Increased Reactive Oxygen Species Generation Research Articles

Novel role of dual oxidase 2 as a mediator of podocyte injury in the diabetic environment

Glomerular injury is a prominent pathological feature of diabetic nephropathy (DN). In glomerular epithelial cells, or podocytes, hyperglycemia alters slit diaphragm proteins and causes foot process effacement, apoptosis and cell detachment. Oxidative stress has emerged as an important pathogenic mechanism in the development of glomerular injury in DN. However, the mechanisms by which these factors exert their action remain poorly understood. We provide evidence that the NADPH oxidase of the Nox family, Dual oxidase 2 (Duox2), is present in cultured glomerular cells, including podocytes. Exposure of cultured podocytes to high concentrations of glucose (HG) elicited a rapid upregulation of Duox2 protein expression. Inhibition of Duox2 with specific siRNA prevented the HG-induced increase in intracellular reactive oxygen species (ROS) generation and hydrogen peroxide (H2O2) production in cultured podocytes. In additional experiments, we established a functional link between Duox2-derived ROS generation and podocyte injury in response to HG. Impairment of Duox2 function nearly abolished HG-mediated DNA fragmentation, apoptosis and decrease in/reorganization of slit diaphragm protein expression in podocytes, indicating that Duox2 is required for the deleterious effects of glucose in podocytes. Our study demonstrates, for the first time, that Duox2 is responsible for increased ROS generation and subsequent alteration of podocyte function in response to HG. Our work serves as proof of concept to demonstrate the utility of targeting Duox2 as a future therapeutic intervention to reduce diabetes-mediated glomerular lesions. Research reported in this poster was supported by the University of the Incarnate Word Offce of Research and Graduate Studies and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC2GM136569. The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AM-18002, a derivative of natural anmindenol A, enhances radiosensitivity in mouse breast cancer cells.

Natural anmindenol A isolated from the marine-derived bacteria Streptomyces sp. caused potent inhibition of inducible nitric oxide synthase without any significant cytotoxicity. This compound consists of a structurally unique 3,10-dialkylbenzofulvene skeleton. We previously synthesized and screened the novel derivatives of anmindenol A and identified AM-18002, an anmindenol A derivative, as a promising anticancer agent. The combination of AM-18002 and ionizing radiation (IR) improved anticancer effects, which were exerted by promoting apoptosis and inhibiting the proliferation of FM3A mouse breast cancer cells. AM-18002 increased the production of reactive oxygen species (ROS) and was more effective in inducing DNA damage. AM-18002 treatment was found to inhibit the expansion of myeloid-derived suppressor cells (MDSC), cancer cell migration and invasion, and STAT3 phosphorylation. The AM-18002 and IR combination synergistically induced cancer cell death, and AM-18002 acted as a potent anticancer agent by increasing ROS generation and blocking MDSC-mediated STAT3 activation in breast cancer cells.

De Novo Designed Ru(II) Metallacycle as a Microenvironment-Adaptive Sonosensitizer and Sonocatalyst for Multidrug-Resistant Biofilms Eradication.

Albeit sonodynamic therapy (SDT) has achieved encouraging progress in microbial sterilization, the scarcity of guidelines for designing highly effective sonosensitizers and the intricate biofilm microenvironment (BME), substantially hamper the therapeutic efficacy against biofilm infections. To address the bottlenecks, we innovatively design a Ru(II) metallacycle-based sonosensitizer/sonocatalyst (named Ru-A3-TTD) to enhance the potency of sonotherapy by employing molecular engineering strategies tailored to BME. Our approach involves augmenting Ru-A3-TTD's production of ultrasonic-triggered reactive oxygen species (ROS), surpassing the performance of commercial sonosensitizers, through a straightforward but potent π-expansion approach. Within the BME, Ru-A3-TTD synergistically amplifies sonotherapeutic efficacy via triple-modulated approaches: (i) effective alleviation of hypoxia, leading to increased ROS generation, (ii) disruption of the antioxidant defense system, which shields ROS from glutathione consumption, and (iii) enhanced biofilm penetration, enabling ROS production in deep sites. Notably, Ru-A3-TTD sono-catalytically oxidizes NADPH, a critical coenzyme involved in antioxidant defenses. Consequently, Ru-A3-TTD demonstrates superior biofilm eradication potency against multidrug-resistant Escherichia coli compared to conventional clinical antibiotics, both in vitro and in vivo. To our knowledge, this study represents the pioneering instance of a supramolecular sonosensitizer/sonocatalyst. It provides valuable insights into the structure-activity relationship of sonosensitizers and paves a promising pathway for the treatment of biofilm infections.

