Messenger RNA for glutamine synthetase: its partial purification, translation in a cell-free system and its regulation by hydrocortisone.

We have verified that the enzymatic hydroxylation of carcinogenic aromatic hydrocarbons produces product molecules in electronically excited states. Introduction of the carcinogen benzo[α]pyrene into liver microsomes from 3-methylcholanthrene-induced rats results in a significant chemiluminescence which is shown for the first time to be correlated with the enzymatic hydroxylation of the parent carcinogen. The kinetics of the chemiluminescence implicate the intermediate epoxide as the precursor to the excited state product molecules.     

Co-oxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H2O2

The reaction between metmyoglobin and H2O2 proceeds with oxidation of the hemo-protein iron to a higher valence state and consumption of the peroxide. This reaction is further associated with (a) O2 evolution; (b) hydroxylation of the aromatic compound salicylate to yield a set of dihydroxybenzoic acid derivatives (analyzed by HPLC with electrochemical detection); (c) autoxidation of cholesterol with formation of 3 beta-hydroxy-5-alpha-cholest-6-ene-5-hydroperoxide; and (d) formation of electronically excited states detected by low-level chemiluminescence. The heterolytic scission of the O-O bond of hydroperoxides by metmyoglobin causes the formation of an oxidizing equivalent capable of promoting peroxidation of linoleate and arachidonate (as indicated by the parallel formation of thiobarbituric acid-reactive material and an enhancement of chemiluminescence intensity). The identity of the oxidizing equivalent(s) is discussed in terms of the formation of a relatively stable higher state of oxidation of heme Fe (FeIV-OH or FeV = O) as well as on possible intermediate species derived during the decomposition of H2O2 by metmyoglobin, such as HO.and 1O2. These species might be involved either simultaneously or sequentially in the peroxidation of fatty acids as well as in the tissue damage associated with the formation of H2O2 in ischemic-reperfusion states.     

Studies on the interaction of bleomycin A2 with rat lung microsomes. III. Effect of exogenous iron on bleomycin-mediated DNA chain breakage

The interaction of bleomycin A₂ with rat lung microsomes results in bleomycin-mediated DNA chain breakage due to the mixed-function oxidase catalyzed activation of bleomycin. This study demonstrates that the addition of exogenous Fe³⁺ significantly enhances the bleomycin-mediated cleavage of DNA deoxyribose, that the enhancing effect of Fe³⁺ is maximum when a 1:1 ratio of bleomycin to Fe³⁺ is achieved and that either NADPH or NADH can serve as pyridine cofactors for this reaction. Since the activation of bleomycin can be facilitated by iron in the Fe²⁺ form, these observations support the hypothesis that the mixed-function oxidase system may serve to maintain either adventitious or exogenous iron in the Fe²⁺ form. In the absence of DNA, the interaction of bleomycin with rat lung microsomes results in the self-inactivation of bleomycin, a reaction which is also enhanced by the addition of exogenous Fe³⁺. Thus, the microsomal mixed-function oxidase system represents an efficient biological system for the ‘activation-inactivation’ of bleomycin.     

Chemienergized aromatic aldehydes from the peroxidase catalyzed oxidation of pyruvates: excited vanillin from vanylpyruvate.

Oxidation by molecular oxygen of vanylpyruvate in dimethylsulfoxide containing potassium t-butoxide results in formation of emissive, electronically excited (singlet) vanillin and of oxalate, that is, the products expected from the cleavage of a dioxetane intermediate. The reaction is a model for the peroxidase and laccase catalyzed processes that occur during lignin degradation by fungi. It is inferred that vanillin formed in the latter processes is generated in an electronically excited state, not necessarily emissive. This view is strengthened by (i) the emission, albeit very weak, observed from the enzyme system, and (ii) the alteration of the enzyme as a result of the reaction, the spectral changes being similar to those induced by uv irradiation of the enzyme alone. Also other peroxidase catalyzed oxidations of aromatic pyruvates should produce the electronically excited aldehyde.     

