Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH⁻ substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

Non-invasive analysis of reactive oxygen species generated in rats with water immersion restraint-induced gastric lesions using in vivo electron spin resonance spectroscopy

Reactive oxygen species (ROS) are reportedly associated with gastric ulcer. We previously reported the use of an in vivo 300-MHz electron spin resonance (ESR) spectroscopy/nitroxyl probe technique to detect _•OH generation in the stomachs of rats with gastric ulcers induced by NH₄OH. However, this is an acute ulcer model, and the relationship between in vivo ROS generation and lesion formation remains to be clarified. To address this question, the same technique was applied to a sub-acute water immersion restraint (WIR) model. A nitroxyl probe that was less membrane-permeable was orally administered to WIR-treated rats, and the spectra in the gastric region were obtained by in vivo ESR spectroscopy. The signal intensity of the orally administered probe was clearly changed in the WIR group, but no change occurred in the control group. Both enhanced signal decay and neutrophil infiltration into mucosa were observed 2 h after WIR with little formation of any mucosal lesions. The enhanced signal decay was caused by _•OH generation, based on the finding that the decay was suppressed by mannitol, desferrioxamine and catalase. Intravenous treatment with either anti-neutrophil antibody or allopurinol also suppressed the enhanced signal decay, and allopurinol depressed neutrophil infiltration into the mucosa. In rats treated with WIR for 6 h, lesion formation was suppressed by 50% with all antioxidants used in this experiment except anti-neutrophil antibody. These findings suggest that _•OH, which is generated in the stomach via the hypoxanthine/xanthine oxidase system upon neutrophil infiltrated into the mucosa, induces mucosal lesion formation, but that it accounts for only half the cause of lesion formation.     

Spin-label electron spin resonance studies of micellar dispersions of PEGs-PEs polymer-lipids

Conventional electron spin resonance (ESR) spectroscopy of different positional isomers of phosphatidylcholine spin labels (n-PCSL; n=5, 7, 10, 12, 14, and 16) has been used to study micellar dispersions made of poly(ethylene glycol)s-phosphatidylethanolamines (PEGs-PEs) polymer-lipids. Such aggregates are currently used as long circulating drug delivery systems "in vivo." We varied both the hydrocarbon chain length and the polymer size of the polymer-lipids. The dependence of the lipid-chain packing density on temperature and on label position as well as the flexibility and polarity profiles with position of chain labeling have been established for the PEGs-PEs micellar dispersions. The results show both similarity and differences either with common micellar dispersions of single chained lyso-palmitoylphosphatidylcholine (C(16)Lyso-PC) or with lamellar dispersions of double chained dipalmitoylphosphatidylcholine (DPPC). Well defined chain flexibility gradients of the same overall shape are obtained in the considered dispersions. However, the mobility of the first acyl chain segments is appreciable higher in micelles of polymer-lipids than in bilayers of DPPC and it becomes indistinguishable at the chain termini. A trend of decreasing polarity on moving toward the bilayer interior is seen in DPPC bilayers, whereas biphasic polarity profiles are obtained in micelles of polymer-lipids and C(16)Lyso-PC. Moreover, the properties of the PEGs-PEs micelles do not depend on the length of the hydrocarbon chain of the polymer-lipids but are slightly influenced by the size of the polymer.     

Free radicals in quinone containing antitumor agents: The nature of the diaziquone (3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) free radical

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.     

Binding of actinomycin D to DNA revealed by DNase I footprinting

We have analyzed the specificity of the actinomycin D-DNA interaction. The 'footprint' method has been used in this investigation. It is shown that: (i) The presence of dinucleotide GC or GG is required for binding of a single drug molecule. (ii) The strong binding sites are encoded by tetranucleotide XGCY; where X not equal to G and Y not equal to C in accordance with RNA elongation hindrance sites [1]. (iii) There is a positive cooperativity in binding of actinomycin D with DNA.     

Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity

Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 μg ml^?1) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections.     