De Novo Designed Ru(II) Metallacycle as a Microenvironment‐Adaptive Sonosensitizer and Sonocatalyst for Multidrug‐Resistant Biofilms Eradication

AbstractAlbeit sonodynamic therapy (SDT) has achieved encouraging progress in microbial sterilization, the scarcity of guidelines for designing highly effective sonosensitizers and the intricate biofilm microenvironment (BME), substantially hamper the therapeutic efficacy against biofilm infections. To address the bottlenecks, we innovatively design a Ru(II) metallacycle‐based sonosensitizer/sonocatalyst (named Ru‐A3‐TTD) to enhance the potency of sonotherapy by employing molecular engineering strategies tailored to BME. Our approach involves augmenting Ru‐A3‐TTD’s production of ultrasonic‐triggered reactive oxygen species (ROS), surpassing the performance of commercial sonosensitizers, through a straightforward but potent π‐expansion approach. Within the BME, Ru‐A3‐TTD synergistically amplifies sonotherapeutic efficacy via triple‐modulated approaches: (i) effective alleviation of hypoxia, leading to increased ROS generation, (ii) disruption of the antioxidant defense system, which shields ROS from glutathione consumption, and (iii) enhanced biofilm penetration, enabling ROS production in deep sites. Notably, Ru‐A3‐TTD sono‐catalytically oxidizes NADPH, a critical coenzyme involved in antioxidant defenses. Consequently, Ru‐A3‐TTD demonstrates superior biofilm eradication potency against multidrug‐resistant Escherichia coli compared to conventional clinical antibiotics, both in vitro and in vivo. To our knowledge, this study represents the pioneering instance of a supramolecular sonosensitizer/sonocatalyst. It provides valuable insights into the structure‐activity relationship of sonosensitizers and paves a promising pathway for the treatment of biofilm infections.

Aquaporin-8 promotes human dermal fibroblasts to counteract hydrogen peroxide-induced oxidative damage: A novel target for management of skin aging.

The skin is subjected to various external factors that contribute to aging including oxidative stress from hydrogen peroxide (H2O2). This study investigated the distribution of aquaporin-8 (AQP8), a protein that transports H2O2 across biological membranes, in skin cells, and its effects in mitigating H2O2-induced oxidative damage. Human dermal fibroblasts were treated with increasing concentrations of H2O2 to evaluate oxidative damage. Cell viability, reactive oxygen species (ROS) generation, and the expression of specific genes associated with skin aging (IL-10, FPR2, COL1A1, KRT19, and Aggrecan) were evaluated and AQP8 expression was assessed via quantitative polymerase chain reaction and western blotting. Small-interfering RNA was used to silence the AQP8 gene and evaluate its significance. The results show that H2O2 treatment reduces cell viability and increases ROS generation, leading to oxidative damage that affects the expression of target molecules. Interestingly, H2O2-treated cells exhibit high levels of AQP8 expression and gene silencing of AQP8 reverses high levels of ROS and low levels of COL1A1, KRT19, and Aggrecan expression in stressed cells, indicating that AQP8 plays a vital role in preventing oxidative damage and consequent aging. In conclusion, AQP8 is upregulated in human dermal fibroblasts during H2O2-induced oxidative stress and may help prevent oxidative damage and aging. These findings suggest that AQP8 could be a potential therapeutic target for skin aging. Further research is necessary to explore the feasibility of using AQP8 as a preventive or therapeutic strategy for maintaining skin health.

