Robert W. Byrnes

The role of redox-active metals in the mechanism of action of bleomycin

Belomycin is a glycopeptide antibiotic routinely used to treat human cancer. It is commonly thought to exert its biological effects as a metallodrug, which oxidatively damages DNA. This review systematically examines the properties of bleomycin which contribute to its reaction with DNA in vitro and may be important in the breakage of DNA in cells. Because strand cleavage results from the reductive activation of dioxygen by metallobleomycins, the mechanism of this process is given primary attention. Current understanding of the structures of the coordination sites of various metallobleomycins, their thermodynamic stabilities, their propensity to form adduct species, and their properties in ligand substitution reactions provide a foundation for consideration of the chemistry of dioxygen activation as well as a basis for thinking about the metal-speciation of bleomycin in biological systems. Oxidation-reduction pathways of iron-bleomycin, copper-bleomycin, and other metal-bleomycin species with O2 are then examined, including information on photochemical activation. With this background, structural and thermodynamic features of the binding interactions of DNA with bleomycin, its metal complexes, and adducts of metallobleomycins are reviewed. Then, the DNA cleavage reaction involving iron-bleomycin is scrutinized on the basis of the preceding discussion. Particular emphasis is placed on the constraints which the presence of DNA places on the mechanism of dioxygen activation. Similarly, the reactions of other metalloforms of bleomycin with DNA are reviewed. The last topic is an analysis of current understanding of the relationship of bleomycin-induced cellular DNA damage to the model developed above, which has evolved on the basis of chemical experimentation. Consideration is given to the question of the importance of DNA strand breakage caused by bleomycin for the mechanism of cytotoxic activity of the drug.

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.

Dna strand breakage in isolated nuclei subjected to bleomycin or hydrogen peroxide

The sources of iron (Fe) and reductant required for DNA strand breakage by the antitumor drug bleomycin (Blm), H2O2 and ascorbate were investigated using nuclei instead of whole cells in order to study a simpler, related system that was subject to better control and easier chemical manipulation. Ehrlich ascites tumor cells were isolated and treated directly on filters, and analysed for DNA damage by alkaline and nondenaturing elution. Extraction and treatment buffers were depleted of trace Fe by passage through Mg(OH)2 gel. Nuclei were treated for 1 hr at 37 degrees. High levels of single- and double-strand breakage were obtained using Fe(III)Blm in the range 0.01 to 0.08 microM. In contrast, Blm was effective only at two orders of magnitude greater concentration. Cu(II)Blm was totally ineffective in causing damage. Depletion of nuclear protein thiols with N-ethylmaleimide reduced double-strand breakage at the upper end of the FeBlm concentration-response curve. A 1 mM concentration of NADPH or NADH greatly increased the extent of double-strand breakage by 0.01 microM FeBlm, suggesting roles for cytochrome P450 or cytochrome b5 reductase in strand breakage. Fe(III)ATP (1:20 metal to ligand and 50 microM in Fe) and Fe(III)EDTA (1:2 metal to ligand and 50 microM in Fe) did not cause single-strand breaks. In the absence of added Fe, H2O2 or ascorbic acid (50 microM) caused less than one Gy-equivalent single-strand breakage. Addition of ascorbate plus Fe(III)ATP or Fe(III)EDTA produced breakage beyond the capacity of alkaline elution to analyse (5-6 Gy). Overall, the results indicate that Fe, which may contribute to DNA damage by Blm and forms of activated oxygen within cells, is not strongly bound in the nucleus and that nuclear thiols other than glutathione contribute reducing equivalents to Fe(III)Blm for the DNA damaging chemistry.

Protection of DNA in HL-60 cells from damage generated by hydroxyl radicals produced by reaction of H2O2 with cell iron by zinc-metallothionein.

