Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions

The hypothesis was investigated that axial ligands bound to Fe(III)-bleomycin [Fe(III)Blm] are destabilized at specific 5'-guanine-pyrimidine-3' binding sites but are stable at nonselective dinucleotides. DNA oligomers and calf-thymus DNA were used in reactions with L-Fe(III)Blm, where phosphate and cyanide served as examples of large and small ligands (L). Both ligands underwent dissociation when L-Fe(III)Blm was bound to d(GGAAGCTTCC)2 (I) but not d(GGAAATTTCCC)2 (II) and at large ratios of calf-thymus DNA to drug. Fe(III)Blm is high spin in 20 mM phosphate buffer, signifying the presence of a phosphate adduct. In the titration of HPO4-Fe(III)Blm with calf-thymus DNA, a large excess of DNA was needed to reach the low-spin state, consistent with an equilibrium competition between phosphate and DNA for Fe(III)Blm. Equilibrium constants for binding Fe(III)Blm and CN-Fe(III)Blm to calf-thymus DNA (6.8x10(5) M(-1) and 5.9x10(4) M(-1), respectively, in HEPES buffer at 25 degrees C and pH 7.4) showed that the CN- ligand also reduced the affinity of DNA for the drug. The kinetics of dissociation of CN- from CN-Fe(III)Blm-DNA were slow and first order in bound drug. The reversible nature of these dissociation reactions was shown using 1H NMR spectroscopy of Fe(III)Blm-I in the absence and presence of large excesses of CN- or phosphate. The results are discussed in terms of a two-state hypothesis for the binding of L-Fe(III)Blm to specific and nonspecific dinucleotides. It is proposed that steric restrictions at specific sites inhibit binding of these ligands.     

The reaction of ferric- and ferrous salts with bleomycin.

1. A comparative study shows that ferrous ions give a much better yield of Fe(III)-bleomycin than ferric ions, when iron salt is added to bleomycin in a buffer solution (pH 7.2). 2. The amount of Fe(III)-bleomycin formed after addition of ferric ions was markedly increased in the presence of ferric ion binding compounds (BSA, citrate) or reducing agents (ascorbate, cysteine).     

Microsome-stimulated activation of ferrous bleomycin in the presence of DNA

The inhibition of Fe(II)-bleomycin activation, by a large excess of DNA, is overcome by rat liver microsomes in the presence of NADPH. This release of inhibition, as indicated by increased yields of base propenal from DNA scission, is enhanced by menadione, is inhibited by superoxide dismutase, and is therefore dependent on superoxide anion. Microsomal activation of Fe(II)-bleomycin doubles the stoichiometry of base propenal yield compared to that obtained upon self-activation of the drug; 0.5 mol of base propenal is formed and 0.5 mol of NADPH is oxidized per mol of Fe(II)-bleomycin. In the presence of a large excess of DNA, Cu(II)-bleomycin is not reduced and Fe(III)-bleomycin is neither reduced nor activated by microsomes in cases where activation of Fe(II)-bleomycin is maximal. We suggest that in vivo, electron transport enzymes at or near the nucleus can stimulate the activation of Fe(II)-bleomycin under conditions where self-activation does not readily occur.     

Bleomycin may be activated for DNA cleavage by NADPH-cytochrome P-450 reductase

In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.     

OH radical formation by photolysis of aqueous porphyrin solutions. A spin trapping and e.s.r. study

Radical production during the photolysis of deaerated aqueous alkaline solutions (pH 11) of some water-soluble porphyrins was investigated. Metal-free and metallo complexes of tetrakis (4-N-methylpyridyl)porphyrin (TMPyP) and tetra (4-sulphonatophenyl)porphyrin (TPPS4) were studied. Evidence for the formation of OH radicals during photolysis at 615, 545, 435, 408 and 335 nm of Fe(III) TPPS4 is presented. Fe(III) TMPyP, Mn(III) TPPS4 and Mn(III) TMPyP also gave OH radicals but only during photolysis at 335 nm. The method of spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN) combined with e.s.r. was used for the detection of OH, H and hydrated electrons. With the spin trap DMPO, photolysis generated DMPO-OH adducts under certain conditions but no DMPO-H adducts could be observed. With POBN, no POBN-H adducts were found. The formation of OH was confirmed by studying competition reactions for OH between the spin traps and OH scavengers (formate, isopropanol) and the concomitant formation of the CO-2 adduct and the (CH3)2COH adduct with both DMPO and POBN. The photochemical generation of OH radicals was pH dependent; at pH 7.5 no OH radicals could be detected. Photolysis (615-335 nm) of dicyanocomplexes of the Fe(III) porphyrins did not produce OH radicals. When corresponding Cu(II), Ni(II), Zn(II) and metal-free porphyrins were photolysed at 615 and 335 nm, no OH radicals could be spin trapped. These results tend to associate the well-known phenomenon of photoreduction of Fe(III) and Mn(III) porphyrins with the formation of OH radicals. This process is described mainly as the photoreduction of the metal ion by the ligand-bound hydroxyl ion via an intramolecular process.     