Morin Inhibits Cell Proliferation and Induces Caspase-mediated Apoptotic Cell Death in Glioma C6 Cell Line

Background Glioma is a recurrent form of primary malignant cancer that occurs in the brain and central nervous system. The adults are the major victims of gliomas, and the men are mostly affected. At present, gliomas are treated with surgical resection followed by radiation and the administration of chemodrugs such as temozolomide. The invasive and high recurrence rate of gliomas often reduces the effectiveness of treatment, which leads to poor recovery. Hence, more potent drugs without side effects on long-term treatment need to be discovered to treat gliomas. Morin is one such promising phytochemical that possesses immense pharmacological properties. It is proven to exert anti-inflammatory, antioxidant, anticancer, antibacterial, antidiabetic, and neuroprotective properties. The current research focuses on examining the growth inhibition and apoptosis-inducing potency of morin against glioma C6 cells. Materials and Methods Rat C6 glioma cells were treated with different concentrations of morin and analyzed for cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The intracellular reactive oxygen species (ROS) production and alteration of mitochondrial membrane potential (MMP) by morin in glioma cells were examined with dichlorodihydrofluorescein diacetate (DCHF-DA) and rhodamine 123 staining. The apoptotic induction in glioma cells was analyzed with dual staining and it was confirmed by quantifying the apoptotic protein using the enzyme-linked immunosorbent assay (ELISA) technique. The anti-inflammatory property of morin was assessed by the inflammatory cytokines tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) using the ELISA technique. Results Our MTT results show that morin treatment significantly reduced the cell viability of rat C6 glioma cells. It also significantly increased ROS generation and decreased MMP in rat C6 glioma cells. Morin treatment also effectively increased caspase-3 and caspase-9, the proapoptotic protein Bax, and decreased the antiapoptotic protein Bcl2. The induction of apoptosis by morin in glioma cells was evident with our acridine orange/ethidium bromide (AO/EtBr) staining results. Morin also decreased the inflammatory cytokine levels in rat C6 glioma cells. Conclusion Our findings have proven that morin significantly induces apoptosis by increasing the levels of apoptotic protein via the generation of ROS. It also effectively reduced inflammatory cytokine levels, thereby exerting anticancer activity against glioma cells. Therefore, morin can be subjected to further research to be designed as a potent anticancer drug to treat gliomas.

Enhanced Photodynamic Therapy Synergizing with Inhibition of Tumor Neutrophil Ferroptosis Boosts Anti-PD-1 Therapy of Gastric Cancer.

For tumor treatment, the ultimate goal in tumor therapy is to eliminate the primary tumor, manage potential metastases, and trigger an antitumor immune response, resulting in the complete clearance of all malignant cells. Tumor microenvironment (TME) refers to the local biological environment of solid tumors and has increasingly become an attractive target for cancer therapy. Neutrophils within TME of gastric cancer (GC) spontaneously undergo ferroptosis, and this process releases oxidized lipids that limit T cell activity. Enhanced photodynamic therapy (PDT) mediated by di-iodinated IR780 (Icy7) significantly increases the production of reactive oxygen species (ROS). Meanwhile, neutrophil ferroptosis can be triggered by increased ROS generation in the TME. In this study, a liposome encapsulating both ferroptosis inhibitor Liproxstatin-1 and modified photosensitizer Icy7, denoted LLI, significantly inhibits tumor growth of GC. LLI internalizes into MFC cells to generate ROS causing immunogenic cell death (ICD). Simultaneously, liposome-deliver Liproxstatin-1 effectively inhibits the ferroptosis of tumor neutrophils. LLI-based immunogenic PDT and neutrophil-targeting immunotherapy synergistically boost the anti-PD-1 treatment to elicit potent TME and systemic antitumor immune response with abscopal effects. In conclusion, LLI holds great potential for GC immunotherapy.

In vitro anti-cancer potency of Mortierella elongata lipids against MCF 7 cells through induction of apoptosis and cell cycle arrest

