Lisinopril Mitigates Radiation-Induced Mitochondrial Defects in Rat Heart and Blood Cells.

The genetic bases and disparate responses to radiotherapy are poorly understood, especially for cardiotoxicity resulting from treatment of thoracic tumors. Preclinical animal models such as the Dahl salt-sensitive (SS) rat can serve as a surrogate model for salt-sensitive low renin hypertension, common to African Americans, where aldosterone contributes to hypertension-related alterations of peripheral vascular and renal vascular function. Brown Norway (BN) rats, in comparison, are a normotensive control group, while consomic SSBN6 with substitution of rat chromosome 6 (homologous to human chromosome 14) on an SS background manifests cardioprotection and mitochondrial preservation to SS rats after injury. In this study, 2 groups from each of the 3 rat strains had their hearts irradiated (8 Gy X 5 fractions). One irradiated group was treated with the ACE-inhibitor lisinopril, and a separate group in each strain served as nonirradiated controls. Radiation reduced cardiac end diastolic volume by 9-11% and increased thickness of the interventricular septum (11-16%) and left ventricular posterior wall (14-15%) in all 3 strains (5-10 rats/group) after 120 days. Lisinopril mitigated the increase in posterior wall thickness. Mitochondrial function was measured by the Seahorse Cell Mitochondrial Stress test in peripheral blood mononuclear cells (PBMC) at 90 days. Radiation did not alter mitochondrial respiration in PBMC from BN or SSBN6. However, maximal mitochondrial respiration and spare capacity were reduced by radiation in PBMC from SS rats (p=0.016 and 0.002 respectively, 9-10 rats/group) and this effect was mitigated by lisinopril (p=0.04 and 0.023 respectively, 9-10 rats/group). Taken together, these results indicate injury to the heart by radiation in all 3 strains of rats, although the SS rats had greater susceptibility for mitochondrial dysfunction. Lisinopril mitigated injury independent of genetic background.

Water-soluble cationic boronate probe based on coumarin imidazolium scaffold: Synthesis, characterization, and application to cellular peroxynitrite detection.

Peroxynitrite (ONOO-) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. Here, the development of a new water-soluble and cationic boronate probe based on a coumarin-imidazolium scaffold (CI-Bz-BA) for the fluorescent detection of ONOO- in cells is reported. The chemical reactivity of the CI-Bz-BA probe toward selected oxidants known to react with the boronate moiety was characterized, and the suitability of the probe for the direct detection of ONOO- in cell-free and cellular system is reported. Oxidation of the probe results in the formation of the primary hydroxybenzyl product (CI-Bz-OH), followed by the spontaneous elimination of the quinone methide moiety to produce the secondary phenol (CI-OH), which is accompanied by a red shift in the fluorescence emission band from 405 nm to 481 nm. CI-Bz-BA reacts with ONOO- stoichiometrically with a rate constant of ∼1 × 106 M-1s-1 to form, in addition to the major phenolic product CI-OH, the minor nitrated product CI-Bz-NO2, which is not formed by other oxidants tested or via myeloperoxidase-catalyzed oxidation/nitration. Both CI-OH and CI-Bz-NO2 products were also formed in the presence of cogenerated fluxes of nitric oxide and superoxide radical anion produced during decomposition of a SIN-1 donor. Using RAW 264.7 cells, we demonstrate the ability of the probe to report endogenously produced ONOO-via fluorescence measurements, including plate reader real time monitoring and two-photon fluorescence imaging. Liquid chromatography/mass spectrometry analyses of cell extracts and media confirmed the formation of both CI-OH and CI-Bz-NO2 in macrophages activated to produce ONOO-. We propose the use of a combination of real-time monitoring of probe oxidation using fluorimetry and fluorescence microscopy with liquid chromatography/mass spectrometry-based product identification for rigorous detection and quantitative analyses of ONOO- in biological systems.

Two-photon fluorescent probe for cellular peroxynitrite: Fluorescence detection, imaging, and identification of peroxynitrite-specific products.

