Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.     

Deglyco-peplomycin metal complexes on DNA fibers: a role of the sugar moiety for the stability and the orientation of the complexes

Binding structures of metal complexes of deglyco-peplomycin (dPEP) on DNA were investigated by comparing dPEP complexes with those of bleomycin (BLM) using DNA fiber EPR spectroscopy. A low spin species of Fe(III)dPEP observed in the DNA pellet changed irreversibly to several high spin species after the fabrication of the DNA fibers. The g values of the high spin species were different from those of Fe(III)BLM. The high spin species could not be nitrosylated reductively to ON-Fe(II)dPEP, suggesting that some nitrogen atoms coordinated to the Fe(III) were displaced on the DNA fibers. On the other hand, O(2)-Co(II)dPEP remained intact on the fibers similarly to O(2)-Co(II)BLM but with an increased randomness in the orientation on the DNA. In contrast to Cu(II)BLM, a considerable amount of Cu(II)dPEP bound almost randomly on B-form DNA fibers. These results indicated that the sugar moiety in peplomycin or bleomycin is playing an important role in enhancing the stability of the metal-binding domain and in the stereospecificity of the binding on DNA.     

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.     

Copper(I) . bleomycin. A structurally unique oxidation-reduction active complex

Cu(I) and Cu(II) form stable 1:1 complexes with bleomycin (BLM). The affinity of both metals for the drug is greater than that of Fe(II). Cu(I) . BLM A2 binds to calf thymus DNA with about the same affinity as Fe(II) . BLM, as judged by DNA-induced fluorescence quenching of the bithiazole moiety of BLM. Based on 1H NMR and potentiometric titration data, the Cu(I) complexes of BLM are shown to have geometries very different than those of other BLM . metal(II) complexes studied thus far. As Cu(I) . BLM is oxidation-reduction active, its geometry is of importance in defining the structural requirements for BLM activity.     

Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding

The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.     

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.     

Copper-dependent cleavage of DNA by bleomycin

DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixture of Cu(I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)--bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)--bleomycin to Cu(I)--bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuproine when the experiment was carried out under conditions consistent with Cu chelation by bathocuproine on the time scale of the experiment. Remarkably, it was found that the extent of DNA degradation obtained with bleomycin in the presence of Fe and Cu was greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in sites of DNA cleavage and in the extent of cleavage at sites shared in common. For deglycoblemycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itself, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)--bleomycin.     

A role for ferrous ion and oxygen in the degradation of DNA by bleomycin.

An interaction between bleomycin and low concentrations of Fe(II) in the degradation of DNA is reported. Complete conversion of simian virus 40 DNA to acid-soluble products occurs at approximately equimolar levels of Fe(II), bleomycin, and DNA; Fe(III) does not substitute for Fe(II) in this reaction. Anaerobiosis inhibits the observed DNA degradation by bleomycin and Fe(II). Optical spectral studies reveal that an oxygen-labile complex is formed between bleomycin and Fe(II).     

Effect of chelating agents and metal ions on the degradation of DNA by bleomycin.

The degradation of DNA by bleomycin was studied in the absence and in the presence of added reducing agents, including 2-mercaptoethanol, dithiothreitol, reduced nicotinamide adenine dinucleotide phosphate, H2O2, and ascorbate, and in the presence of a superoxide anion generating system consisting of xanthine oxidase and hypoxanthine. In all cases, breakage of DNA was inhibited by low concentrations of chelators; where examined in detail, deferoxamine mesylate was considerably more potent than (ethylenedinitrilo)tetraacetic acid. Iron was found to be present in significant quantities in all reaction mixtures. Thus, the pattern of inhibition observed is attributed to the involvement of contaminating iron in the degradation of DNA by bleomycin. Cu(II), Zn(II), and Co(II) inhibit degradation of DNA by bleomycin and Fe(II) in the absence of added reducing agents. A model is proposed in which the degradation of DNA in these systems is dependent on the oxidation of an Fe(II)-bleomycin-DNA complex.     