Globally, cancer remains the second-whacking cause of mortality. Several studies, like in vitro cell studies, clinical trials, in vivo studies, and cohort studies, have authenticated the anticancer potency of omega-3 polyunsaturated fatty acids (PUFA) against various cancer types. The present study reports the in vitro anticancer potency of PUFA produced by Mortierella elongata (Accession No. OK402027) against Michigan Cancer Foundation-7 (MCF-7) breast cancer cell lines. The biocompatibility and cytotoxicity of M. elongata lipids were evaluated against human embryonic kidney and MCF-7 cells, respectively. The anti-proliferative activity of M. elongata lipids was examined at three different concentrations based on the inhibitory concentration (IC50) value. The apoptotic activity of M. elongata lipids was analyzed by fluorescence microscopy by implementing three different staining methods, such as acridine orange/ethidium bromide, 4,6-diamidino-2-phenylindole, and propidium iodide. Further, the biological activity of M. elongata on apoptosis induction, cell cycle progression, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) generation were evaluated. The MTT assay revealed the anti-proliferative activity of M. elongata lipids, and the IC50 value of M. elongata lipids at 24 h was found to be 28 ± 1.3 μg/mL. A significant decrease in the percentage of live cells with 57.36% of apoptotic and 14.10% of necrotic cells was revealed in cells treated with 100 μg/mL of M. elongata lipids. Anti-proliferative activity is associated with increased ROS generation and the loss of MMP. The lipids of M. elongata induced a dose-dependent G1 arrest and were found to be more effective at 100 μg/ml, accumulating 54.49% of MCF-7 cells in the G1 phase. Taken together, the present study has confirmed the in vitro anticancer potency of M. elongata lipids via apoptosis and cell cycle arrest.

Biogenic Zinc Oxide Nanoparticles synthesized from Tinospora Cordifolia induce oxidative stress, mitochondrial damage and apoptosis in Colorectal Cancer.

Cancer chemotherapy remains a serious challenge, and new approaches to therapy are urgently needed to build novel treatment regimens. The methanol extract of the stem of Tinospora Cordifolia was used to synthesize biogenic zinc oxide nanoparticles (ZnO-NPs) that display anticancer activities against colorectal cancer. Biogenic ZnO-NPs synthesized from methanol extract of Tinospora Cordifolia stem (ZnO-NPs TM) were tested against HCT-116 cell lines to assess anticancer activity. UV-Vis, FTIR, XRD, SEM, and TEM analysis characterized the biogenic ZnO-NPs. To see how well biogenic ZnO-NPs fight cancer, cytotoxicity, AO/EtBr staining, Annexin V/PI staining, mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) analysis, and caspase cascade activity analysis were performed to assess the anticancer efficacy of biogenic ZnO-NPs. The IC50 values of biogenic ZnO-NPs treated cells (HCT-116 and Caco-2) were 31.419 ± 0.682μg/ml and 36.675 ± 0.916μg/ml, respectively. qRT-PCR analysis showed that cells treated with biogenic ZnO-NPs Bax and P53 mRNA levels increased significantly (p ≤ 0.001). It showed to have impaired MMP and increased ROS generation. In a corollary, our in vivo study showed that biogenic ZnO-NPs have an anti-tumour effect. Biogenic ZnO-NPs TM showed both in vitro and in vivo anticancer effects that could be employed as anticancer drugs.

Leishmanicidal Activity of Moringa oleifera (L.) Leaf Protein Extract on the Indian Strain of Leishmania donovani: An In Vitro Study

IntroductionRecurring incidents of drug resistance and the health hazards associated with the chemotherapeutic treatment of visceral leishmaniasis necessitate the search for safer yet equally effective biologically available alternate therapeutics. Plant products are being emphasised in this regard. Thus, Moringa oleifera leaf was chosen here, and an in vitro study was undertaken to determine its antileishmanial activity against visceral leishmaniasis. MethodsThe protein in M. oleifera leaf extract (Mo-EX) was identified by denaturing polyacrylamide gel electrophoresis. The antipromastigote activity and cytotoxicity of the proteinaceous Mo-EX were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Phase contrast microscopy and scanning electron microscopy analyses confirmed the morphological alterations of the promastigotes. The anti-amastigote activity of Mo-EX was determined by microscopic intracellular counting. Apoptosis, reactive oxygen species (ROS) generation, and thiol depletion were assessed by flow cytometric analysis, and nitric oxide production was determined by colorimetric assays. ResultsM. oleifera leaf extract displayed 50% inhibitory concentration valuesagainst Leishmania donovani promastigotes and intracellular amastigotes of 11.85 ± 0.08 and 8.86 ± 0.03 μg/ml, respectively, with a 50% lethal concentration in RAW 264.7 macrophages of 2 036.25 ± 0.04 μg/ml. Mo-EX reduced thiol production, significantly increased ROS generation, and induced apoptosis in L. donovani promastigotes while altering their morphology. Mo-EX evoked ROS and nitric oxide generation in parasite-infected host cells ex vivo. ConclusionsThe findings of the present study elucidate the leishmanicidal activity of protein-rich Mo-EX in L. donovani (MHOM/IN/1983/AG83) promastigotes and amastigotes, and Mo-EX was found to be moderately safe in RAW 264.7 host macrophages in vitro.

