Bleomycin-iron can degrade DNA in the presence of excess ethylenediaminetetraacetic acid in vitro

The antineoplastic drug bleomycin, when complexed to Fe(II), causes both single- and double-stranded lesions in DNA in vitro. EDTA is commonly used to inhibit the reaction of bleomycin-Fe with DNA, presumably by removing the metal cofactor. In this study, we utilized a simple assay involving the conversion of supercoiled plasmid DNA to the nicked or linear forms to further investigate the ability of bleomycin-Fe to degrade DNA in the presence of EDTA. We found that a 1:1 complex of bleomycin and Fe can degrade plasmid DNA even in the presence of a 10(6) molar excess of EDTA over bleomycin. Furthermore, we found that the half-life for inactivation of bleomycin-Fe by excess EDTA is about 1.5 h. Finally, we demonstrate that excess bleomycin associated with the outer plasma membranes of cells can damage DNA after the cells are lysed in buffers containing EDTA and SDS. These results suggest that EDTA may not be an efficient inhibitor of the reaction of bleomycin-Fe with DNA.     

The iron chelating cardioprotective prodrug dexrazoxane does not affect the cell growth inhibitory effects of bleomycin

The clinical use of bleomycin is limited by a dose-dependent pulmonary toxicity. Bleomycin is thought to be growth inhibitory by virtue of its ability to oxidatively damage DNA through its complex with iron. Our previous preclinical studies showed that bleomycin-induced pulmonary toxicity can be reduced by pretreatment with the doxorubicin cardioprotective agent dexrazoxane. Dexrazoxane is thought to protect against iron-based oxygen radical damage through the iron chelating ability of its hydrolyzed metabolite ADR-925, an analog of ethylenediaminetetraacetic acid (EDTA). ADR-925 quickly and effectively displaced either ferrous or ferric iron from its complex with bleomycin. This result suggests that dexrazoxane may have the potential to antagonize the iron-dependent growth inhibitory effects of bleomycin. A study was undertaken to determine if dexrazoxane could antagonize bleomycin-mediated cytotoxicity using a CHO-derived cell line (DZR) that was highly resistant to dexrazoxane through a threonine-48 to isoleucine mutation in topoisomerase IIalpha. Dexrazoxane is also a cell growth inhibitor that acts through its ability to inhibit the catalytic activity of topoisomerase II. Thus, the DZR cell line allowed us to examine the cell growth inhibitory effects of bleomycin in the presence of dexrazoxane without the confounding effect of dexrazoxane inhibiting cell growth. The cell growth inhibitory effects of bleomycin were unaffected by pretreating DZR cells with dexrazoxane. These results suggest that dexrazoxane may be clinically used in combination with bleomycin as a pulmonary protective agent without adversely affecting the antitumor activity of bleomycin.     

DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage

Summary. Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.     

In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.     

Effects of extracts of rat tissues on the action of bleomycin.

The cell sap (105 000 times g supernatant) of various tissues of rats caused DNA degradation in the presence of bleomycin. The activity was fractionated into two peaks by column chromatography on Sephadex G-25. The activity in Peak A (excluded fraction) appeared to be due to some proteinaceous entity, while that recovered in Peak B (retarded fraction), constituting about 90% of the total activity, seemed to be due to ascorbic acid, judging by results of further gel filtration and the effect of treatment with ascorbate oxidase. Incubation of bleomycin with Peak A or B caused loss of the ability of the antibiotic to degrade DNA. It is proposed that the action of bleomycin on DNA, and its inactivation by tissue extracts, depend, at least in part, on the presence of ascorbic acid.     

The radioprotective agent WR1065 protects cells from radiation damage by regulating the activity of the Tip60 acetyltransferase

Background: The aminothiol WR1065 is a highly effective free radical scavenger which can protect cells from the cytotoxic effects of ionizing radiation. Currently, WR1065 is used clinically to protect patients from radiation injury occurring during radiation therapy protocols. However, it is becoming increasingly clear that WR1065 can alter radiosensitivity through a mechanism which is independent of its ability to function as a free radical scavenger. Here, we examined the ability of WR1065 to directly regulate signaling pathways involved in the DNA damage response. Methodology: The ability of WR1065 to enhance the survival of irradiated bone marrow cells and primary cultures was established. DNA damage signaling was monitored by measuring activation of the ATM kinase by western blot analysis and activation of Tip60 using an in vitro acetylation assay. Tip60 function was abrogated by expression of a catalytically inactive Tip60, and the effect on radiosensitivity evaluated. Principal findings: Treatment of cells with WR1065 led to a small but significant increase in the kinase activity of ATM. Further, WR1065 robustly activated the Tip60 acetyltransferase, which is a key upstream regulator of the ATM kinase. In addition, WR1065 directly activated the acetyltransferase activity of purified Tip60 in vitro, indicating a direct interaction between WR1065 and Tip60. Finally, cells with reduced levels of Tip60 activity exhibited a significant reduction in radioprotection by WR1065. Conclusions: Direct regulation of Tip60''s acetyltransferase activity by WR1065 makes a significant contribution to the radioprotective effects of WR1065. Activators of Tip60 may therefore make effective clinical radioprotectors.     

DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.     