Redox potential of the active bleomycin-Fe(III) complex by cyclic voltammetry

The redox potential of the active Fe(III) complex of bleomycin (BLM), which is a DNA cleaving species, was measured by cyclic voltammetry at 25 °C under a hydrogen atmosphere. The cyclic voltammogram showed the reversible one-electron Fe(III)/Fe(II) coupled redox reaction at −0.225 V versus SCE. Under the same conditions the redox potentials of the iso-BLM—Fe(III) complex and the deglyco-BLM—Fe(III) complex were also observed, but the cyclic voltammogram for the inactive Fe(III) complex of BLM could not be obtained.     

Suppression of somatostatin levels in the hepatic portal and systemic plasma of the rat by synthetic human pancreatic polypeptide.

The 1:1 Cu(II), Co(II), Co(II)-O₂, Fe(II)-NO, and Fe(III) complexes of depBLM have been investigated by ESR spectroscopy and compared with the corresponding metal complexes of BLM. DepBLM which lacks the α-amino group of β-aminoalanine portion in BLM molecule, forms the metal complexes different from BLM with regard to the fifth axial donor. In addition, the formation of hydroxyl radical by the depBLM-Fe(II) complex is remarkably lower than that by the BLM-Fe(II) complex. This study indicates an important effect of fifth axial nitrogen on metal coordination and oxygen activation of BLM.     

Effect of metals on nucleoside hydroperoxide, a product of ionizing radiation in DNA

Ionizing radiation causes formation of thymine hydroperoxides in DNA. Their decomposition generates more stable products and active oxygen species which may oxidize other DNA bases. We have determined the effects of free and chelated metal ions on the degradation of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU). Two products were formed as analyzed by HPLC: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU). Sn(II) and Fe(II) caused instantaneous HPMdU degradation; Sn(II) generated only HMdU, whereas Fe(II) formed about equal amounts of both. Sn(IV) and Fe(III) were inactive. Cu(I), Cu(II), and Co(II) caused a time-dependent formation of both products, with FdU predominating. In the presence of Cu(I), Cu(II), and Fe(II), formate inhibited formation of HMdU but enhanced that of FdU. EDTA abolished Cu(I)-induced decomposition of HPMdU but only decreased that which was mediated by Cu(II). In contrast, EDTA enhanced the activity of Fe(III) with a time-dependent formation of FdU. EDTA and diethylenetriaminepentaacetic acid (DTPA) caused an instantaneous Fe(II)-mediated decomposition of HPMdU to FdU. Only desferal partially inhibited the activity of Fe(II), whereas the activities of Cu(I), Cu(II), and Fe(III) were blocked by desferal and DTPA. Possible mechanisms of HPMdU degradation by metal ions in the absence or presence of formate or chelators as well as formation of the .OH are discussed.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.     

Synergistic effects of flavonoids and ascorbate on enhancement in DNA degradation induced by a bleomycin-Fe complex

Flavonoids were examined for synergistic effects with ascorbate on enhancement of DNA degradation induced by a bleomycin(BLM)-Fe complex. The synergistic effects of flavonoids and ascorbate on DNA degradation induced by the BLM-Fe complex were observed to be greater with flavonoids such as isorhamnetin, kaempferol and morin, which accelerated oxidation more markedly in the presence, than in the absence of BLM. Conversely, myricetin and fisetin, which showed oxidation barely accelerated by the addition of BLM, inhibited DNA degradation promoted by ascorbate. Consequently, there was a good correlation between oxidation of flavonoids accelerated by BLM and the extent of DNA degradation promoted synergistically with ascorbate. Our previous studies indicated that oxidation of flavonoids accelerated by BLM and DNA degradation promoted by flavonoids were not correlated with Fe(III)-reducing activity of flavonoids. Those results suggest that Fe(III)-reducing activity of flavonoids is not the only factor determining DNA degradation-promoting activity induced by the BLM-Fe complex. On the other hand, in a Fenton reaction, degradation of 2-deoxy-d-ribose promoted by flavonoids was correlated to the Fe(III)-reducing activity of flavonoids. However, there was not a synergistic interaction between flavonoids and ascorbate in the degradation of 2-deoxy-d-ribose. Therefore, it is suggested that the synergistic DNA degradation caused by flavonoids and ascorbate in the BLM-Fe redox cycle arose from the difference in the reductive processes in which flavonoids and ascorbate mainly act.     

Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).     

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.     

Iron-bleomycin-deoxyribonucleic acid system. Evidence of deoxyribonucleic acid interaction with the alpha-amino group of the beta-aminoalanine moiety

The Fe(III) complex of bleomycin (BLM) is, at pH 4, in the high-spin form. At pH 7, the coordination of the alpha-amino group of the beta-aminoalanine moiety of BLM converts it to a low-spin species: BLM X Fe(III) X alpha NH2. The conversion of the high-spin species to the low-spin one can also take place at pH 4 (i) by addition of ligands L such as N3-, S2O3(2-), and SCN- or (ii) through interaction with DNA. Moreover, the addition, at pH 7, of DNA to BLM X Fe(III) that has been previously complexed with one of these ligands L displaces this latter from its position. These results suggest that (i) the ligand L occupies the same site of coordination as the alpha-amino group and (ii) an interaction occurs between the beta-aminoalanine moiety of BLM and DNA that lowers the pKd of the alpha-amino group, promoting its coordination to iron.     

Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

The unwinding of plasmid DNA by bleomycin A2 (BLM A2) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu2+ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A2 at concentrations where cupric ions alone had no effect on DNA supercoiling. The concentration of BLM A2 required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu2+. A plot of [Cu2+] vs extent of DNA unwinding in the presence of 10(-4) M BLM A2 gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu2+ + BLM demethyl A2, the latter of which differs from BLM A2 only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II).BLM demethyl A2 was found to be much less effective than Cu(II).BLM A2 as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.     

Metal coordination core of bleomycin : comparison of metal complexes between bleomycin and its biosynthetic intermediate.

On the basis of electron spin resonance results, the 1:1 Cu(II), Co(II), Co(II)-O₂, and Ni(III) complexes of bleomycin(BLM) have been compared with the corresponding metal complexes of its biosynthetic intermediate(P-3A). The present study suggests that (1) P-3A is an useful ligand for the clarification of metal-binding sites of BLM; (2) the secondary amine, pyrimidine ring nitrogen, deprotonated peptide nitrogen of histidine residue, and histidine imidazole groups as planar ligand donors, and the α-amino group as axial donor, are substantially important for metal-coordination of BLM; and (3) the sugar and bithiazole portions of BLM probably contribute to stabilization of Co(II)-O₂ adduct complex and axial sixth coordination of Cu(II) and Ni(III) complexes.     

Deglyco-bleomycin. Degradation of DNA and formation of a structurally unique Fe(II) . CO complex

In analogy with bleomycin, deglyco-bleomycin B2 has been found to form a stable, diamagnetic complex with Fe(II) and CO. Although the stoichiometry of this complex appeared to be the same as that formed with bleomycin, the geometry of the deglyco-bleomycin complex was fundamentally different, especially as regards orientation of the beta-aminoalanine moiety. In the presence of Fe(II) and O2, deglyco-bleomycin A2 and deglyco-bleomycin B2 were found to release [3H]thymine from radiolabeled PM-2 DNA; when employed at limiting concentrations, deglyco-bleomycin A2 and B2 gave about half as much [3H]thymine release as the respective bleomycins. In view of the spectral evidence (Burger, R. M., Horwitz, S. B., Peisach, J., and Wittenberg, J. B. (1979) J. Biol. Chem. 254, 12299-12302) that Fe(II) . bleomycin . CO has the same geometry as the complex formed by initial association of bleomycin, Fe(II), and O2, the accumulated data suggest strongly that all metal complexes of bleomycin (derivatives) capable of DNA degradation need not have the same geometry.