Survey of Activated Cleaning Chemistries for Low-Stress Post-Chemical Mechanical Planarization Cleaning of Silicon Carbide

Integrated Circuit (IC) technologies have begun transitioning to wide band gap (WBG) substrates (i.e., Silicon carbide (SiC)) due to the intrinsic properties (i.e., high capacitance, thermal stability, and wear resistance) of these semiconducting materials. This has led to the development of Chemical Mechanical Planarization (CMP) processes that use aggressive chemical conditions and increasing mechanical forces to reach optimal material removal rates (MRR). The harsh CMP process parameters often result in contamination/defectivity (i.e., organic residue, abrasive particles, etc.) left on post-polish substrate surfaces and thus posing difficulty within the post-CMP cleaning process. Traditionally, post-CMP cleaning utilizes a polyvinyl alcohol (PVA) brush scrubbing method on the substrate surface. This has been shown to cause secondary defects (i.e., scratches, particle agglomerations, etc.) that inevitably lead to overall device failure. Alternative post-CMP methods utilize non-contact cleaning via megasonic energy to target removing defects (i.e., organic residue, pad debris, particle agglomerations, etc.) and avoid secondary defect generation. This research surveys post-CMP cleaning processes (contact and non-contact) on SiC substrates with various cleaning chemistries (i.e., supramolecular surfactants, catalytic complexes for generating reactive oxygen species (ROS), non-metal catalyst). Initial results show that supramolecular surfactants can produce high particle removal efficiency (PRE) due to their ability to be broken down into their monomeric units and encapsulate any removed residue under both brush and megasonic conditions Additionally, increased ROS generation disrupts the non-covalently adsorbed defects to the SiC surface and leads to an increased cleaning efficiency while decreasing the overall variability of particle removal results. Combining the ROS systems' disruptive power with surfactants' encapsulation mode, a highly efficient clean with low variability can be achieved under low-stress conditions.

The Impact of PSR™ (Plant Small RNA Technology), Tea Extract, and Its Principal Components on Mitochondrial Function and Antioxidant Properties in Skin Cells

Objective: This study explored the impact of a black tea extract obtained through (plant small RNA) PSRTM technology, characterized by its abundance of small molecules, particularly citric acid—an antioxidant and tricarboxylic acid (TCA) cycle contributor—on mitochondrial health. The primary focus was to assess whether this extract could counteract reactive oxygen species (ROS)-induced mitochondrial alterations associated with aging, which lead to impaired mitochondrial function, reduced ATP production, and increased ROS generation. Methods: The PSRTM extraction method was employed to obtain a high content of polyphenols and small molecules, particularly citric acid. Results: In comparison with a conventional extract, the PSRTM extract demonstrated significant enhancements in aconitase activity, an ROS-sensitive enzyme in the TCA cycle, as well as basal respiration and ATP synthesis in fibroblast cells and skin biopsies. Moreover, the PSRTM extract effectively reduced ROS production by safeguarding this critical enzyme within the Krebs cycle and displayed superior capabilities in scavenging free radicals when exposed to UV-induced stress. When administered post-UV exposure, the PSRTM extract protected nuclear DNA by reducing the formation of cyclobutane pyrimidine dimers (CPDs) and promoting DNA repair mechanisms. Furthermore, the extract exhibited beneficial effects on the extracellular matrix, characterized by a reduction in matrix metalloprotease 1 (MMP1) and an increase in fibrillin 1 expression. Conclusions: These findings collectively suggest that the PSRTM extract holds promising antiaging potential, potentially functioning as a mitochondrial nutrient/protector due to its multifaceted benefits on mitochondrial function, nuclear DNA integrity, and the extracellular matrix.

