Synthetic transformations of higher triterpenoids. XXX. Synthesis and cytotoxic activity of betulonic acid amides with fragments of nitroxyl radicals

Interaction of betulonic acid chloride with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, and 3-aminomethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl yielded the corresponding triterpene amides. The synthesized derivatives of betulonic acid were shown to exhibit a cytotoxic activity on models of the CEM-13, U-937, and MT-4 tumor cells. The concentration of the most active N-[3-oxo-28-norlup-20(29)-en-17-carbamoyl-(2,2,6,6-tetramethylpiperidine-4-yl)-1-oxyl that inhibited survival of the tumor cells by 50% (CCID₅₀) proved to be 5.7–33.1 μM.     

Spin-labeling of porcine pepsin and rhizopus chinensis acid protease by diazoketone reagents

The active site of porcine pepsin and that of rhizopus chinensis acid protease were labeled with diazoketone type spin labels, 4-(3-diazo-2-oxopropylidene)-2,2,6,6-tetramethylpiperidine-1-oxyl (I) and 3-(4-diazo-3-oxo-cis-1-butenyl)-2,2,5,5-tetramethylpyrroline-1-oxyl (II), respectively. The values of τ_c showed that the nitroxide motion was only slightly restricted in the I bound enzymes. The trans isomer of II bound to another site of the enzymes. Addition of pepstatin reduced the nitroxide motion in all the labeled enzymes.     

Prevention of Hyperoxia-Induced Alterations in Synaptosomal Membrane-Associated Proteins by N-tert-Butyl-α-Phenylnitrone and 4-Hydroxy-2,2,6,6-Tetramethylpiperidin-1-oxyl (Tempol)

Abstract: Hyperoxia has been considered a model of free radical reactive oxygen species production in aging and age-related disorders. Previously, we studied the membrane protein alterations that occur during hyperoxia; we found that exposure of young animals to 24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane proteins. We reasoned that free radical oxidation was involved in this protein oxidation. In accordance, in the current study we investigated the protective nature of two known free radical scavengers, N-tert-butyl-α-phenylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol), against 24-h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPR) protein-specific spin labeling, assay of the activity of the oxidatively sensitive enzyme glutamine synthetase (GS), and measurement of protein carbonyl content. Before hyperoxia, gerbils received intraperitoneal injections of varying concentrations of either of the two free radical scavengers. After 30 min, the gerbils were exposed to 90–100% O₂ for 24 h. For the spin labeling experiments, cortical synaptosomes were isolated from gerbils. The membrane proteins were spin labeled with the thiol-specific label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl). As in our earlier study, the EPR spectral parameter of MAL-6-labeled membranes, the W/S ratio, decreased with hyperoxia (p < 0.00001). This effect was lessened significantly with administration of PBN (p < 0.0003) or Tempol (p < 0.00003). For the GS and protein carbonyl assays, cortical proteins were used. The activity of the GS decreased with hyperoxia (p < 0.000005), and this effect likewise was lessened with administration of PBN (p < 0.004) or Tempol (p < 0.002). The protein carbonyl content increased with hyperoxia (p < 0.0002), and there was a protective effect found with Tempol (p < 0.000001). The optimum doses for PBN and Tempol were 20 and 5 mg/kg, respectively. The results are discussed with reference to the use of free radical scavengers as potential antiaging agents.     

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry

Abstract

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H₂O₂) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H₂O₂ were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H₂O₂. Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Prooxidative effects of TEMPO on human erythrocytes

The prooxidative effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were observed in human erythrocytes. Incubation of red blood cells with the membrane-permeable TEMPO leads to a decrease in the concentration of intracellular reduced glutathione, accompanied by the reduction of TEMPO. Extracellular ferricyanide inhibited the loss of glutathione and reduction of TEMPO. TEMPO induced glutathione release from the cells and oxidation of hemoglobin to methemoglobin; ferricyanide prevented these effects. These results indicate that TEMPO may act as an oxidant to erythrocytes, whilst extracellular ferricyanide protects against its effects.     

Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?