Activated bleomycin. A transient complex of drug, iron, and oxygen that degrades DNA

"Activated bleomycin" is an oxygenated iron drug complex which embodies the drug's DNA-cleaving activity. This activity is exercised on DNA, if present, but if DNA is absent, the drug itself is inactivated. Hyperfine interactions in the EPR spectra of activated bleomycin prepared with 57Fe(II) and 17O2 demonstrate the presence of iron as Fe(III) and of bound oxygen originating in dioxygen. Bleomycin can also be activated with Fe(III) and either H2O2 or ethyl hydroperoxide. These latter reactions do not produce a ferrous intermediate nor do they require O2. But O2 is required for the reaction of activated bleomycin with DNA to yield the malondialdehyde-like chromogens used to monitor DNA degradation. The attack on DNA is quantitatively concurrent with the decay of activated bleomycin, however generated.     

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.     

Synthetic analogues and biosynthetic intermediates of bleomycin. Metal-binding, dioxygen interaction, and implication for the role of functional groups in bleomycin action mechanism

In order to clarify the role of bleomycin functional groups in action mechanism, the metal-binding, dioxygen activation, and DNA cleavage of several synthetic analogues and biosynthetic intermediates of bleomycin have been investigated. The present results support that 1) the beta-aminoalaninepyrimidine-beta-hydroxyhistidine portion of the bleomycin molecule substantially participates in the Fe(II) and dioxygen interactions, 2) the transposition of the pyrimidine (or pyridine) and imidazole groups in the Fe(II)-coordination is essential for the effective binding and activation of molecular oxygen by the bleomycin ligands, and 3) the gulose-mannose moiety plays an important role as an environmental factor for the efficient dioxygen reduction and DNA cleavage, although the sugar portion does not contribute significantly to the nucleotide specificity in the DNA strand scission. Certain oligopeptides are able to mimic the metal-binding and dioxygen activation by bleomycin, but not induce the effective DNA cleavage. Probably, the bithiazole DNA interaction site of bleomycin delivers the iron/dioxygen chemistry to particularly the DNA (formula, see text) nucleotide sequences.     

Activation of chemiluminescence accompanying lipid peroxidation in monolayer liposome suspension

In the presence of rhodamine J the chemiluminescence intensity of monolayer liposomes induced by ferrous ions is increased by three orders. There is no accumulation of malonic dialdehyde. It is suggested that chemiluminescence activation is related to rhodamine J interaction with the products of lipid peroxidation whose molecules are in the excited state.     

Ferrous Ions Detected in Iron-Overloaded Cord Blood Plasma From Preterm and Term Babies: Implications for Oxidative Stress

Redox active iron chelatable to bleomycin is often present in the plasma of cord blood samples taken from preterm and term babies. The low caeruloplasmin and high ascorbate levels in plasma at birth may allow this iron to exist in the reduced ferrous state. In support of this postulate thirteen cord blood samples showing the presence of low molecular mass iron were able to degrade DNA in the presence of bleomycin and plasma.     

Ferrous Ions Detected in Iron-Overloaded Cord Blood Plasma From Preterm and Term Babies: Implications for Oxidative Stress

Redox active iron chelatable to bleomycin is often present in the plasma of cord blood samples taken from preterm and term babies. The low caeruloplasmin and high ascorbate levels in plasma at birth may allow this iron to exist in the reduced ferrous state. In support of this postulate thirteen cord blood samples showing the presence of low molecular mass iron were able to degrade DNA in the presence of bleomycin and plasma.     

Light-induced nicking of deoxyribonucleic acid by cobalt(III) bleomycins

The anticancer drug bleomycin is a glycopeptide that causes strand scission of DNA both in vivo and in vitro. Cleavage of DNA by bleomycin has been studied extensively in vitro, with the findings that ferrous ion and molecular oxygen must be present and that addition of reducing agents greatly enhances the reaction. To date, only iron has been shown to be an effective metal cofactor for the cleavage of DNA by bleomycin. Here it is reported that two stable cobalt(III) complexes of bleomycin are strikingly effective in causing single-strand breaks (nicks) in supercoiled DNA in the presence of ultraviolet or visible radiation. For example, 366-nm light from an 18-W long-wavelength mercury lamp for 1 h causes 10(-6) M cobalt(III) bleomycin to completely convert supercoiled phi X174 DNA (10(-8) M DNA, 10(-4) M phosphate) into the nicked circular form. Furthermore, numerous alkali-labile sites are produced on the DNA during this treatment. The observed reactions are not caused by adventitious iron, and they occur only in the presence of cobalt(III) bleomycin and light.     