Empagliflozin prevents oxidative stress in human coronary artery endothelial cells via the NHE/PKC/NOX axis

BackgroundEmpagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca2+), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation. MethodsHuman coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca2+ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na+/NCX/Ca2+ in the ROS inhibitory capacity of EMPA. ResultsCompared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-β knockdown inhibits the NOX activation induced by Ca2+ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca2+ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch. ConclusionEMPA reduced NOX activity via attenuation of the NHE/Na+/NCX/Ca2+/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.

Agonists and hydrogen peroxide mediate hyperoxidation of β2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to β2-agonists in constrictive airway disorders

Asthma and other airway obstructive disorders are characterized by heightened inflammation and excessive airway epithelial cell reactive oxygen species (ROS), which give rise to a highly oxidative environment. After decades of use, β2-adrenergic receptor (β2AR) agonists remain at the forefront of treatment options for asthma, however, chronic use of β2-agonists leads to tachyphylaxis to the bronchorelaxant effects, a phenomenon that remains mechanistically unexplained. We have previously demonstrated that β2AR agonism increases ROS generation in airway epithelial cells, which upholds proper receptor function via feedback oxidation of β2AR cysteine thiolates to Cys-S-sulfenic acids (Cys-SOH). Our previous results also demonstrate that prevention of normal redox cycling of this post-translational oxi-modification back to the thiol prevents proper receptor function. Given that Cys-S-sulfenic acids can be irreversibly overoxidized to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participation in redox reactions, we hypothesized that β2-agonist tachyphylaxis may be explained by hyperoxidation of β2AR to S-sulfinic acids. Here, using airway epithelial cell lines and primary small airway epithelial cells from healthy and asthma-diseased donors, we show that β2AR agonism generates H2O2 in a receptor and NAPDH oxidase-dependent manner. We also demonstrate that acute and chronic receptor agonism can facilitate β2AR S-sulfination, and that millimolar H2O2 concentrations are deleterious to β2AR-mediated cAMP formation, an effect that can be rescued to a degree in the presence of the cysteine-donating antioxidant N-acetyl-L-cysteine. Our results reveal that the oxidative state of β2AR may contribute to receptor functionality and may, at least in part, explain β2-agonist tachyphylaxis.

OR06-04 Environmental Pollutants Perfluoroalkyl Acids Potently Stimulated Aldosterone Biosynthesis

Abstract Disclosure: B. Caroccia: None. T.M. Seccia: None. G. Pallafacchina: None. M. Piazza: None. I. Caputo: None. R. Rizzuto: None. G. Rossi: None. A large environmental contamination of drinking water by perfluoroalkyl substances (PFAS) markedly increased by 8-fold the plasma levels of pentadecafluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in a Northern Italy population with that shows a high prevalence of arterial hypertension and cardiovascular disease. As the link between PFAS and arterial hypertension is unknown, we investigated if they could enhance the biosynthesis of aldosterone, the main mineralocorticoid hormone that has pressor effects. We exposed human adrenocortical carcinoma HAC15 cells to 1 µM or 10 µM concentration of PFOA and PFOS, alone or in combination, for 24, 48 or 72 hours and we measured changes in cell viability, aldosterone synthase (CYP11B2) mRNA, cell and mitochondrial reactive oxygen species (ROS), and aldosterone production in the absence or presence of the superoxide scavenger tempol. Experiments were also performed in the presence of 10 or 100 nM Ang II. We found that PFAS increased CYP11B2 mRNA by 3-fold and aldosterone secretion and mitochondrial ROS production by 2-fold over controls (p < 0.01 for all). PFAS also enhanced the effects of the aldosterone secretagogue Ang II on CYP11B2 mRNA and aldosterone secretion: 10 nM or 100 nM Ang II increased CYP11B2 mRNA by 12- and 52-fold, respectively, compared to vehicle (p < 0.001). Moreover, 1 µM PFAS potentiated the effect of 10 nM or 100 nM Ang II on CYP11B2 gene expression by 30- and 80-fold, respectively (p < 0.001). As the ROS scavenger tempol, which decreased ROS, significantly blunted the CYP11B2 gene expression induced by either PFAS alone, or by concomitant exposure to Ang II and PFAS, the effect of PFAS likely occurs via increased ROS generation. These results indicate that at concentrations mimicking those found in human plasma of exposed individuals, PFAS exert a prooxidant effect and act as potent disruptors of human adrenocortical cell function, thus implicating them as causative factors of human arterial hypertension due to aldosteronism. Presentation: Thursday, June 15, 2023