The aim of the research was to study the role played by extracellular O ₂ ^.- radicals, which are implicated in cardiac cell damage and the protective effect by cell-permeable, nitroxide, superoxide dismutase-mimics. Cardiomyocytes cultures from 1-day-old rats served as the test-system. Experiments were performed since 5th day in culture when >80% of the cells were beating myocardial cells. Oxidative damage was induced by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM glucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate damages were spontaneous beating, lactate dehydrogenase release and ATP level. The rhythmic pulsation was followed microscopically. To determine the kinetics of cytosolic enzyme release from the cells, media samples were collected at various points of time and assayed for enzyme activity. To determine the cellular ATP, cells were washed with sodium phosphate buffer, scraped off and boiled for 3 min with sodium phosphate buffer. Following centrifugation the supernatant was collected and ATP was determined by the chemiluminogenic assay using firefly tails. The present results indicate that nitroxide stable free radicals, in the millimolar concentration range, provide full protection without toxic side-effect. Unlike exogenously added SOD that failed to protect, exogenous catalase provided almost full protection. In addition, the metal-chelating agent dipyridyl, but not diethylene-triamine-pentaacetate or desferrioxamine, protected the cultured cells. The present results suggest that H₂O₂ is the predominant toxic species mediating the oxidative damage whereas extracellular superoxide radical does not contribute to cultured cardiomyocyte damage. Since nitroxides do not remove H₂O₂ they can protect the cells possibly by oxidizing the metal ions and inhibiting the Fenton reaction. The superoxide dismutase-mimic activity of nitroxides does not seem to underlie their protective effect, however, the involvement of intracellular O ₂ ^.- cannot be excluded.Abbreviations CHDO 2-spirocyclohexane doxyl (2-cyclohexane-5,5-demethyl-3-oxazolidinoxyl) - DF desferrioxamine - DTPA diethylene-triamine-pentaacetate - EPR electron paramagnetic resonance - HX hypoxanthine - LDH lactate dehydrogenase - SOD superoxide dismutase - SEM standard error of mean: TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl - TEMPAMINE 4-amino-2,2,6,6-tetramethyl-piperidinoxyl - XO xanthine oxidase - CAT catalase     

Effect of proline on the production of singlet oxygen

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.     

CW EPR and 9 GHz EPR imaging investigation of stable paramagnetic species and their antioxidant activities in dry shiitake mushroom (Lentinus edodes)

We investigated the antioxidant activities and locations of stable paramagnetic species in dry (or drying) shiitake mushroom (Lentinus edodes) using continuous wave (CW) electron paramagnetic resonance (EPR) and 9?GHz EPR imaging. CW 9?GHz EPR detected paramagnetic species (peak-to-peak linewidth (ΔH_pp)?=?0.57?mT) in the mushroom. Two-dimensional imaging of the sharp line using a 9?GHz EPR imager showed that the species were located in the cap and shortened stem portions of the mushroom. No other location of the species was found in the mushroom. However, radical locations and concentrations varied along the cap of the mushroom. The 9?GHz EPR imaging determined the exact location of stable paramagnetic species in the shiitake mushroom. Distilled water extracts of the pigmented cap surface and the inner cap of the mushroom showed similar antioxidant activities that reduced an aqueous solution of 0.1?mM 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. The present results suggest that the antioxidant activities of the edible mushroom extracts are much weaker than those of ascorbic acid. Thus, CW EPR and EPR imaging revealed the location and distribution of stable paramagnetic species and the antioxidant activities in the shiitake mushroom for the first time.     

An ionophoretic model for the study of calcium-calcium exchange

A Pressman cell was used to study the Br-X537A-mediated translocation of ⁴⁵Ca from one aqueous phase into another across an immiscible organic phase. In this system, an increase in the concentration of ⁴⁰Ca in one chamber (mimicking the extracellular compartment) resulted in an increased efflux of ⁴⁵Ca added in trace amount to the other chamber (simulating the cytosolic compartment). Such a Ca-Ca exchange process was also observed when the concentration of ionophore was increased, and could be blocked by the organic Ca-antagonist suloctidil. However, no Ca-Ca exchange occurred when the downhill rate of ⁴⁰Ca translocation was increased by use of a pH gradient. This ionophoretic model also suggests that a low intracellular concentration of Ca tends to maintain Ca influx at a low rate, and that the asymetrical distribution of Ca facilitates rather than impeeds the ionophoretic extrusion of intracellular Ca against its chemical gradient.     