Effect of deoxyribonucleic acid on the production of reduced oxygen by bleomycin and iron

The binding of bleomycin to DNA in the presence and absence of ferric iron was measured by fluorescence spectroscopy. In millimolar concentrations of tris(hydroxymethyl)aminomethane, pH 7.5, approximately 80% of the bleomycin binds to DNA. Ferric iron seems to have no significant effect on the binding of DNA to bleomycin. The induction of oxygen uptake by ferrous iron and bleomycin was monitored in the presence and absence of DNA. DNA has no effect on the rate of oxygen uptake. Therefore, the iron binding site and the DNA binding site appear to be independent of each other. Under conditions where 80% of the bleomycin is bound to DNA, the ferrous iron-bleomycin-induced reduction of oxygen follows Michaelis-Menten kinetics. Ferrous iron autoxidation produces ethylene from methional. The addition of bleomycin greatly increases ethylene production. DNA, under conditions where 80% of the bleomycin is bound to DNA, inhibits ethylene production. Since ethylene is a measure of hydroxyl radical production, we conclude that DNA is able to compete with methional for the hydroxyl radical. We postulate a mechanism for DNA double-strand breaks in which the bleomycin selectively binds to DNA and recurrently produces the hydroxyl radical at that site. The localized generation of many hydroxyl radicals as provided by the proposed oxidation-reduction cycle mechanism may cause multiple strand breaks taking place on both strands of the DNA duplex leading to double-strand breaks. Since catalase, but not superoxide dismutase, is able to inhibit ferrous iron-bleomycin-induced products of the hydroxyl radical, hydrogen peroxide, but not the superoxide radical, is the immediate precursor of the hydroxyl radical.     

Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways.

In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and RAD6-dependent DNA repair. Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration. Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin. The most important mechanisms involved are recombination and postreplication repair. The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the RAD6- and RAD52-dependent repair processes. Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations. RAD3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin. The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates. On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.     

Reaction of FeBlm with DNA: Fe(II)Blm-NO.

The structure of the iron bleomycin nitric oxide complex is altered in the presence of calf thymus DNA as determined from epr studies. This altered structure predominates for one iron bleomycin nitric oxide molecule per coil of the DNA helix. In the absence of nitric oxide, as the pH is lowered, iron bleomycin dissociates in two steps, supporting the hypothesis that in-plane nitrogens may be easily perturbed.     

On the mechanism of peroxyoxalate chemiluminescence. Quenched chemiluminescence as a detection method in HPLC

Several analytes such as the inorganic anions bromide, iodide, sulphite and nitrite and organic compounds as substituted anilines and sulphur compounds cause quenching of peroxyoxalate chemiluminescence. A detection method for liquid chromatography based on the quenching phenomenon has been developed. It makes use of an immobilized luminophore, i.e. 3-aminofluoranthene covalently bound via an alkyl-spacer on controlled pore glass, packed in the detector cell. The mechanism behind the quenching has been elucidated by investigating the roles of luminophores (both in the liquid and in solid state) and oxalates in peroxylate CL with respect to quenchers. Most probably the quencher destroys the radical ion pair produced after electron transfer in the last stage of the CIEEL reaction scheme, thus preventing the formation of electronically excited luminophore.     