Antifungal Activity of ZnO Nanobullets against Schizosaccharomyces pombe

In this study, an enhanced antifungal response of ZnO nanobullets (NBs) against Schizosaccharomyces pombe is reported. The ZnO NBs were prepared by alkali precipitation method and confirmed by microscopic, morphological and optical studies using SEM, EDX, TEM, HRTEM and photoluminescence (PL) spectroscopic techniques. Growth kinetics and MIC studies were conducted following the growth inhibition percentage studies. Colony forming assay, well diffusion, disc diffusion, N-acetyl cysteine (NAC) effect on S. pombe growth, trypan blue study, cellular reactive oxygen species (ROS) quantification using H2DCFDA dye, Bradford assay, DNA fragmentation and all other relevant protocols were performed in antifungal studies. ZnO nanobullets (NBs) were shown by SEM and TEM examinations to have an average size of 50 nm. The hexagonal wurtzite structure of ZnO NBs was confirmed by HRTEM’s lattice fringe findings. Defectrelated visible emissions at 412, 436, 457 and 564 nm were confirmed via PL analysis. It was found that ZnO NBs resulted in complete growth inhibition of S. pombe at 200 μg/mL. When S. pombe was treated with ZnO NBs, the Bradford assay revealed enhanced protein leakage, but the TBARS assay revealed lipid peroxidation brought on by reactive oxygen species (ROS). When S. pombe was exposed to ZnO NBs, the H2DCFDA assay revealed increased ROS generation, whilst the trypan blue assay revealed increased cell membrane fusion and lower viability. According to present study, the treatment with ZnO NBs caused S. pombe to develop damaged cell walls, leaky proteins and DNA breakage.

Helicobacter pylori infection induces autophagy via ILK regulation of NOXs-ROS-Nrf2/HO-1-ROS loop.

Reactive oxygen species (ROS) can regulate the occurrence of autophagy, and effective control of the balance between ROS and autophagy may be an important strategy for Helicobacter pylori induced gastric-related diseases. In this study, infection with H. pylori led to a lower level of ILK phosphorylation and increased ROS generation. Knockdown of ILK enhanced total ROS generation, and upregulated NADPH oxidase (NOX) subunit p22-phox levels. Inhibition of NOXs affected total ROS generation. The inhibition of NOX and ROS generation reduced Nrf2 and HO-1 levels, and knockdown of ILK significantly enhanced Nrf2 levels in H. pylori-infected GES-1 cells. Activation of Nrf2 by DMF decreased ROS levels. Therefore, NOX-dependent ROS production regulated by ILK was essential for activation of Nrf2/HO-1 signaling pathways in H. pylori-infected GES-1 cells. Beclin1, ATG5 and LC3B-II levels were higher both in H. pylori-infected and ILK-knockdown GES-1 cells. In NAC-pretreated GES-1 cells infected with H. pylori, the LC3B-II level was decreased compared to that in cells after H. pylori infection alone. Stable low expression of ILK with further knockdown of Beclin1 or ATG5 significantly reduced LC3B-II levels in GES-1 cells, while with the addition of the autophagy inhibitor chloroquine (CQ), LC3B-II and p62 protein levels were both remarkably upregulated. H. pylori accelerated the accumulation of ROS and further led to the induction of ROS-mediated autophagy by inhibiting ILK levels. Together, these results indicate that H. pylori infection manipulates the NOX-ROS-Nrf2/HO-1-ROS loop to control intracellular oxygen stress and further induced ROS-mediated autophagy by inhibiting ILK levels.

Mitochondrial displacement loop region single nucleotide polymorphisms and mitochondrial DNA copy number associated with risk of ankylosing spondylitis.