A new naphthalene-based boronate probe, NAB-BE, for the fluorescence-based detection of inflammatory oxidants, including peroxynitrite, hypochlorous acid, and hydrogen peroxide, is reported. The chemical reactivity and fluorescence properties of the probe and the products are described. The major, phenolic oxidation product, NAB-OH, is formed in case of all three oxidants tested. This product shows green fluorescence, with a maximum at 512 nm, and can be excited either at 340 nm or in the near infrared region (745 nm) for two-photon fluorescence imaging. Peroxynitrite is the fastest of the oxidants tested and, in addition to the phenolic product, leads to the formation of a nitrated product, NAB-NO2, which can serve as a fingerprint for peroxynitrite. The probe was applied to detect peroxynitrite in activated macrophages using fluorimetry and two-photon fluorescence microscopy, and both NAB-OH and NAB-NO2 products were detected in cell extracts by liquid chromatography-mass spectrometry. The combined use of fluorometric high-throughput analyses, fluorescence imaging, and liquid chromatography-mass spectrometry-based product identification and quantitation is proposed for most comprehensive and rigorous characterization of oxidants in biological systems.

Mitochondria-targeted magnolol inhibits OXPHOS, proliferation, and tumor growth via modulation of energetics and autophagy in melanoma cells.

INTRODUCTION: Melanoma is an aggressive form of skin cancer for which there are no effective drugs for prolonged treatment. The existing kinase inhibitor antiglycolytic drugs (B-Raf serine/threonine kinase or BRAF inhibitors) are effective for a short time followed by a rapid onset of drug resistance. PRESENTATION OF CASE: Here, we show that a mitochondria-targeted analog of magnolol, Mito-magnolol (Mito-MGN), inhibits oxidative phosphorylation (OXPHOS) and proliferation of melanoma cells more potently than untargeted magnolol. Mito-MGN also inhibited tumor growth in murine melanoma xenografts. Mito-MGN decreased mitochondrial membrane potential and modulated energetic and mitophagy signaling proteins. DISCUSSION: Results indicate that Mito-MGN is significantly more potent than the FDA-approved OXPHOS inhibitor in inhibiting proliferation of melanoma cells. CONCLUSION: These findings have implications in the treatment of melanomas with enhanced OXPHOS status due to metabolic reprogramming or drug resistance.

HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion.

Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

High-Throughput Screening of NOX Inhibitors.

Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.

Embedding cyclic nitrone in mesoporous silica particles for EPR spin trapping of superoxide and other radicals.

The generation of superoxide radical anion in biological systems is one of the major initiating events in the redox biology of NADPH oxidases and mitochondrial redox signalling. However, the pallette of chemical tools for superoxide detection is very limited, hampering progress in understanding the chemical biology of superoxide. Although EPR spin trapping is regarded as the most rigorous technique for superoxide detection, rapid reduction of the EPR-active superoxide spin adducts to EPR-silent hydroxylamines, or to hydroxyl radical adducts by bioreductants, significantly limits the applicability of this technique in biological systems. To overcome these limitations, in this work, we report the synthesis and characterization of a new mesoporous silica functionalized with a phosphorylated cyclic spin trap (DIPPMPO nitrone). The DIPPMPO-grafted silica is a versatile spin-trap agent enabling the identification of a wide range of carbon or oxygen-centered transient radicals in organic and in aqueous media. Moreover, superoxide was efficiently trapped under in vitro conditions in both cell-free and cellular systems. The generated superoxide adduct exhibited an exceptional half-life of 3.5 h and a resistance toward bioreductant agents such as glutathione for several hours.

Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis.