Excited-state properties of Escherichia coli DNA photolyase in the picosecond to millisecond time scale

Escherichia coli DNA photolyase contains a stable flavin radical that is readily photoreduced in the presence of added electron donors. Picosecond, nanosecond, and conventional flash photolysis technique have been employed to investigate the events leading to photoreduction from 40 ps to tens of milliseconds following flash excitation. Direct light absorption by the flavin radical produces the first excited doublet state which undergoes rapid (within 100 ps) intersystem crossing to yield the lowest excited quartet (n pi*) state. In contrast, light absorption by the folate chromophore produces a new intermediate state via interaction of the folate excited singlet state with the ground-state flavin radical, leading to an enhanced yield of the excited radical doublet state and hence quartet state. Subsequent reaction of the excited quartet state involves hydrogen atom abstraction from a tryptophan residue. Secondary electron transfer from added electron donors occurs to the oxidized tryptophan radical with rate constants ranging from 10(4) (dithiothreitol) to 4 x 10(6) M-1 s-1 (n-propyl gallate). The low value of the latter rate compared to reduction of the tryptophan radical in lysozyme suggests that the reactive tryptophan is highly buried in photolyase. A redox potential diagram has been constructed for the ground and excited states involved. It is concluded that the one-electron reduction potential of the excited quartet state of the flavin radical must be at least 1.23 V more positive than the ground state, in agreement with the value of delta E greater than 1.77 V calculated from spectroscopic data.     

Photochemical-like effects in DNA caused by enzynically energized triplet carbonyl cmpounds

The same circular dichroism spectrum as that of DNA conformationally altered by UV irradiation is observed when native DNA is added to an enzymic system which produces an electronically excited triplet carbonyl compound.     

Substituent effects on the decomposition of chemiluminescent tricyclic aromatic dioxetanes

Three tricyclic 1,2‐dioxetane derivatives, 1a, 2a and 3a were synthesized from their corresponding 1,4‐dioxin acenaphthylene compounds, 1, 2 and 3, by reaction with singlet‐oxygen (¹O₂) in dichloromethane. Evidence for the formation of the dioxetanes 1a, 2a and 3a is provided by the chemiluminescence (CL) that corresponds to the emission from the electronically excited diesters 1b*, 2b* and 3b*, which are decomposed thermally from the dioxetanes 1a, 2a and 3a, respectively. The highly strained 1,2‐dioxetane ring decomposes from a twisted geometry by simultaneous cleavages of the O–O and C–C bonds, producing the electronically excited diester that emits CL. It was observed that the CL from compound 2a is red‐shifted relative to that of compounds 1a and 3a suggesting a higher degree of stabilization for the excited state by the electron‐donating methoxy group. Also, a study of the solvent effect on fluorescence shows a significant red‐shift in compound 2b, indicating a more polar excited state. The kinetics of the thermal decomposition of the 1,2‐dioxetanes clearly demonstrate that the CL characteristics of compound 2a are quite different from those of compounds, 1a and 3a. These results are consistent with the proposed intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism which is triggered by the electron‐donating group of compound 2a. Copyright © 2013 John Wiley & Sons, Ltd.     

A comparative study of the interactions of bleomycin with nuclei and purified DNA

Fe(III)-bleomycin associates strongly with rat liver nuclei and binds to nuclear DNA. Metal-free and Cu(II)-bleomycin, however, do not bind to nuclei. The treatment of nuclei with activated iron-bleomycin results in nucleic base and base propenal release from the DNA, and also gives membrane peroxidation. Isolation and quantitation of the base propenals and free bases released subsequent to activated bleomycin treatment reveal an alteration in the stoichiometry of these products compared to those released from purified DNA. With nuclei, significantly less propenal is formed, although the yield of free base is equivalent to that from purified DNA. The membrane peroxidation products from nuclei are the same as those obtained from microsomal membranes treated with activated bleomycin. Superoxide dismutase inhibits the membrane peroxidation but has no effect on the DNA breakage reactions. The results implicate a role for iron in mediating the in vivo action of bleomycin and also reveal a potentially toxic effect, membrane peroxidation, separate from DNA damage.     

On the reaction of iron bleomycin with thiols and oxygen.

Iron(III)bleomycin undergoes a redox reaction with thiols. Evidence from epr and UV-visible absorbance spectra indicate that the metal complex forms an intermediate with sulfhydryl groups presumably by adding a sixth ligand. In the presence of oxygen iron bleomycin cycles between its two oxidation states to catalyze the generation of oxygen free radicals using thiols as a source of electrons.