Diphenyleneiodonium Treatment Inhibits the Development of Severe Herpes Stromal Keratitis Lesions.

Reactive oxygen species (ROS) play an important role in tissue inflammation. In this study, we measured the intracellular level of ROS in herpes stromal keratitis (HSK) corneas and determined the outcome of manipulating ROS level on HSK severity. Our results showed the predominance of ROS generation in neutrophils but not CD4 T cells in HSK corneas. NADPH oxidase 2 (NOX2) enzyme is known to generate ROS in myeloid cells. Our results showed baseline expression of different NOX2 subunits in uninfected corneas. After corneal herpes simplex virus-1 (HSV-1) infection, an enhanced expression of NOX2 subunits was detected in infected corneas. Furthermore, flow cytometry results showed a higher level of gp91 (Nox2 subunit) protein in neutrophils from HSK corneas, suggesting the involvement of NOX2 in generating ROS. However, no significant decrease in ROS level was noticed in neutrophils from HSV-1-infected gp91-/- mice than in C57BL/6J (B6) mice, suggesting NOX2 is not the major contributor in generating ROS in neutrophils. Next, we used diphenyleneiodonium (DPI), a flavoenzyme inhibitor, to pharmacologically manipulate the ROS levels in HSV-1-infected mice. Surprisingly, the neutrophils from peripheral blood and corneas of the DPI-treated group exhibited an increased level of ROS than the vehicle-treated group of infected B6 mice. Excessive ROS is known to cause cell death. Accordingly, DPI treatment resulted in a significant decrease in neutrophil frequency in peripheral blood and corneas of infected mice and was associated with reduced corneal pathology. Together, our results suggest that regulating ROS levels in neutrophils can ameliorate HSK severity. IMPORTANCE Neutrophils are one of the primary immune cell types involved in causing tissue damage after corneal HSV-1 infection. This study demonstrates that intracellular ROS production in the neutrophils in HSK lesions is not NOX2 dependent. Furthermore, manipulating ROS levels in neutrophils ameliorates the severity of HSK lesions. Our findings suggest that excessive intracellular ROS in neutrophils disrupt redox homeostasis and affect their survival, resulting in a decrease in HSK lesion severity.

ROS-initiated chemiluminescence-driven payload release from macrocycle-based Azo-containing polymer nanocapsules.

Reactive oxygen species (ROS) overproduction is involved in many pathological processes, particularly in inflammatory diseases. Therefore, ROS-responsive nanocarriers for specific drug release have been highly sought after. Herein we developed a ROS-responsive drug delivery system based on covalently self-assembled polymer nanocapsules (Azo-NCs) formed via crosslinking macrocyclic cucurbit[6]urils by a photo-sensitive azobenzene derivative (Azo). Luminol, a chemiluminescent molecule activatable by ROS, was co-loaded into Azo-NCs together with a therapeutic payload. When exposed to high ROS concentration that is typically encountered in inflammatory cells or tissues, the ROS-initiated blue chemiluminescence of luminol drives photoisomerization of the Azo groups within Azo-NCs, leading to Azo-NCs' surface transformation and distortion of the nanostructure, and subsequent payload release. As a proof-of-concept, ROS-responsive payload release from luminol-loaded Azo-NCs in inflammatory cells and zebrafish was demonstrated, showing promising anti-inflammatory effects in vitro and in vivo.

NADPH oxidase 2 as a potential therapeutic target for protection against cognitive deficits following systemic inflammation in mice.