Lung cancer often has a poor prognosis, with brain metastases a major reason for mortality. We modified lonidamine (LND), an antiglycolytic drug with limited efficacy, to mitochondria-targeted mito-lonidamine (Mito-LND) which is 100-fold more potent. Mito-LND, a tumor-selective inhibitor of oxidative phosphorylation, inhibits mitochondrial bioenergetics in lung cancer cells and mitigates lung cancer cell viability, growth, progression, and metastasis of lung cancer xenografts in mice. Mito-LND blocks lung tumor development and brain metastasis by inhibiting mitochondrial bioenergetics, stimulating the formation of reactive oxygen species, oxidizing mitochondrial peroxiredoxin, inactivating AKT/mTOR/p70S6K signaling, and inducing autophagic cell death in lung cancer cells. Mito-LND causes no toxicity in mice even when administered for eight weeks at 50 times the effective cancer inhibitory dose. Collectively, these findings show that mitochondrial targeting of LND is a promising therapeutic approach for investigating the role of autophagy in mitigating lung cancer development and brain metastasis.

Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future.

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

Molecular elements of pheromone detection in the female moth, Heliothis virescens.

Pheromones play pivotal roles in the reproductive behavior of moths, most prominently for the mate finding of male moths. Accordingly, the molecular basis for the detection of female-released pheromones by male moths has been studied in great detail. In contrast, little is known about how females can detect pheromone components released by themselves or by conspecifics. In this study, we assessed the antenna of female Heliothis virescens for elements of pheromone detection. In accordance with previous findings that female antennae respond to the sex pheromone component (Z)-9-tetradecenal, we identified olfactory sensory neurons that express its cognate receptor, the receptor type HR6. All HR6 cells coexpressed the "sensory neuron membrane protein 1" (SNMP1) and were associated with supporting cells expressing the pheromone-binding proteins PBP1 and PBP2. These features are reminiscent to male antennae and point to congruent mechanisms for pheromone detection in the two sexes. Further analysis of the SNMP1-expressing cells revealed a higher number in females compared to males. Moreover, in females, the SNMP1 neurons were arranged in clusters, which project their dendrites into a common sensillum, whereas in males there were only solitary SNMP1-neurons and only 1 per sensillum. Not all SNMP1 positive cells in female antennae expressed HR6 but instead the putative pheromone receptors HR11 and HR18, respectively. Neurons expressing 1 of the 3 receptor types were assigned to different sensilla. Together the data indicate that on the antenna of females, sensory neurons in a subset of sensilla trichodea are equipped with molecular elements, which render them responsive to pheromones.

Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases.

NADPH oxidases are a family of enzymes capable of transferring electrons from NADPH to molecular oxygen. A major function of NADPH oxidases is the activation of molecular oxygen into reactive oxygen species. Increased activity of NADPH oxidases has been implicated in various pathologies, including cardiovascular disease, neurological dysfunction, and cancer. Thus, NADPH oxidases have been identified as a viable target for the development of novel therapeutics exhibiting inhibitory effects on NADPH oxidases. Here, we describe the development of new assays for measuring the activity of NADPH oxidases enabling the high-throughput screening for NADPH oxidase inhibitors.

On the use of peroxy-caged luciferin (PCL-1) probe for bioluminescent detection of inflammatory oxidants in vitro and in vivo - Identification of reaction intermediates and oxidant-specific minor products.

Peroxy-caged luciferin (PCL-1) probe was first used to image hydrogen peroxide in living systems (Van de Bittner et al., 2010 [9]). Recently this probe was shown to react with peroxynitrite more potently than with hydrogen peroxide (Sieracki et al., 2013 [11]) and was suggested to be a more suitable probe for detecting peroxynitrite under in vivo conditions. In this work, we investigated in detail the products formed from the reaction between PCL-1 and hydrogen peroxide, hypochlorite, and peroxynitrite. HPLC analysis showed that hydrogen peroxide reacts slowly with PCL-1, forming luciferin as the only product. Hypochlorite reaction with PCL-1 yielded significantly less luciferin, as hypochlorite oxidized luciferin to form a chlorinated luciferin. Reaction between PCL-1 and peroxynitrite consists of a major and minor pathway. The major pathway results in luciferin and the minor pathway produces a radical-mediated nitrated luciferin. Radical intermediate was characterized by spin trapping. We conclude that monitoring of chlorinated and nitrated products in addition to bioluminescence in vivo will help identify the nature of oxidant responsible for bioluminescence derived from PCL-1.

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide.

High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases.

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.

Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses.

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.