The binding of actinomycin D and adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides

The effect of actinomycin D and adriamycin on synthetic polynucleotides, single-stranded viral DNA and supercoiled DNA has been studied employing the fluorescent probe, terbium. Marked displacement of the probe was observed when any deoxyribose-containing polynucleotide was pretreated with either drug. With supercoiled DNA, an unwinding of the supercoil was observed at very low drug concentrations (at approx. 1:500 molar ratio of drug:DNA) prior to the displacement of the terbium. This unwinding was visualized by agarose gel electrophoresis at molar ratios of approx. 1:200. The effect was more apparent and occurred at lower drug:DNA ratios with actinomycin D than with adriamycin. Unlike cis-dichlorodiammine platinum(II), actinomycin D did not protect pBR322 DNA from cleavage at its BamHI site. The hydrolysis of Φχ174 DNA by a series of G-C-specific restriction nucleases (including HhaI, HpaII and HaeIII) was also not affected by prior treatment of the DNA with actinomycin D.     

The role of the DMPO-hydrated electron spin adduct in DMPO-*OH spin trapping

Time-resolved in situ radiolysis ESR (electron spin resonance, equivalently EPR, electron paramagnetic resonance) studies have shown that the scavenging of radiolytically produced hydroxyl radical in nitrous oxide-saturated aqueous solutions containing 2 mM DMPO is essentially quantitative (94% of the theoretical yield) at 100 micros after the electron pulse [1]. This result appeared to conflict with earlier results using continuous cobalt-60 gamma radiolysis and hydrogen peroxide photolysis, where factors of 35 and 33% were obtained, respectively [2,3]. To investigate this discrepancy, nitrogen-saturated aqueous solutions containing 15 mM DMPO were cobalt-60 gamma irradiated (dose rate = 223 Gy/min) for periods of 0.25-6 min, and ESR absorption spectra were observed approximately 30 s after irradiation. A rapid, pseudo-first-order termination reaction of the protonated DMPO-hydrated electron adduct (DMPO-H) with DMPO-OH was observed for the first time. The rate constant for the reaction of DMPO-H with DMPO-OH is 2.44 x 10(2) (+/- 2.2 x 10(1)) M(-1) s(-1). In low-dose radiolysis experiments, this reaction lowers the observed yield of DMPO-OH to 44% of the radiation-chemical OH radical yield (G = 2.8), in good agreement with the earlier results [2,3]. In the absence of the DMPO-H radical, the DMPO-OH exhibits second-order radical termination kinetics, 2k(T) = 22 (+/- 2) M(-1) s(-1) at initial DMPO-OH concentrations > or = 13 microM, with first-order termination kinetics observed at lower concentrations, in agreement with earlier literature reports [4].     

Electron spin resonance studies of chromanoxyl radicals derived from tocopherols

Electron spin resonance measurements were performed for the chromanoxyl radicals obtained from -, β-, γ-, δ-tocopherol, tocol and their model compounds by oxidizing the phenol precursors with PbO₂ in toluene. The proton hyperfine coupling constants were determined, and the ‘experimental’ spin densities were evaluated from the hyperfine coupling constants. From the results, the methyl-substitution effects on the unpaired spin distribution and molecular structure of the chromanoxyl radicals have been studied.     

TNF-alpha-mediated apoptosis in chondrocytes sensitized by MG132 or actinomycin D

The mechanism of TNF-alpha-mediated chondrocyte apoptosis in human articular cartilage was investigated. First passage OA chondrocytes were treated with actinomycin D or MG132 in combination with TNF-alpha to facilitate cell death. The patterns of apoptosis-related proteins, NF-kappaB activation, and IkappaB degradation were analyzed. Cell death was increased by 0.2 microg/ml of actinomycin D or 20 microM MG132 in combination with TNF-alpha. Apoptosis potentiated by MG132 was more effectively inhibited by caspase inhibitors than that by actinomycin D. MG132 or actinomycin D both led to a significant increase in p53, but the expressions of the p53 response proteins increased only in MG132 treated chondrocytes. TNF-alpha induced chondrocyte IkappaB phosphorylation was unaffected by either MG132 or actinomycin D. MG132, but not actinomycin D, inhibited the chondrocyte IkappaB degradation induced by TNF-alpha and NF-kappaB activation. Our results suggest that MG132 and actinomycin D exert different influences upon TNF-alpha-mediated chondrocyte apoptotic signaling.     