BACKGROUND: Research indicates that sepsis increases the risk of developing cognitive impairment. After systemic inflammation, a corresponding activation of microglia is rapidly induced in the brain, and multiple neurotoxic factors, including inflammatory mediators (e.g., cytokines) and reactive oxygen species (e.g., superoxide), are also released that contribute to neuronal injury. NADPH oxidase (NOX) enzymes play a vital role in microglial activation through the generation of superoxide anions. We hypothesized that NOX isoforms, particularly NOX2, could exhibit remarkable abilities in developing cognitive deficits induced by systemic inflammation. METHODS: Mice with deficits of NOX2 organizer p47phox (p47phox-/-) and wild-type (WT) mice treated with the NOX inhibitor diphenyleneiodonium (DPI) were used in this study. Intraperitoneal lipopolysaccharide (LPS) injection was used to induce systemic inflammation. Spatial learning and memory were compared among treatment groups using the radial arm maze task. Brain tissues were collected for evaluating the transcript levels of proinflammatory cytokines, whereas immunofluorescence staining and immunoblotting were conducted to determine the percentage of activated glia (microglia and astroglia) and damaged neurons and the expression of synaptic proteins and BDNF. RESULTS: Cognitive impairment induced by systemic inflammation was significantly attenuated in the p47phox-/- mice compared to that in the WT mice. The p47phox-/- mice exhibited reduced microglial and astroglial activation and neuronal damage and attenuated the induction of multiple proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and CCL2. Similar to that observed in the p47phox-/- mice, the administration of DPI significantly attenuated the cognitive impairment, reduced the glial activation and brain cytokine concentrations, and restored the expression of postsynaptic proteins (PSD-95) and BDNF in neurons and astrocytes, compared to those in the vehicle-treated controls within 10 days after LPS injection. CONCLUSIONS: This study clearly demonstrates that NOX2 contributes to glial activation with subsequent reduction in the expression of BDNF, synaptic dysfunction, and cognitive deficits after systemic inflammation in an LPS-injected mouse model. Our results provide evidence that NOX2 might be a promising pharmacological target that could be used to protect against synaptic dysregulation and cognitive impairment following systemic inflammation.

A Tumor Agnostic Therapeutic Strategy for Hexokinase 1-Null/Hexokinase 2-Positive Cancers.

Since Warburg's observation that most cancers exhibit elevated glycolysis, decades of research have attempted to reduce tumor glucose utilization as a therapeutic approach. Hexokinase (HK) activity is the first glycolytic enzymatic step; despite many attempts to inhibit HK activity, none has reached clinical application. Identification of HK isoforms, and recognition that most tissues express only HK1 while most tumors express HK1 and HK2, stimulated reducing HK2 activity as a therapeutic option. However, studies using HK2 shRNA and isogenic HK1+HK2- and HK1+HK2+ tumor cell pairs demonstrated that tumors expressing only HK1, while exhibiting reduced glucose consumption, progressed in vivo as well as tumors expressing both HK1 and HK2. However, HK1-HK2+ tumor subpopulations exist among many cancers. shRNA HK2 suppression in HK1-HK2+ liver cancer cells reduced xenograft tumor progression, in contrast to HK1+HK2+ cells. HK2 inhibition, and partial inhibition of both oxidative phosphorylation and fatty acid oxidation using HK2 shRNA and small-molecule drugs, prevented human liver HK1-HK2+ cancer xenograft progression. Using human multiple myeloma xenografts and mouse allogeneic models to identify potential clinical translational agents, triple therapies that include antisense HK2 oligonucleotides, metformin, and perhexiline prevent progression. These results suggest an agnostic approach for HK1-HK2+ cancers, regardless of tissue origin.

Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome.

The increased levels of intracellular reactive oxygen species (ROS) in granulosa cells (GCs) may affect the pregnancy results in women with polycystic ovary syndrome (PCOS). In this study, we compared the in vitro fertilization and embryo transfer (IVF-ET) results of 22 patients with PCOS and 25 patients with tubal factor infertility and detected the ROS levels in the GCs of these two groups. Results showed that the PCOS group had significantly larger follicles on the administration day for human chorionic gonadotropin than the tubal factor group (P < 0.05); however, the number of retrieved oocytes was not significantly different between the two groups (P > 0.05). PCOS group had slightly lower fertilization, cleavage, grade I/II embryo, clinical pregnancy, and implantation rates and higher miscarriage rate than the tubal factor group (P > 0.05). We further found a significantly higher ROS level of GCs in the PCOS group than in the tubal factor group (P < 0.05). The increased ROS levels in GCs caused GC apoptosis, whereas NADPH oxidase 2 (NOX2) specific inhibitors (diphenyleneiodonium and apocynin) significantly reduced the ROS production in the PCOS group. In conclusion, the increased ROS expression levels in PCOS GCs greatly induced cell apoptosis, which further affected the oocyte quality and reduced the positive IVF-ET pregnancy results of women with PCOS. NADPH oxidase pathway may be involved in the mechanism of ROS production in GCs of women with PCOS.

Biological evaluation of diphenyleneiodonium chloride (DPIC) as a potential drug candidate for treatment of non-tuberculous mycobacterial infections.