Scavenging of hydroxyl radicals (.OH) by the zinc form of metallothionein (ZnMT) was studied in HL-60 cells and in nuclei from such cells previously treated with ZnCl2 (ZnMT cells). Cells were grown for 48 h to label DNA for alkaline elusion experiments. During the last 24 h 0.1 mM ZnMT was included to induce ZnMT. Generation of DNA single-strand breaks (SSBs) by H2O2 in cells (5 x 10(5)/ml) treated at 4 degrees was increased by approximately 70% in Zn-treated cells by comparison with control cells. These cells had grown from an initial concentration of 5 x 10(5)/ml to a concentration at harvest of 16 x 10(5)/ml. Cells started at 6 x 10(5)/ml and growing to a final concentration of 20 x 10(5)/ml did not exhibit a similar increase in SSBs. This elevation in SSBs was traced to an increase in cell Fe content which exhibited a sharp dependence upon concentrations of cells and of ZnCl2 at the time of addition. The diffusion distance (d) from Fe to DNA of ZnMT cells treated with H2O2 was found to be 3.4 nm. This compares with a distance of 6.1 nm in control cells. SSB generation by hydroxyl radicals formed by 137Cs-gamma rays in Zn-treated cells decreased by 12%, accompanied by a decrease in d from 4.8 nm to 2.9 nm. Thus, ZnMT preferentially reacts with OH formed at some distance from DNA. In nuclei isolated from ZnMT cells started at 5 x 10(5)/ml, SSB generation by H2O2 increased by 60%. The d in these nuclei was 4.9 nm, similar to the distance in control nuclei reported previously. These data suggest that, in addition to altering the scavenging environment, treatment of cells with Zn leads to an increase in reactive Fe in cells and in isolated nuclei which can generate DNA damage through reaction with H2O2.

Inhibition of bleomycin-induced cellular DNA strand scission by 1,10-phenanthroline

Inhibition by 1,10-phenanthroline of cellular DNA strand scission induced by the antitumor antibiotic bleomycin in Ehrlich ascites tumor cells was studied. DNA alkaline elution was performed on cells after 1-hr bleomycin treatments. Pretreatment for 24 hr with initial 1,10-phenanthroline concentrations of 0.2 nmol/10(5) cells, which depletes cells of ferritin iron by 80%, had no consistent effect on bleomycin strand breakage. However, simultaneous treatment with 3.1 nmol of 1,10-phenanthroline/10(5) cells and with bleomycin concentrations from 5 to 25 microM decreased both apparent double-stranded breaks and random breakage. When cells were treated with both 3.1 nmol of 1,10-phenanthroline/10(5) cells and 25 microM bleomycin, washed free of both drugs, and incubated at 35 degrees for 1 hr, the resulting breakage was equivalent to that found in cells treated with bleomycin only. When the combination treatment was extended to 4 hr, cell washing and reincubation resulted in increased strand scission, as compared with strand scission in cells treated with bleomycin only. Growth inhibition by bleomycin was not affected appreciably by temporary suppression of DNA strand breakage activity.

Evidence for involvement of multiple iron species in DNA single-strand scission by H2O2 in HL-60 cells

Some of the properties of cellular iron species which react with H2O2 to cause DNA single-strand breaks in HL-60 cells were characterized in control cells and in cells made deficient of iron using 4,7-phenylsulfonyl-1,10-phenanthroline (bathophenanthroline disulfonic acid or BPS) and ascorbate. Single-strand breaks were measured using alkaline elution of DNA of cells treated at 4 degrees to minimize repair during treatment. Strand breakage in the presence of 10% serum was only 40% of that in the absence of serum. This effect was traced to reaction of H2O2 with metals, most likely iron, in serum. Dimethyl sulfoxide (Me2SO) inhibited a maximum of 65% of breaks in control cells. The diffusion distance from the site of generation of hydroxyl radicals to the site of reaction with DNA for the Me2SO-inhibitable fraction was 6.9 nm. There was no significant alteration in the fraction of Me2SO-inhibitable strand breaks or in diffusion distance in iron-deficient cells, though total strand breaks decreased by 70%. When the effect of extracellular iron in serum was taken into account, 60 microM orthophenanthroline (OP) inhibited a maximum of 85% of strand breaks. In cells pretreated with 60 microM OP, the Me2SO-inhibitable fraction of the remaining strand breaks decreased to 32%, while the diffusion distance decreased to 4.1 nm. These data indicate the existence of a number of different iron species, as characterized by overlapping but not coincidental inhibition by OP and Me2SO, and by differing diffusion distances.