Sequence-specific inhibition of RNA elongation by actinomycin D

We have developed a method to localize specific sites where RNA elongation is arrested due to DNA-bound ligands. The method was used to determine apparent binding sites for actinomycin D. We have found 14 strong RNA hindrance sites along nucleotide sequence of T7 and D111 T7 DNA of 380 nucleotides full length under low actinomycin D concentration conditions. Nucleotide sequence of all the sites is described by general formula XGCY where X ‡ G and Y ‡ C.     

Protein kinase B inhibits apoptosis induced by actinomycin D in ECV304 cells through phosphorylation of caspase 8

Actinomycin D (act-D) anchors itself into DNA-base pairs by intercalation and thereby inhibits mRNA synthesis. It has been well established that act-D elicits apoptosis in various cell types involving endothelial cells. However, the regulatory mechanisms of actinomycin D-induced apoptotic cell death still remain unclear. Here, we investigated apoptotic cell death and its underlying regulatory mechanisms elicited by actinomycin D in ECV304. Act-D induced typical apoptotic features including chromatin condensation and translocation of phosphatidylserine. Since the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, it was of interest to determine if this pathway could protect against apoptosis induced by act-D. Inhibition of PI3K/PKB significantly increased act-D-induced apoptosis. Moreover, act-D-induced cell death was physiologically linked to PKB-mediated cell survival through caspase-8. These results suggest that cross-talk between the PKB and caspase-8 pathways may regulate the balance between cell survival and cell death in ECV304.     

Naturally occurring quinols and quinones studied as semiquinones by electron spin resonance

A simple procedure using electron spin resonance techniques was applied to detect, identify and quantify quinones and quinols in crude plant extracts. Hydroquinone was determined in Pyrus, plumbagin in Drosera and Ceratostigma, and hydrojuglone in Juglandaceae. Hydrojuglone is found in markedly higher concentrations in Pterocarya and in Juglans than in Carya. Plastoquinol has been observed in 500 of 700 plant extracts studied. Esters of phenolic acids are easily detected and distinguished, e.g. chlorogenic and rosmarinic acids. Esters of homoprotocatechuic and of dihydrocaffeic acid occur widely in the Oleaceae. The limitations of the method are discussed.     

Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies

Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.     

The prosthetic group of methanol dehydrogenase from Hyphomicrobium X: electron spin resonance evidence for a quinone structure.

Partial reduction of the isolated prosthetic group of methanol dehydrogenase yields a free radical with the same characteristics as the one contained in the enzyme. The Electron Spin Resonance spectrum in alkaline aqueous solution displays hyperfine structure and is interpreted in terms of an isotropic g-value, hyperfine coupling constants and nuclear spins. The magnitudes of these parameters indicate that the prosthetic group is a quinone containing two nitrogen atoms.     

Effect of anaesthesia-induced alterations in haemodynamics on in vivo kinetics of nitroxyl probes in electron spin resonance spectroscopy

Although the advent of in vivo electron spin resonance (ESR) spectroscopy has allowed analysis of the redox status of living animals, whether the haemodynamic condition affects the signal decay rate remains unknown. Three kinds of haemodynamic conditions were generated by changing the anaesthetic dosage in mice. Haemodynamics was analysed (n=6 each) and in vivo ESR was performed to measure the signal decay rates of three nitroxyl spin probes (carbamoyl-, carboxy- and methoxycarbonyl-PROXYL) at the chest and head regions (n=6 for each condition and probe). Haemodynamic analysis revealed negative inotropic and chronotropic effects on the cardiovascular system depending on the depth of anaesthesia. Although signal decay rates differed among three probes, they were not affected by heart rate alteration. In this study we report the haemodynamics-independent signal decay rate of nitoxyl probes.