BACKGROUND: Novel drug discovery against non-tuberculous mycobacteria is beset with a large number of challenges including the existence of myriad innate drug resistance mechanisms as well as a lack of suitable animal models, which hinders effective translation. In order to identify molecules acting via novel mechanisms of action, we screened the Library of Pharmacologically Active Compounds against non-tuberculous mycobacteria to identify such compounds. METHODS: Whole-cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested for cytotoxicity against Vero cells to determine the selectivity index, and time-kill kinetics were determined against Mycobacterium fortuitum. The compound's ability to synergize with amikacin, ceftriaxone, ceftazidime and meropenem was determined using fractional inhibitory concentration indexes followed by its ability to decimate mycobacterial infections ex vivo. Finally, the in vivo potential was determined in a neutropenic murine model mimicking mycobacterial infection. RESULTS: We have identified diphenyleneiodonium chloride (DPIC), an NADPH/NADH oxidase inhibitor, as possessing potent antimicrobial activity against non-tuberculous mycobacteria. DPIC exhibited concentration-dependent bactericidal activity against M. fortuitum and synergized with amikacin, ceftriaxone, ceftazidime and meropenem. When tested in a murine neutropenic M. fortuitum infection model, DPIC caused a significant reduction in bacterial load in kidney and spleen. The reduction in bacterial count is comparable to amikacin at a 100-fold lower concentration. CONCLUSIONS: DPIC exhibits all properties to be repositioned as a novel anti-mycobacterial therapy and possesses a potentially new mechanism of action. Thus, it can be projected as a potential new therapeutic against ever-increasing non-tuberculous mycobacterial infections.

Diphenyleneiodonium Mitigates Bupivacaine-Induced Sciatic Nerve Damage in a Diabetic Neuropathy Rat Model by Attenuating Oxidative Stress.

BACKGROUND: Increased oxidative stress has been linked to local anesthetic-induced nerve injury in a diabetic neuropathy (DN) rat model. The current study explores the effects of diphenyleneiodonium (DPI) chloride, an NADPH oxidase (NOX) inhibitor, on bupivacaine-induced sciatic nerve injury in DN rats. METHODS: A rat DN model was established through high-fat diet feeding and streptozotocin injection. The model was confirmed via testing (i) blood glucose, (ii) hindpaw allodynia responses to von Frey (VF) monofilaments, (iii) paw withdrawal thermal latency (PWTL), and (iv) nerve conduction velocity (NCV). Bupivacaine (Bup, 0.2 mL, 5 mg/mL) was used to block the right sciatic nerve. DPI (1 mg/kg) was injected subcutaneously 24 hours and 30 minutes before the sciatic block. At 24 hours after the block, NCV, various reactive oxygen species, and Caspase-3 were evaluated to determine the extent of sciatic nerve injury. RESULTS: The DN rat model was successfully established. Compared with the DN control group, the postblock values of VF responses (DN-Con, 16.5 ± 1.3 g; DN + Bup, 19.1 ± 1.5 g, P < .001) and PWTL significantly increased (DN-Con, 13.3 ± 1.1 seconds; DN + Bup, 14.6 ± 1.1 seconds, P = .028); the NCV of sciatic nerve was significantly reduced (DN-Con, 38.8 ± 2.4 m/s, DN + Bup, 30.5 ± 2.0 m/s, P = .003), and sciatic nerve injury (as indicated by axonal area) was more severe in the bupivacaine-treated DN group (DN-Con, 11.6 ± 0.3 µm, DN + Bup, 7.5 ± 0.3 µm, P < .001). In addition, DPI treatment significantly improved nerve function (VF responses, 17.3 ± 1.3 g; PWTL, 13.4 ± 1.1 seconds; NCV, 35.6 ± 3.1 m/s) and mitigated loss of axonal area (9.6 ± 0.3 µm). Compared to the DN + Bup group (without DPI), the levels of lipid peroxides and hydroperoxides, as well as the protein expression of NOX2, NOX4, and Caspase-3, were significantly reduced in the DN + Bup + DPI group (P < .05). CONCLUSIONS: Subcutaneous injection of DPI appears to protect against the functional and neurohistological damage of bupivacaine-blocked sciatic nerves in a high-fat diet/streptozotocin-induced DN model.

NADPH oxidase: its potential role in promotion of pulmonary arterial hypertension.