Stimulatory effect of phosphate, ATP and ADP on iron transfer from Fe(III)-bleomycin to apotransferrin

1. The rate of ferric ion transfer from Fe(III)-bleomycin to apotransferrin was increased in the presence of orthophosphate, ATP and ADP, while AMP was without effect. 2. Ortho phosphate activation probably involves formation of a Fe(III)-bleomycin-phosphate complex. The optical absorption of Fe(III)-bleomycin at 450 nm is enhanced in the presence of phosphate. 3. ATP and ADP remove the ferric ion from the iron-drug complex; thus making the ferric ion readily available for uptake by apotransferrin. 4. Low concentrations of ATP, ADP and AMP, also enhance the 450 nm absorption of the iron-drug complex. Higher ATP and ADP concentrations reduce both the 450 and 384 nm absorption of Fe(III)-bleomycin.     

Stimulatory effect of ascorbate on iron transfer from bleomycin to apotransferrin

The chemotherapeutic agent, bleomycin, forms a 1:1complex with both Fe(III) and Fe(II). The rate offerric ion transfer from bleomycin toapotransferrin is rather slow. However, when ascorbate was added toFe(III)-bleomycin priorto exposure to apotransferrin, the transfer rate was markedly increased. Ascorbatereadilyreduces Fe(III)-bleomycin to Fe(II)-bleomycin. A second order rate constant of 2.4 mM min wasestimated for this reaction. Fe(II)-bleomycinimmediately combines with O 2 , generating the so-called'acti-vatedbleomycin' complex. The data suggest that a reduced form of iron-bleomycin more readilydonatesits iron ion to apotransferrin. Reoxidation of ferrous ions, andFe(III)-transferrin formation occur rapidly.     

Spin trapping study on the kinetics of Fe2+ autoxidation: formation of spin adducts and their destruction by superoxide.

The oxidation of Fe2+ was investigated by electron spin resonance spin trapping techniques with N-t-butyl-alpha-phenylnitrone (PBN) and dimethyl sulfoxide. Under pure oxygen, the spin adduct PBN/.OCH3 was rapidly generated by the addition of Fe2+ (0.2-1.2 mM) into phosphate buffer containing ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide, and PBN at pH 7.4, but it decayed. The decay process of PBN/.OCH3 consists of two components. The fast decay was dependent on Fe2+ concentration. Another was due to destruction of the spin adduct by superoxide anion (.O2-), because superoxide dismutase (SOD) markedly prevented the decay. Catalase decreased the yield of PBN/.OCH3. When EDTA was replaced by diethylenetriaminepentaacetic acid (DTPA), both the generation and decay process of PBN/.OCH3 were slow. SOD and catalase effects were similar to those in EDTA. Fe2+ produced PBN/.OCH3 even in the absence of chelators. We could estimate the kinetic parameters by computer simulation, comparing the Fe2+ oxidation in EDTA with that in DTPA. These results demonstrate that Fe2+ reacts with O2 to generate .O2- and then H2O2, which produces .CH3 by reaction with Fe2+ and dimethyl sulfoxide.(.)OCH3 results from the reaction between .CH3 and O2. The adduct PBN/.OCH3 decays by reaction with Fe2+ and .O2-.     

Interaction of citrate with Fe(III)-bleomycin

1. Citrate binds to Fe(III)-bleomycin, removing the ferric ion from the iron-drug complex; a reaction that may be of physiological significance. 2. Low concentrations of citrate markedly enhance the rate of iron transfer from Fe(III)-bleomycin to apotransferrin; an iron binding plasma protein.     

