Biomedical subjects

W E Antholine

Publications and source records attributed to W E Antholine.

At least 55 records · Page 3Linked to original sources

PubMed1992

Reaction of DNA-bound Co(II)bleomycin with dioxygen.

The aerobic oxidation of Co(II)bleomycin bound to calf thymus DNA has been investigated in relation to the mechanism of reaction in solution in the absence of DNA. Kinetics of dioxygenation of the Co(II) complex were followed by spectrophotometric and electron spin resonance spectroscopy as well as dioxygen analysis. The reaction is slower than when carried out in solution; its rate is inversely related to the ratio of DNA base pairs to Co(II)bleomycin. The subsequent oxidation reaction, observed spectrophotometrically and by dioxygen analysis, is second order in cobalt complex. The calculated second order rate constant is also inversely related to the base pair to metal complex ratio. Once this ratio exceeds three, the reaction rate slows significantly with each additional increment of DNA added to the starting reaction mixture. Taking advantage of the high stability of O(2)-Co(II)bleomycin bound to greater than a 3-fold excess of DNA base pairs, it could be demonstrated that the rate constant for oxidation of two O(2)-Co(II)bleomycin molecules is much slower than that for O(2)-Co(II)bleomycin plus Co(II)bleomycin. With the same technique it was observed that the metal centers of O(2)-Co(II)bleomycin and Fe(II)bleomycin also undergo oxidation. The binding to DNA of both solution products of the oxidation of Co(II)bleomycin by O2 was examined by 1H NMR spectroscopy. Peroxy-Co(III)bleomycin, Form I, binds with higher affinity than Co(III)bleomycin, Form II. At lower ionic strength, the size of the DNA binding site for each form is about 2 base pairs/molecule of drug.

Animals↗

PubMed1992

The influence of gender on developing copper deficiency and on free radical generation of rats fed a fructose diet.

The present investigation was conducted to determine whether differences in copper and iron status between male and female rats can be detected during the development of copper deficiency. These differences may explain the protection of the female against the severity of copper deficiency. In addition, the livers of all rats were exposed to electron-spin resonance (ESR) spectroscopy for the presence of free radicals. Male and female rats were fed from weaning either copper-deficient or -adequate diets containing fructose for 31 days. Rats were killed at day 0, 8, 16, 24, and 31 of the study. Throughout the study, copper-deficient males exhibited the same organ copper concentrations as copper-deficient female rats. However, only in the male did copper deficiency cause a reduction in body weight and an increase in liver and heart sizes but a decrease in pancreas size. In contrast, organ iron concentrations were different between males and females. Only copper-deficient males were anemic. Only the livers of copper-deficient males showed the presence of free radicals. Although the livers of copper-deficient female rats exhibited higher concentrations of hepatic iron than their male counterparts, their livers did not show the presence of free radicals. The data of the present study suggest that changes in organ sizes and the severity of copper deficiency are not solely due to the total concentrations of iron and/or copper. The type of iron compound and the presence of free radicals may be involved in the pathology of copper deficiency of the male.

Analysis of Variance↗

PubMed1992

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1991

Orientation of non-blue cupric complexes on DNA fibers.

Three different orientations of non-blue, type 2 cupric complexes on DNA fibers are obtained from EPR data. The cupric complex of bleomycin, CuBlm, binds as described previously (Shields, H., McGlumphy,C., and Hamrick, P., J., Jr. (1982) Biochim. Biophys. Acta 697, 113-120), except possibly with more restricted motion. The square plane of CuBlm makes an angle of about 65 degrees with the fiber axis. The tridentate complex 2-formylpyridine monothiosemicarbazonato Cu2+ binds with its planar structure perpendicular to the fiber axis. In contrast, other tridentate cupric complexes of tripeptides, CuGHK and CuGHG, bind with the square plane parallel to the fiber axis. The bound forms of Cu(GHK) and Cu(GHG) are determined mostly by the GH moiety in the complex; the contribution of lysine in defining the orientation of the copper moiety is minimal. Thus, the structure of the ligand determines the orientation of these complexes on DNA.