NADPH oxidases (NOXs) are a group of enzymes for superoxide anion (O2·- ) generation through transferring electrons from NADPH to molecular oxygen, which is rapidly converted into hydrogen peroxide (H2O2). There are seven members in NOX family, including NOX1 to NOX5, dual oxidase1, and dual oxidase 2. Recent studies have demonstrated that NOX subtypes may have different functions in different types of pulmonary arterial hypertension (PAH). The NOX-derived reactive oxygen species (ROS) are key factors that are involved in promoting the processes of pulmonary vascular remodeling, such as endothelial dysfunction, proliferation of pulmonary arterial smooth muscle cells (PASMCs), and cellular trans-differentiation, which are the basic pathologic characteristics of PAH. Inhibition of NOX shows beneficial effect on prevention of PAH development. Thus, NOX might be a potential target for PAH therapy. The main purpose of this review is to summarize recent findings on the role of NOX, particularly the NOX subtypes, in promotion of PAH development and to list recent progress regarding the NOX-based intervention for PAH.

The combined effect of resveratrol and diphenyleneiodonium on irradiation-induced injury to the hematopoietic system.

Both resveratrol(Res) and diphenyleneiodonium(DPI) have been shown to have radioprotective effects on hematopoietic system injury. However, the cooperative effect of Res and DPI are unknown. In this study, we explored the radioprotective effect of the combination of Res and DPI both in vitro and in vivo. Our results showed that the combined treatment of Res and DPI was more effective in protecting irradiated BMMNCs in terms of cell viability, colony-forming ability, and reconstitution ability in vitro compared with Res or DPI treatment alone. However, in mice, the combination of Res and DPI had no enhanced protection on 4Gy total body irradiation (TBI)-induced hematopoietic system injury, including TBI-induced myelosuppression, induction of the splenic index, and increases in HSC/HPC numbers and the colony-forming ability of BMCs,compared to Res or DPI alone. An exception was the number of BMCs. These studies illustrated the inconsistency between experiments carried out in vitro and in vivo and suggest an interaction between Res or DPI in vivo.

Inhibition of NADPH oxidase protects against metastasis of human lung cancer by decreasing microRNA-21.

The objective of this study was to detect the effect of NADPH oxidase (NOX) inhibition on metastasis of lung cancer. Primary human lung cancer cells were isolated from surgical tissues using the Cancer Cell Isolation Kit. Invasion was detected using the BD Biocoat Matrigel Invasion Chamber assay. Expressions of microRNA-21 (miR-21), PTEN, MMP9, and p47 were detected by qPCR. Groups of nude mice were challenged with A549 cells with or without DPI and detected for tumor metastasis and survival. NOX inhibition in human lung cancer cells significantly reduced their invasive potential in vitro. NOX inhibition in vivo led to decreased metastasis of human lung cancer and prolonged the survival time of tumor-bearing nude mice. Further, NOX inhibition resulted in decreased expression of miR-21 in human lung cancer cells. Increased expression of miR-21 abrogated the effect of NOX inhibitor on metastasis of human lung cancer in vitro and in vivo. Decreased expression of miR-21 facilitated the effect of NOX inhibitor on metastasis of human lung cancer in vitro and in vivo. Furthermore, increased expression of PTEN and decreased expression of MMP9 were observed in human lung cancer cells in response to NOX inhibition. Finally, close correlations of miR-21 expression levels with NADPH oxidase expression level and differentiation state of tumor cells were observed in lung cancer patients. Inhibition of NADPH oxidase protected against metastasis of human lung cancer cells by decreasing miR-21 expression, which could facilitate the understanding of lung cancer pathogenesis and provided clues for the development of novel therapeutics for lung cancer patients.

Design, synthesis and biological evaluation of mitochondria targeting theranostic agents.

Dual mitochondria targeting fluorescent F16-TPP analogues were designed and synthesized. Uptake and cytotoxicity studies indicate that FF16 and FF16-TPP, two compounds discovered in this study, are promising mitochondria targeting theranostic agents.

Suppression of the inflammatory response by diphenyleneiodonium after transient focal cerebral ischemia.

Diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, reduces production of reactive oxygen species (ROS) and confers neuroprotection to focal cerebral ischemia. Our objective was to investigate whether the neuroprotective action of DPI extends to averting the immune response. DPI-induced gene changes were analyzed by microarray analysis from rat brains subjected to 90 min of middle cerebral artery occlusion, treated with NaCl (ischemia), dimethylsulfoxide (DMSO), or DMSO and DPI (DPI), and reperfused for 48 h. The genomic expression profile was compared between groups using ingenuity pathway analysis at the pathway and network level. DPI selectively up-regulated 23 genes and down-regulated 75 genes more than twofold compared with both DMSO and ischemia. It significantly suppressed inducible nitric oxide synthase signaling and increased the expression of methionine adenosyltransferasesynthetase 2A and adenosylmethionine decarboxylase 1 genes, which are involved in increasing the production of the antioxidant glutathione. The most significantly affected gene network comprised genes implicated in the inflammatory response with an expression change indicating an overall suppression. Both integrin- and interleukin-17A-signaling pathways were also significantly associated and suppressed. In conclusion, the neuroprotective effects of DPI are mediated not only by suppressing ischemia-triggered oxidative stress but also by limiting leukocyte migration and infiltration.

Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats.

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-ß-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-ß-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.

Roles of NADPH oxidase in occurrence of gastric damage and expression of cyclooxygenase-2 during ischemia/reperfusion in rat stomachs.

NADPH oxidase is an enzyme that converts molecular oxygen into reactive oxygen species, which cause severe damage in several organs. Cyclooxygenase (COX)-2 is an inducible enzyme that is important in gastric mucosal defense and repair processes. It is unclear whether NADPH oxidase is related to COX expression in the gastric mucosa, so we investigated the correlation. Under urethane anesthesia, a male Sprague Dawley rat stomach was mounted in an ex-vivo chamber, and ischemia/reperfusion (I/R) was performed through a cannula in the femoral vein. I/R significantly increased NADPH oxidase activity, H(2)O(2) production, and myeloperoxidase (MPO) activity. In contrast, ischemia alone clearly enhanced both NADPH oxidase activity and H(2)O(2) production but not MPO activity. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) suppressed I/R-induced mucosal damage. On the other hand, the selective COX-2 inhibitor rofecoxib exhibited a tendency to enhance the severity of gastric damage induced by I/R, although the selective COX-1 inhibitor SC-560 and the nonselective COX inhibitor indomethacin had no effect. I/R also increased the expression of COX-2, and this increase was suppressed by pretreatment with DPI. These findings suggest that the increase in NADPH oxidase activity is involved in the occurrence of gastric mucosal damage induced by I/R and that this enzyme activity may be causally related to the upregulation of COX-2 during I/R.

[Antiedematous effect of diphenyliodinium salts on experimental toxic lung edema].

A series of diphenyliodinium (DPI) salts (chloride, sulfate, acetate and iodide) were synthesized and studied as preventive and therapeutic agents in the model toxic lung edema induced by thiourea and phosgene in rats. All DPI salts exhibited high antiedematous activity. The preventive action of drugs was more effective than their curative effect. The duration of the antiedematous action of DPI iodide exceeded 24 h. The effective dose of DPI salts in the toxic lung edema was within 0.2-0.5 LD50.

Diphenyleneiodonium and dimethylsulfoxide for treatment of reperfusion injury in cerebral ischemia of the rat.

Diphenyleneiodonium (DPI) is an inhibitor of the free radical producing NAD(P)H-oxidase. We tested whether DPI shows neuroprotective properties after focal cerebral ischemia and we used dimethylsulfoxide (DMSO), a nonspecific free radical scavenger, as a solvent. In male Wistar rats middle cerebral artery occlusion (1.5 h) and subsequent reperfusion (48 h) (MCAO/R) was induced with the filament model. Immediately after reperfusion the animals received either 0.25 ml normal saline, DMSO, or a combination of DMSO and DPI; each group consisted of 10 animals. MRI was performed at different times after reperfusion. Gelatine zymography of brain tissue for MMP-2 and MMP-9 was performed. The infarct sizes and BBB damage showed a significant difference between controls and the DPI/DMSO group for almost all points in time in all sequences. The activity of MMP-2 and MMP-9 was significantly reduced by DPI/DMSO but not by DMSO alone. DMSO treatment alone resulted in a protective effect with reduced lesion sizes measured by MRI at selected points of time, consistent with its known free radical scavenger effect. The combination of DMSO with DPI partly augmented this effect, presumably due to the additional inhibition of MMP-2 and MMP-9 by DPI. Moreover, the neurological outcome in both therapeutic groups was improved compared to controls with a significant difference between the therapeutic groups in favour of DPI and DMSO. The combination of DPI and DMSO reduced the activity of MMP-2 and MMP-9, attenuated the postischemic blood-brain barrier damage and improved neurological outcome. This was most likely due to reduced oxidative stress.

Development of lipophilic cations as therapies for disorders due to mitochondrial dysfunction.