Binding of nicotinamide nucleotides to dihydrolipoamide dehydrogenase measured with spin-labeled analogs

Binding of NAD and NADH to dihydrolipoamide dehydrogenase fromEscherichia coli and from pig heart was measured using the spin-labeled analogsN ⁶-(2,2,6,6-tetramethylpiperidine-4-yl-1-oxyl)-NAD and -NADH. A decrease in the peak amplitudes of the respective EPR spectra results after adding enzyme to the cofactor analogs. With the bacterial enzyme normal hyperbolic saturation behavior with the NAD analog and one binding site per subunit (K _s =0.51 mM) are observed, while the NADH analog reveals a sigmoidal binding characteristic. A high-affinity and a low-affinity site (K _s =0.087 and 0.33 mM) are found for binding of the NAD analog to the pig heart enzyme and only one type of binding site is observed for the NADH analog (K _s =22 µM).     

Revealing of the Spin-Spin Interaction Due to Incorporating a Spin Label in the Carbohydrate Parts of Immuniglobulins M and G

Abstract

The EPR spectra of the preparations produced by spin labeling of the carbohydrate parts in monoclonal IgM and normal IgG with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl as the spin label indicate the existence of a rapid spin-spin exchange interaction between two spin labels. In the case of spin-labeled IgM, the carrier of such a spectrum is shown to be a glycopeptide noncovalently bound to IgM; it includes two spin labels and may be detached from the macromolecule by a combination of dialysis and gel filtration.     

Genetic susceptibility to intracellular infections: Nramp1, macrophage function and divalent cations transport

Nramp1 is one of the few host resistance genes that have been characterized at the molecular level. Nramp1 is an integral membrane protein expressed in the lysosomal compartment of macrophages and is recruited to the membrane of bacterial phagosomes where it affects intracellular microbial replication. Nramp1 is part of a very large gene family conserved from bacteria and man that codes for transporters of divalent cations transporters. We propose that Nramp1 affects the intraphagosomal microbial replication by modulating divalent cations content in this organelle. Both mammalian and bacterial transporters may compete for the same substrate in the phagosomal space.     

Novel glycosylation of the nitroxyl radicals with peracetylated glycosyl fluorides using a combination of BF(3) x OEt(2) and an amine base as promoters

Glycosylation of the nitroxyl radicals, 4-acetoxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-acetoxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrollin-1-oxyl (3-carbamoyl-PROXYL) with peracetylglycosyl fluoride as the glycosyl donor, in the presence of boron trifluoride diethyl etherate (BF(3) x OEt(2)) and an amine base afforded the corresponding hydroxylamine-O-glycosides in 25-100% yields.     

Esterified trityl radicals as intracellular oxygen probes

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O₂ concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O₂ concentrations and the effects of menadione and KCN on the rates of O₂ consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O₂ concentration and consumption.     

Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.     

The oxidizing agent menadione induces an increase in the intracellular molecular oxygen concentration in K562 and A431 cells: Direct measurement using the new paramagnetic EPR probe fusinite

The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10⁶ cells in controls to 3 ng O/min/10⁶ cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.     

Detecting,Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H₂O₂ production, respectively.     

Extracellular redox environments regulate adipocyte differentiation

Oxidized extracellular redox states have been associated with many diseases related to obesity, including heart disease and diabetes, but relatively little is known about the relationship between extracellular redox states and obesity. In 3T3-L1 preadipocytes, oxidizing extracellular redox potentials (E_h) increased intracellular and mitochondrial reactive oxygen species (ROS) production. 3T3-L1 adipocytes showed a greater response to extracellular E_h, producing more intracellular ROS, than preadipocytes. 3T3-L1 adipocytes also produced more extracellular ROS and re-regulated the extracellular E_h to a more oxidizing state than preadipocytes. During 3T3-L1 differentiation, cellular glutathione and mitochondrial thioredoxin-2 become oxidized, suggesting that adipogenesis may be enhanced under conditions promoting intracellular and mitochondrial compartment oxidation. Under various extracellular E_h, 3T3-L1 adipogenesis, as determined by lipid accumulation and the expression of early genetic markers of adipogenesis, was sensitive to the extracellular redox environment, where it was enhanced under oxidizing conditions and lower under reducing conditions. Using a diet-induced obesity mouse model, plasma was collected before and after the 8 week diet regimens. Plasma GSH E_h was unchanged as a consequence of weight gain but plasma cystiene (Cys) E_h was significantly oxidized in overweight animals. Data presented here show that adipocytes/excessive adipose preferentially alter extracellular E_h to a more oxidized state in vivo and in vitro and may promote further adipogenesis.