Bleomycin↗

PubMed1991

Inhibition of iron uptake in HL60 cells by 2-formylpyridine monothiosemicarbazonato Cu(II).

The electron paramagnetic resonance (EPR) signal of the tyrosyl radical attributed to ribonucleoside diphosphate reductase decreases after treatment of promyelocytic leukemic HL60 cells with 2-formylpyridine thiosemicarbazonato copper(II) (CuL). According to EPR studies, CuL forms adducts with both histidine and cysteine-like Lewis bases associated with isolated membranes from HL60 cells. After the addition of CuL, the EPR signal for the cysteine-like adduct decreases substantially over a 15-min period. The reduction of signal is consistent with oxidation of thiols as shown by an analysis of sulfhydryl content. It is hypothesized that receptor-mediated transferrin internalization is inhibited by oxidation of critical thiols. Since the uptake of 59Fe-transferrin is greatly inhibited by the treatment of HL60 cells with CuL, the reduced uptake of iron by cells, in the presence of CuL, may lead to decreased iron availability for the activity of the M2 subunit of ribonucleotide reductase and a subsequent decrease in the tyrosyl radical signal of the enzyme. Moreover, the intact subunit M2 is no longer detected by EPR, even after the addition of excess iron.

Biological Transport, Active↗

PubMed1991

The severity of copper deficiency can be ameliorated by deferoxamine.

The present study was undertaken in order to determine whether hepatic iron overload plays a role in the exacerbation of copper deficiency. Weanling male Sprague-Dawley rats were fed a copper-deficient (0.6 microgram Cu/g) diet containing 62% fructose for 5 weeks. Some of the copper-deficient rats were injected daily with deferoxamine (DFX), an iron chelator that has been widely used to reduce iron overload. DFX reduced hepatic iron concentrations, which in turn ameliorated the pathology of copper deficiency when compared with nontreated copper-deficient animals. It is suggested that hepatic iron overload in a reduced environment plays a major role in the exacerbation of copper deficiency. Once the concentration of hepatic iron is reduced, the severity of the deficiency should be improved.

Animals↗

PubMed1991

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.

Bleomycin↗

PubMed1990

Oxidation of low-density lipoprotein by Cu2+ and lipoxygenase: an electron spin resonance study.

The aim of this work was to obtain spectroscopic evidence for free radicals formed during copper ion- and lipoxygenase-catalyzed oxidation of the low-density lipoprotein. During the initial oxidation phase, a free-radical metabolite derived from the endogenous alpha-tocopherol present in the low-density lipoprotein was detected by the electron spin resonance technique. The divalent copper ions were bound to the residual EDTA present in the low-density lipoprotein and to the protein. Production of the alpha-tocopherol radical was suppressed in the presence of spin traps. Evidence for the low-density lipoprotein-lipid derived radicals was obtained by ESR-spin trapping methods. Implications of these findings in the oxidative modification of the low-density lipoprotein are discussed.

Copper↗

PubMed1990

Microimmiscibility and three-dimensional dynamic structures of phosphatidylcholine-cholesterol membranes: translational diffusion of a copper complex in the membrane.

Saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes have been studied, with a special attention paid to fluid-phase immiscibility in cis-unsaturated phosphatidylcholine (PC)-cholesterol membranes as previously proposed and to the three-dimensional structure of the membrane. The investigation was carried out with dual probes: a membrane-soluble, square-planar copper complex, (3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato]copper(II) (CuKTSM2), and one of several nitroxide radical lipid-type spin-labels. Bimolecular collision rates between metal ion and spin-label were determined by measuring the nitroxide spin-lattice relaxation times (T1's) in the presence and absence of CuKTSM2 by use of saturation-recovery ESR techniques, and from these measured rates, translational diffusion coefficients of CuKTSM2 were estimated. Profiles of the collision rate across the membrane bilayer were obtained with Tempocholine phosphatidic acid ester, 5-doxylstearic acid, 16-doxylstearic acid, and cholesterol-type spin-labels as a function of cholesterol mole fraction, length and unsaturation of acyl chains, and temperature. In the liquid-crystalline phase of saturated PC membranes, incorporation of cholesterol decreases the collision rate at all depths in the membrane, and the effect of cholesterol is smallest in the middle of the bilayer. In trans-unsaturated PC membranes, a cholesterol-induced decrease of the collision rate was also observed, except in the head-group regions. In cis-unsaturated PC membranes, virtually no effect of cholesterol was observed on the collision rate, either with phospholipid-type spin-labels or with cholesterol-type spin-labels. This result is in clear contrast with our previous observation, in which the effect of cholesterol in cis-unsaturated PC membranes is small on the alkyl-chain motion of phospholipid-type spin-labels but large on the wobbling rotational diffusion of cholesterol-type spin-labels [Pasenkiewicz-Gierula, M., Subczynski, W. K., & Kusumi, A. (1990) Biochemistry 29, 4059-4069]. A model is proposed to explain these results in which the fluid-phase immiscibility is prevalent in cis-unsaturated PC-cholesterol membranes, but where cholesterol-rich (cholesterol oligomeric) domains are small (several lipids) and/or of short lifetime (10(-9) s to less than 10(-7) s). It is suggested that this microimmiscibility arises from the structural nonconformability between the rigid cholesterol ring structure and the rigid bend at the cis double bonds in PC alkyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstanes↗

PubMed1990

Development of drug resistance to gallium nitrate through modulation of cellular iron uptake.

We have shown that transferrin-gallium (Tf-Ga) blocks DNA synthesis through inhibition of cellular iron incorporation and a diminution in the activity of the iron-dependent M2 subunit of ribonucleotide reductase. To examine the mechanisms of drug resistance to gallium, we developed a subline of HL60 cells (R cells) which is 29-fold more resistant to growth inhibition by gallium nitrate than the parent line (S cells). R cells displayed a 2.5-fold increase in transferrin (Tf) receptor expression, without a change in receptor affinity for Tf. The uptake and release of 67Ga were similar for both S and R cells. The uptake of 59Fe-Tf by S cells was inhibited by gallium nitrate over 24-48 h of incubation. In contrast, 59Fe-Tf uptake by R cells, although initially inhibited by gallium nitrate at 24 h, was no longer inhibited at 48 h of incubation. 59FeCl3 uptake by R cells was significantly greater than that of S cells, regardless of the time in culture. Despite the increase in 59Fe uptake by R cells, the ferritin content of these cells was lower than that of S cells. The ribonucleotide reductase electron spin resonance signal of R cells was comparable to that of S cells. R cells were not cross-resistant to Adriamycin, vincristine, cis-platinum or hydroxyurea. Resistance to gallium nitrate in this subline of HL60 cells results primarily from the ability of cells to overcome the gallium-induced block in iron incorporation. In addition, intracellular iron in R cells appears to traffic preferentially to a non-ferritin compartment.

Antineoplastic Agents↗

PubMed1990

Oxidative stress induced by a copper-thiosemicarbazone complex.

Copper thiosemicarbazones cause considerable oxidative stress. This effect may be related to their cytotoxicity. In the present work, the chemical and cellular properties of a new ligand, pyridoxal thiosemicarbazone (H2T), and its copper(II) chelate (CuT) are assessed. CuT is toxic to cultured Ehrlich ascites tumor cells, producing nearly complete cell kill at drug/cell ratios of 2.5-4 nmol/10(5) cells in a monolayer culture over a 48-h treatment period. This concentration is at least 1 order of magnitude lower than those required for a similar degree of cytotoxicity by H2T or CuCl2. The following observations support the view that activated oxygen species are generated by interaction of CuT with Ehrlich cells: (1) Room-temperature electron spin resonance spectroscopy and atomic absorption measurements show rapid cellular uptake and CuT-thiol adduct formation. (2) Cellular thiol content is reduced. (3) High levels of DNA strand scission result from 1-h treatments of cells by concentrations of CuT that cause growth inhibition and toxicity. (4) The extent of strand scission can be increased by addition of superoxide dismutase and decreased by catalase or DMSO in the treatment medium. Catalase and DMSO do not inhibit the toxic effect of CuT. This suggests that DNA damage is not responsible for inhibition of cell proliferation by CuT.

