytic performance. In this work, Pd@UiO-66-on-ZIF-L nanomaterials were successfully synthesised onto a self-supported flexible carbon cloth (Pd@UiO-66-on-ZIF-L/CC) through a novel strategy called MOF-on-MOF. Then, Au nanoparticles were electrodeposited onto Pd@UiO-66-on-ZIF-L/CC to obtain Au-Pd@UiO-66-on-ZIF-L/CC, which can serve as an excellent electrocatalyst for the reduction of hydrogen peroxide (H2O2). The obtained flower-like Pd@UiO-66-on-ZIF-L/CC hybrid MOF changes the structure of the monomeric MOF alone and adds more attachment sites. The synergy of the bimetals greatly improved the catalytic performance of the as-developed sensor. Electrochemical experiment results show that the proposed sensor based on Au-Pd@UiO-66-on-ZIF-L/CC has an extended linear range from 1 μM to 19.6 mM with a sensitivity of 390 μA mM-1 cm-2, and a low limit of detection (LOD) of 21.2 nM (S/N = 3). Moreover, it has good anti-interference, reproducibility, repeatability and excellent stability. Furthermore, the real-time in situ detection of H2O2 secreted from human adenocarcinomic alveolar basal epithelial cells (A549 cells) was achieved by culturing cells on Au-Pd@UiO-66-on-ZIF-L/CC, which indicates the potential of the sensor for applications in cancer pathology. Both the synthesis strategy and the sensor design provide new methods and ideas for the production of ultrasensitive H2O2 electrochemical sensors.

Glutathione peroxidase mimics based on conformationally-restricted, peri-like, 4,5-disubstituted fluorene dichalcogenides.

Glutathione peroxidase (GPx) regulates cellular peroxide levels through glutathione oxidation. GPx-mimics based on 4,5-disubstituted fluorene diselenides, their oxides, and ditellurides show catalytic activities consistent with conformational restriction about the dichalcogen bond.

DUOX2 participates in skin aging induced by UVB in HSF2 cells by activating NF-κB signaling.

Skin and in particular photoaging or premature aging, are caused by a variety of factors, including hormone imbalance and exposure to ultraviolet radiation. The aim of the present study was to explore the roles of Dual oxidase 2 (DUOX2) and related NF-κB signals in skin photoaging. Cell models of photoaging were constructed by irradiating human skin fibroblast lines (HSF2) with ultraviolet B (UVB) of different doses (0, 15, 30 and 60 mj/cm2). The cell counting kit-8 (CCK8) was used to determine cell proliferation. Flow cytometry was used to determine the production of reactive oxygen species (ROS). A biochemical method was to determine the content of hydrogen peroxide, and the quantitative PCR (qPCR) was used to determine the expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), Col-Ⅰ and α-SMA in the cells. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Western blot analysis was performed to determine the expression of DUOX2, p65 and p-p65. The results showed that,UVB irradiation dose- and time-dependently inhibited the proliferation of HSF2 cells. Cellular inflammatory response, ROS production and hydrogen peroxide increase was promoted. Col-Ⅰ and α-SMA were downregulated, MMP2 and MMP9 were upregulated, and the phosphorylation of NF-κB p65 was promoted. The above indicators were all reversed by interference with DUOX2. Overexpression of DUOX2 has an effect that is similar to UVB irradiation, but the effects can be significantly weakened by NF-κB inhibitor, NAC. Upregulation of DUOX2 expression plays a crucial role in UVB-induced aging of HSF2 cells. The specific mechanism is related to the promotion of ROS production and cellular inflammatory response and activation of NF-κB signals.

Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

Chemoresistive Nanostructured Sensors for the Analysis of Human Tumor Tissue Exhalations

One of the greatest goals in medicine is early-stage tumor detection. An effective prevention is crucial to allow physicians and surgeons to intervene on tumor affected patients with the available therapies and surgical techniques with a high probability of success. The purpose of this research relies on the identification of cancer presence by detecting the volatile organic biomarkers directly produced by cancer cells. Cancer cells produce different biomarkers with respect to healthy cells, mainly due to the altered metabolism and cellular membrane peroxidation. This difference can be recognized by using an innovative device manufactured in the Sensor Laboratory of the University of Ferrara, hosting a specific sensor array chosen for biological sample investigation. The sensor array is composed by four chemoresistive metal-oxide nanostructured sensors, able to detect gases in low concentration (up to 1 ppm) with excellent stability and repeatability. In this study, a fast-responding, reliable and reproducible sensing device, named SCENT B1[1], showed to be capable of discriminating cancerous cells from healthy ones, proving to be an efficient and low-invasive screening system.The device combines a specific electronic and pneumatic system to a specifically selected sensor array. The sample is inserted inside a sample box through a support composed by Petri dishes, while a stabilized ambient airflow conveys the exhalations to sensors. Sensor responses are then statistically analyzed by principal component analysis (PCA).Measurements have been performed on cancer and healthy tissues, extracted during surgery from human colon and rectum, with the future aim of extending the study to the other tumor types.Sensors chosen are the following: a mixture of tin and titanium oxides with addition of gold (ST25Au), tungsten oxide (W11), a mixture of tin, titanium and niobium oxides (STN) and a solid solution of titanium oxide, tantalum and vanadium (TiTaV). Sensors have been selected after a feasibility study performed on immortalized cells of diverse types, where sensors responses have been correlated with cell type and cell initial plating concentration of the samples; the measures have been performed after 24, 48, 72 hours of incubation.The sensor output signal is a voltage directly proportional to the sensor conductance; it depends on the chemical redox reactions taking place on sensor surface [2,3]. In Figure (a) the response R=ÄG/G for each sensor is reported, where ÄG is the difference between the sensor conductance with and without the metabolites expelled by the cells of a tissue. All four sensors gave larger responses (although with different amplitudes) to the tumor tissue with respect to the healthy one. Smaller and reproducible responses were given by the breeding ground (DMEM) only, confirming that it does not alter the measurement.The results are consistent with the stronger metabolism of tumor cells with respect to the healthy ones, because the former emits larger amounts of volatile biomarkers [4,5]. Other tests proved also that different initial plating concentrations (250k, 500k and 1M) of cell give increasing responses, as shown in Figure (b). From this study what emerges is that the device is capable of distinguishing different cell samples basing on their health status and concentrations. The encouraging results of this study are the basis of a deepen study for the clinical validation of the device as a oncologic screening device.