The pathogenesis of ankylosing spondylitis (AS) seems to be associated with genetics, the environment, heredity, and oxidative stress. Single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) region of mitochondrial DNA (mtDNA) and mtDNA copy number were investigated for their correlation with AS patients. This study included 83 AS patients and 100 healthy controls from the Second Hospital of Hebei Medical University. DNAs were extracted from blood samples for polymerase chain reaction analysis and quantitative real-time polymerase chain reaction analysis. Plasma reactive oxygen species (ROS) levels were measured by fluorescent probe technology. The distribution frequencies of the minor alleles of nucleotides 16304C (p = .037), 16311C (p = .027), and 152C (p = .034) were remarkably higher in AS patients than in healthy controls, which indicated that the16304C, 16311C, and 152C alleles were correlated with an increased risk of AS. Simultaneously, mtDNA copy number was statistically higher in patients with AS compared with controls (1.450 ± 0.876 versus 0.835 ± 0.626, p < .001). We also observed an increased ROS generation in AS patients compared with controls (27 066.169 ± 18 364.819 versus 14 758.330 ± 5854.946, p < .001) subsequently. In addition, the AS susceptible SNP 16311C is associated with high ROS levels (35 065.177 ± 26 999.934 vs. 25 005.818 ± 14 999.495, p = .043). Our study demonstrated that SNPs in the mtDNA D-loop could be AS risk biomarkers with the potential to promote oxidative stress levels; mtDNA copy number-induced mitochondrial dysfunction may also be involved in AS pathogenesis.

Antibacterial Properties of Mesoporous Silica Nanoparticles Modified with Fluoroquinolones and Copper or Silver Species.

Antibiotic resistance is a global problem and bacterial biofilms contribute to its development. In this context, this study aimed to perform the synthesis and characterization of seven materials based on silica mesoporous nanoparticles functionalized with three types of fluoroquinolones, along with Cu2+ or Ag+ species to evaluate the antibacterial properties against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, including clinical and multi-drug-resistant strains of S. aureus and P. aeruginosa. In addition, in order to obtain an effective material to promote wound healing, a well-known proliferative agent, phenytoin sodium, was adsorbed onto one of the silver-functionalized materials. Furthermore, biofilm studies and the generation of reactive oxygen species (ROS) were also carried out to determine the antibacterial potential of the synthesized materials. In this sense, the Cu2+ materials showed antibacterial activity against S. aureus and E. coli, potentially due to increased ROS generation (up to 3 times), whereas the Ag+ materials exhibited a broader spectrum of activity, even inhibiting clinical strains of MRSA and P. aeruginosa. In particular, the Ag+ material with phenytoin sodium showed the ability to reduce biofilm development by up to 55% and inhibit bacterial growth in a "wound-like medium" by up to 89.33%.

Differential Pattern of Cell Death and ROS Production in Human Airway Epithelial Cells Exposed to Quinones Combined with Heated-PM2.5 and/or Asian Sand Dust

The combined toxicological effects of airborne particulate matter (PM), such as PM2.5, and Asian sand dust (ASD), with surrounding chemicals, particularly quinones, on human airway epithelial cells remain underexplored. In this study, we established an in vitro combination exposure model using 1,2-naphthoquinones (NQ) and 9,10-phenanthroquinones (PQ) along with heated PM (h-PM2.5 and h-ASD) to investigate their potential synergistic effects. The impacts of quinones and heated PM on tetrazolium dye (WST-1) reduction, cell death, and cytokine and reactive oxygen species (ROS) production were examined. Results revealed that exposure to 9,10-PQ with h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction at 1 μM compared to the corresponding control while markedly decreasing it at 10 μM. Higher early apoptotic, late apoptotic, or necrotic cell numbers were detected in 9,10-PQ + h-PM2.5 exposure than in 9,10-PQ + h-ASD or 9,10-PQ + h-PM2.5 + h-ASD. Additionally, 1,2-NQ + h-PM2.5 exposure also resulted in an increase in cell death compared to 1,2-NQ + h-ASD and 1,2-NQ + h-PM2.5 + h-ASD. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD significantly increased ROS production, especially with h-PM2.5. Our findings suggest that quinones, at relatively low concentrations, induce cell death synergistically in the presence of h-PM2.5 rather than h-ASD and h-PM2.5 + h-ASD, partially through the induction of apoptosis with increased ROS generation.