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.     

Role of quinone-iron(III) interaction in NADPH-dependent enzymatic generation of hydroxyl radicals.

To study the effect of chelation of iron ions by quinones on the generation of OH radicals in biological redox systems, we have synthesized quinones that can form complexes with Fe(III) ions: 2-phenyl-4-(butylamino)naphtho[2,3-h]quinoline-7,12-dione (Qbc) and 2-phenyl-4-(octylamino)naphtho[2,3-h]quinoline-7,12-dione (Qoc). A quinone with a similar structure without chelating group was synthesized as a control sample: 2-phenyl-5-nitronaphtho[2,3-g]indole-6,11-dione (Qn). Using optical spectroscopy, we determined the stability constant of Qbc with Fe(III) [Ks = (7 +/- 1) x 10(18) M-3] and the stoichiometry of the complex Fe(Qbc)3 in chloroform solutions. One-electron reduction potentials of Qbc, Qn, and adriamycin in dimethyl sulfoxide were measured by cyclic voltammetry. In the presence of Fe(III) the one-electron reduction potentials shifted toward positive values by 0.16 and 0.1 V for Qbc and adriamycin, respectively. Using the spin trap 5,5'-dimethyl-1-pyroline N-oxide (DMPO) and EPR, it was found that Qbc in the Fe(III) complex stimulated the formation of OH radicals in the enzymatic system consisting of NADPH and NADPH-cytochrome P-450 reductase more efficiently than adriamycin and quinone Qn. This is indicated by the absence of a lag period in the spin adduct appearance for Qbc and by a significantly higher rate of the spin adduct production, as well as by a larger absolute concentration of the spin adduct obtained for Qbc in comparison with Qn in the presence of Fe(III).(ABSTRACT TRUNCATED AT 250 WORDS)     

P-3A and (−)-desacetamido P-3A: demonstration and Study of their effective functional cleavage of duplex DNA

A study of the Fe(II) complexes of P-3A (1) and (−)-desacetamido P-3A (2) abilities to cleave duplex DNA was conducted through examination of single-strand and double-strand cleavage of supercoiled φX174 RFI DNA (Form I) in the presence of O₂ to produce relaxed (Form II) and linear (Form III) DNA, respectively. Like Fe(II)-bleomycin A₂ and deglycobleomycin A₂, Fe(II)-1 and 2 effectively produced both single- and double-strand cleavage of supercoiled φX174 DNA. Unlike Fe(II)-bleomycin A₂ or deglycobleomycin A₂, Fe(II)-1 and 2 were found to cleave duplex w794 DNA with no discemible sequence selectively suggesting that the polynucleotide recognition of the C-terminus tetrapeptide S subunit of the bleomycins including the bithiazole may dominate the bleomycin A₂ DNA cleavage selectivity.     

M?ssbauer, EPR and NMR studies of the acid-induced reduction and changes in spin state of ferric bleomycin

Iron-57 M?ssbauer, electron paramagnetic resonance (EPR) and H-1 nuclear magnetic resonance (NMR) studies of iron-bleomycin complexes in the pH range from 1.0 to 6.0 are reported. Sequential protonation of the ligands produces a variety of high-spin and low-spin complexes of the metal. Of particular interest is the reversible equilibrium between Fe(III)- and oxygen-stable Fe(II)-bleomycin. Below pH 3.5 Fe(II) complexes form, with maximal reduction occurring at approximately pH 2. At still lower pH, Fe(III) complexes unassociated with bleomycin become dominant. The observed reduction in the absence of exogenous reducing agents suggests the possible involvement of intramolecular autoreduction in bleomycin-mediated DNA degradation.     

The orientation of the ligands in iron(III)-bleomycin intercalated with DNA

EPR data show that Fe(III)-bleomycin intercalates with DNA, or that the Fe(III) coordination sphere has a fixed geometrical configuration with respect to the DNA helical axis. An analysis of the data from oriented DNA fibers, drawn from a viscous gel, shows that the angle between the fiber axis and the normal to a plane containing the Fe(III) ion and ligands ranges between 15 and 30 degrees. The principal g values for the low-spin Fe(III)-bleomycin-DNA complex at pH 7.5 are 2.45, 2.18 and 1.87     

Spin-trapping studies of the oxidation-reduction reactions of iron bleomycin in the presence of thiols and buffer.