Mitochondrial dysfunction causes or exacerbates a number of diseases. These include genetic disorders such as Friedreich's ataxia where the primary lesion is a defect in a nuclear gene and those diseases caused by mutations to mitochondrial DNA. Mitochondrial damage also contributes to neurodegenerative diseases, diabetes and ischaemia-reperfusion injury. Drug therapies to prevent or alleviate mitochondrial dysfunction use redox active compounds, anti-oxidants or mitochondrial co-factors, however, their effectiveness is limited. A promising approach to increase the selectivity and potency of these compounds is to modify them so that they concentrate within mitochondria. This can be done by incorporating a lipophilic cation which causes the molecules to concentrate several hundred-fold in mitochondria, driven by the membrane potential across the inner membrane. As lipophilic cations cross biological membranes easily, they can be delivered to mitochondria of the heart, brain and skeletal muscle, the organs most affected by mitochondrial damage. Mitochondria-targeted lipophilic cations may lead to improved therapies for diseases involving mitochondrial dysfunction.

Antiinflammatory effects of NADPH oxidase inhibitors.

Proinflammatory cytokines prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice suffering from experimental arthritis so as to attain an activated state, which, upon a second stimulus, releases 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced NADPH oxidase activity deregulates ROS-dependent signal transduction pathways of inflammation, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of two inhibitors of NADPH oxidase, diphenylene iodoniumchloride (DPI) and staurosporine, was tested in male DBA/1 x B10A(4R) hybrid mice suffering from potassium peroxochromate arthritis. Daily doses of 2.8 mumol/kg of DPI or 30 nmol/kg of staurosporine sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 mumol/kg of DPI, and 100 nmol/kg of staurosporine suppressed the arthritis by 85%. The onset, progression, and remission of arthritis correlated to both the activity of phagocytic NADPH oxidase (r = 0.750) and to overt disease symptoms as judged by the arthritis index. Our data support the hypothesis that oxidative stress plays a pivotal role in the pathology of arthritis, which can be therapeutically targeted by NADPH oxidase inhibitors.

Mechanism of inhibition of FaDu hypopharyngeal carcinoma cell growth by tetraphenylphosphonium chloride.

Cationic phosphonium salts are interesting because they inhibit the proliferation of carcinoma cells more than untransformed epithelial cells in vitro. This differential anti-proliferative effect has been used to identify phosphonium salts and other lipophilic cations that later demonstrated effects in animals. Using 6 carcinoma-derived and 2 untransformed epithelial cell lines, tetraphenylphosphonium chloride (TPP) and other cationic aryl phosphonium salts (CAPS) demonstrated a growth inhibition pattern similar to that of cation rhodamine 123, suggesting that CAPS may inhibit mitochondrial function. We tested this hypothesis for the effect of phosphonium salt TPP on FaDu human hypopharyngeal carcinoma cells. TPP inhibited the proliferation of FaDu carcinoma cells at submicromolar concentrations. Uptake of 3H-TPP by FaDu cells was partially inhibited in medium containing high K+ and fully inhibited by valinomycin in this medium, indicating that TPP accumulates preferentially in mitochondria, and to a lesser extent in the cytoplasm. FaDu cells exposed to TPP exhibited damage to mitochondrial inner membranes, reduced ATP/ADP ratios, decreased oxygen uptake rates and decreased mitochondrial membrane potentials. The treated cells secreted lactate more rapidly than untreated controls and exhibited hypersensitivity to 2-deoxyglucose, an inhibitor of glycolysis. TPP's antimitochondrial effects apparently enhance cytoplasmic glycolysis. In conclusion, TPP inhibits FaDu carcinoma cell growth by inhibiting mitochondrial respiration and ATP synthesis. Cationic phosphonium salts that inhibit carcinoma cell growth through antimitochondrial effects might be used to treat solid tumors without the risk of secondary tumors associated with agents affecting nuclear DNA.

[Protective effects of 3, 6-dimethamidodibenzopyriodinium citrate on myocardial injury induced by ischemia and reperfusion in rats].

3, 6-Dimethamidodibenzopyriodonium citrate (I-65) 0.5 and 1 mg.kg-1 pretreatment reduced the size of myocardial infarct after ischemia for 40 min and reperfusion for 120 min. I-65 0.5 mg.kg-1 decreased myocardial creatine kinase, lactate dehydrogenase release, and Ca2+ accumulation after ischemia for 40 min and reperfusion for 120 min. The results show that I-65 prevents cardiac ischemia and reperfusion injury and the effect is considered to be related to inhibition of myocardial Ca2+ accumulation.