Animals↗

PubMed1990

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for gz and gx at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.

Amino Acid Sequence↗

PubMed1990

The copper site in nitrous oxide reductase.

The properties of the novel copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri are described. Multifrequency electron paramagnetic resonance spectroscopy is used to characterize the various forms of the enzyme. The features observed at 2.4, 3.4, 4.5, 9.31 and 35 GHz are explained by a mixed-valence [Cu(1.5)...Cu(1.5)] S = 1/2 species with the unpaired electron delocalized between the two Cu nuclei. This site is also present in the catalytically inactive derivative of nitrous oxide reductase which was obtained from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The resemblance of the low-frequency electron paramagnetic resonance spectra to the spectra for the so-called CuA of cytochrome c oxidase can be taken as a first indication that the CuA may have a structural and electronic arrangement similar to the electron-paramagnetic-resonance-detectable copper in nitrous oxide reductase. Results from oxidation/reduction experiments, and from a quantitative determination of sulfhydryl and disulfide residues in the various forms of nitrous oxide reductase, suggest the involvement of the redox-couple cysteine/cystine in the structural organization of the active site of nitrous oxide reductase.

Binding Sites↗

PubMed1990

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.

Animals↗

PubMed1989

Orientation of dioxygen bound to cobalt(II) bleomycin-DNA fibers.

The molecular oxygen adduct of Co(II)-bleomycin is stable for long periods when bound to salmon sperm DNA at large ratios of polymer to drug. According to ESR studies of orientation of the paramagnetic complex associated with DNA fibers, the oxygen-oxygen bond is restricted to a plane perpendicular to the fiber axis. Thus, one can define three g values for the adduct 2.104, 2.016, and 2.000, one parallel to the fiber axis and two orthogonal to it. There is no change in orientation over the range of 77 K to ambient temperature. Furthermore, there is no difference in results at a series of relative humidities ranging from less than 76% where bulk DNA alone assumes an A conformation to 95% where it is primarily B DNA. A structural model is presented for the geometry of the metal binding domain of O2-Co-bleomycin in relationship to the fiber axis of DNA.

Bleomycin↗

PubMed1989

Activity of bleomycin in iron- and copper-deficient cells.

Three models were used to examine the requirement of bleomycin (Blm) for iron (Fe) to carry out its antitumor or cytotoxic activity. Mice were made iron deficient by dietary means. Animals with depressed iron stores in liver and low plasma and ascites fluid iron supported Ehrlich tumor growth as well as mice maintained on a control diet. Bleomycin was equally effective against this tumor in iron-deficient mice as it was against the tumor in iron-sufficient controls. Likewise, nutrient copper deficiency did not change the efficacy of the drug. Ehrlich cells in culture were treated with a non-growth inhibiting concentration of the chelating agent, 1,10-phenanthroline before or during their exposure to bleomycin. Again, the treated cells were as sensitive to drug as controls, despite the fact that this ligand reduces cellular iron and zinc and can extract iron from Fe(II)Blm. Lastly, it was demonstrated that iron-depleted Euglena gracilis cells growing at reduced rates were as sensitive to growth inhibition by bleomycin as control cells.

Animals↗

PubMed1989

ESR studies of the interaction of copper(II)GHK, histidine, and Ehrlich cells.

The tridentate complex CuGHK does not form ESR detectable adducts upon addition to either glutathione or Ehrlich ascites cells under our conditions. The absence of adducts is consistent with the poor uptake of CuGHK by cells. ESR spectra are used to characterize adduct formation between CuGHK and histidine. The CuGHK-histidine adduct is not stable in the presence of Ehrlich ascites tumor cells. It is argued that a Cu(His)2 complex is formed as a consequence of the interaction of GHK with cells.

Animals↗