The reaction of ferrous bleomycin with dioxygen is reexamined to clarify whether radical species derived from molecular oxygen are generated. Detection of low levels of spin-trapped oxyradicals confirm the production of OH during this reaction when bleomycin is present in excess, but not when iron and drug concentrations are equal. In phosphate buffer, hydroxyl radicals continue to be spin trapped for at least 15 min after Fe(II)bleomycin has been oxidized to Fe(III)bleomycin. In HEPES buffer, detection of a HEPES radical in the absence of spin trap over the same period independently supports the conclusion that reactive radicals are present after the initial oxidation of Fe(II)bleomycin is complete. When glutathione is included in the aerobic reaction mixture, thiyl radical species are spin trapped. The reaction of Fe(III)bleomycin with cysteine produces thiyl radical without spin-trapped hydroxyl radical.     

Oxygen-based free radical generation by ferrous ions and deferoxamine

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH.) was suggested by the formation of the OH. spin trap adduct (DMPO/OH). with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH). adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH. scavengers, but not by superoxide dismutase. Iodination was optimal in 5 x 10(-4) M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10(-4) M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.     

Iron-bleomycin interactions with oxygen and oxygen analogues. Effects on spectra and drug activity.

Despite extensive structural dissimilarities, iron . bleomycin complexes and heme-containing oxygenases display remarkable similarities in binding oxygen antagonists and in spectral properties deriving from bound iron. Fe(II)-bleomycin reversibly forms a complex with either CO or isocyanide (lambda max = 384 and 497 nm, respectively), either of which interfere with its oxygen-dependent cleavage of DNA. A similar but paramagnetic complex forms with NO (lambda max = 470 nm; AN = 24 G). In contrast, cyanide enhances bleomycin activity against DNA. Complexes of bleomycin and FE(III), formed either by direct association or by autoxidation of the Fe(II) . bleomycin complex, exhibit indistinguishable EPR and visible spectra, which change characteristically with pH. At neutral pH, Fe(III) . bleomycin is a low spin complex (g = 2.45, 2.18, 1.89; lambda max = 365, 384 nm) and, at low pH, it is a high spin rhombic complex (geff = 9.4, 4.3; lambda max = 430 nm). These complexes are interconvertible (pK 4.3). Fe(II) . bleomycin oxidation, although reversible by spectral criteria, is accompanied by drug inactivation unless DNA is present.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

Ozone Degrades into Hydroxyl Radical under Physiological Conditions : A Spin Trapping Study

Defining the reactants is a critical step towards elucidating the mechanism of ozone toxicity to biomembranes. To document ozone-induced HO·radicals, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide was used and the resulting spin adduct was monitored with electron spin resonance spectroscopy. Chelexed potassium phosphate buffer (10 millimolar and 0.2 molar) at pH 7.2 and 7.8 was exposed to ozone (1-40 microliters per liter) by directing a stream of ozone over the surface for 60 seconds. Under these conditions, no HO· was detected. Using 0.5 × 10⁻⁴ molar caffeic acid in phosphate buffer, strong DMPO·OH electron spin resonance signals were obtained, indicating HO· production. Air controls yielded no signal. High pH (7.8) enhanced signal strength. Furthermore, with sorbitol (0.4 osmolal final concentration), a net HO· signal loss of 28% was observed, while a carbon-centered sorbitol radical adduct appeared. Although HO· radicals were produced, no breakage of Daucus carota protoplast plasma membranes was observed nor were differences in membrane fluidity observed as determined by 5-doxyl stearic acid.     

The orientation of the ligands in iron(III)-bleomycin intercalated with DNA

EPR data show that Fe(III)-bleomycin intercalates with DNA, or that the Fe(III) coordination sphere has a fixed geometrical configuration with respect to the DNA helical axis. An analysis of the data from oriented DNA fibers, drawn from a viscous gel, shows that the angle between the fiber axis and the normal to a plane containing the Fe(III) ion and ligands ranges between 15 and 30 degrees. The principal g values for the low-spin Fe(III)-bleomycin-DNA complex at pH 7.5 are 2.45, 2.18 and 1.87.