Repair of bleomycin-induced DNA double-strand breakage in Ehrlich ascites tumor cells.

The effect of 1,10-phenanthroline (OP) on repair of bleomycin (Bleo)-induced double-strand breaks in Ehrlich ascites tumor cells was studied using nondenaturing filter elution. 1,10-Phenanthroline is a metal chelator which is believed to inhibit strand breakage by Bleo through competition for intracellular iron. Cells were treated with 25 microM Bleo for 1 h, washed free of unincorporated drug, and then reincubated in the absence or presence of OP. In the absence of OP, relative elution (with respect to cells irradiated with 50 Gy and used as an internal standard) decreased in a first-order process with a half-time for repair of 2.4 h. 1,10-Phenanthroline at 10 nmol/10(5) cells (50 microM) accelerated the net decrease in relative elution, producing a biphasic response with half-times of 5.3 h and less than 30 min for the two components. Thus functionally active Bleo remained in cells after they were washed free of unincorporated drug. 1,10-Phenanthroline at a concentration of 3.1 nmol/10(5) cells did not result in a similar net acceleration of repair of double-strand breakage, though it increased the rate of repair of single-strand breakage as measured by alkaline elution. The differences in repair observed in response to different OP concentrations are discussed in terms of models for double-strand breakage by Bleo. After 4-5 h repair, relative elution from Bleo-treated cells remained at about 40% of that achieved at the end of 1-h Bleo treatment in either the presence or absence of the 10 nmol OP/10(5) cells, demonstrating that some double-strand breaks were resistant to repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Requirement of endogenous iron for cytotoxicity caused by hydrogen peroxide in Euglena gracilis

Abstract It is widely thought that redox-active metals in cells such as iron or copper catalyze the reduction of hydrogen peroxide to toxic hydroxyl radicals or their equivalent. However, this has not been directly demonstrated in vivo . To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intracellular iron content can be modulated by the concentration of iron in the defined growth medium without effect on the proliferation rate of the cells. E. gracilis contains two large storage pools of cytosolic iron which can be monitored to assess cellular iron status. The toxicity of H 2 O 2 in E. gracilis was inversely related to the amount of iron in the extracellular medium. At high levels of external iron, the metal carried out the Fenton reaction with H 2 O 2 outside the cell, producing hydroxyl radicals as detected by electron-spin resonance spin-trapping experiments. This reaction reduced the amount of H 2 O 2 that could diffuse into cells to cause toxicity. When cells with different intracellular iron content were placed in iron-deficient media, the the toxicity of H 2 O 2 , measured by inhibition of proliferation, was directly related to the concentration of internal iron. Using cells deficient in iron, this oxidant did not inhibit proliferation at low concentration but was somewhat effective at higher concentrations. Although the iron chelating agents, 1,10-phenanthroline and desferrioxamine, also depressed cytosolic iron, they were cytotoxic to E. gracilis and so could not be used unambiguously to examine the role of intracellular iron in the toxic effects of hydrogen peroxide.

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

Diffusion distances of known iron complexes in model systems.

Diffusion distances (abbreviated ds), the distances between the sites of generation of presumed hydroxyl radicals (*OH) by low molecular weight forms of Fe and the site of their reaction with substrate, were measured for three model systems for cellular DNA of varying degrees of complexity. Two ds for Fe complexed with each of ethylene diamminetetraaccetic acid (FeEDTA) and nitrilotriacetic acid (FeNTA) were measured for generation of malondialdehyde-type products (MDA) from deoxyribose and of single-strand breaks (SSBs) in the plasmid pBR322. The closer ds for pBR322 SSB generation (5-6 nm) were considerably greater than the ds for MDA generation in the deoxyribose assay (2-3 nm). This is consistent with charge-charge interactions playing an important role in defining d. The ds for FeNTA, FeEDTA, and other Fe species generating SSBs in isolated Ehrlich ascites tumor cell nuclei ranged from 2.1 to 14 nm. Charge-charge interactions, Fe-ligand-specific interactions, and binding to nuclear components were concluded to be important factors affecting d in isolated nuclei. Other factors related to nuclear structure may also play a role.