Stobadine inhibits lysosomal enzyme release in vivo and in vitro

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

Antioxidant potential of aminothiazole derivative and its protective effect on H2O2-induced oxidative damage on pBR322 DNA and RBC cellular membrane

The present work was carried out to evaluate the antioxidant and free radical scavenging activity of aminothiazole derivative by performing various in vitro assays; to study its protective effect on H(2)O(2)-induced oxidative damage on pBR322 DNA and on RBC cellular membrane. The in vitro assays were performed with different concentrations of aminothiazole derivative (6.15, 12.29, 18.44, 24.59, and 30.73 microM) and the results were compared with standards like ascorbic acid and trolox. Our results clearly indicated that aminothiazole derivative at a dose of 18.44 microM exhibited radical scavenging activity greater than that of ascorbic acid and trolox. The DNA protective effect on pBR322 DNA showed that there was a concentration-dependent inhibition of the disappearance of supercoiled (ccc) form of DNA on incubation with 30 mM H(2)O(2) in the presence of different concentrations of aminothiazole derivative. Thus our compound at 1.5 mM prevents the conversion from supercoiled (ccc) form to open circular form (oc) form of pBR322 DNA. Pretreatment with aminothiazole derivative at a dose of 18.44 microM prevents membrane damage and exhibits an IC(50) value, which is the concentration of the sample required to inhibit 50% of the radical formed greater than that of the standards (ascorbic acid and trolox). Thus our compound of interest aminothiazole derivative exhibits antioxidant and free radical scavenging properties greater than that of standards like ascorbic acid and trolox and thereby protects pBR322 DNA and RBC cellular membrane from free radical induced oxidative damage.     

Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans

This brief resume enumerates the multiple actions of melatonin as an antioxidant. This indoleamine is produced in the vertebrate pineal gland, the retina and possibly some other organs. Additionally, however, it is found in invertebrates, bacteria, unicellular organisms as well as in plants, all of which do not have a pineal gland. Melatonin's functions as an antioxidant include: a), direct free radical scavenging, b), stimulation of antioxidative enzymes, c), increasing the efficiency of mitochondrial oxidative phosphorylation and reducing electron leakage (thereby lowering free radical generation), and 3), augmenting the efficiency of other antioxidants. There may be other functions of melatonin, yet undiscovered, which enhance its ability to protect against molecular damage by oxygen and nitrogen-based toxic reactants. Numerous in vitro and in vivo studies have documented the ability of both physiological and pharmacological concentrations to melatonin to protect against free radical destruction. Furthermore, clinical tests utilizing melatonin have proven highly successful; because of the positive outcomes of these studies, melatonin's use in disease states and processes where free radical damage is involved should be increased.     

Ascorbate both activates and inactivates bleomycin by free radical generation.

The chemotherapeutic agent bleomycin (BLM) is activated by reducing agents to break isolated DNA. Paradoxically, these same reducing agents protect cellular DNA from BLM damage. To resolve this paradox, we have examined the reaction of FeIIIBLM with DNA in the presence of ascorbate. As expected, ascorbate augments FeIIIBLM-induced DNA damage. However, when ascorbate is added to FeIIIBLM prior to exposure to DNA, a redox-inactive BLM is produced in a reaction that generates the ascorbyl radical. This reaction occurs in both ascorbate-supplemented buffer and unsupplemented plasma. In buffered solution, this reaction was found to be stoichiometric; for each mole of BLM present, 6.9 mol of ascorbate was oxidized and 4.7 mol of oxygen was consumed. Iron was found to serve only as a catalyst for the reaction. These data suggest that both activation of BLM and the generation of redox-inactive BLM occur via the same reaction and that BLM-induced DNA damage depends upon BLM reaching DNA prior to its interaction with reducing agents.     

Effects of reducing and oxidizing agents on the action of bleomycin.

The effects of reducing agents, such as 2-mercaptoethanol, dithiothreitol, L-ascorbic acid, or sodium borohydride, and oxidizing agents, such as hydrogen peroxide or dehydroascorbic acid, on the in vitro action of bleomycin were investigated. After the incubation of DNA with a low concentration of bleomycin and a reducing or oxidizing agent, single strand breaks were mainly caused in the DNA molecules. The degradation of DNA was largely prevented by the removal of oxygen, or by the addition of divalent cations or of S-(2-aminoethyl)isothiuronium bromide hydrobromide, a radical scavenger, to the incubation mixture. Preincubation of bleomycin with these reducing or oxidizing agents reduced the DNA-degrading activity of the antibiotic. However, this reduction in activity was observed even in the absence of oxygen, or in preincubation mixture supplemented with radical scavenger.     

In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch

Mammalian metallothioneins (MT) have been reported to scavenge free radicals. There is no experimental evidence to show that fish MT has a similar property. In the present study cadmium-induced MT (Cd-MT) from the liver of an Indian freshwater fish Channa punctata Bloch was investigated for its free radical scavenging activity using three different in vitro assays. Exposure to cadmium chloride (0.2 mg/kg body weight; three doses on alternate days) resulted in a marked induction of Cd-MT in liver. Only a single isoform of Cd-MT was found to be induced. Molecular weight of Cd-MT was found to be 14 kDa as deduced by SDS-PAGE analysis. The purified Cd-MT effectively scavenged the following free radicals: superoxide radical (O2*-), 2,2'-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS*+) and 1,1-diphenyl-picrylhydrazyl radical (DPPH*). The radical scavenging effect was found to be concentration-dependent. Also, the purified MT exhibited an inhibitory effect on ferric nitrilotriacetate (Fe-NTA) induced oxidative DNA damage in vitro. The cysteine residues of MT are proposed to be the main candidate for its radical scavenging activity. Findings of the present study strongly suggest a free radical scavenging role for fish MT. Present study adds to the little existing knowledge about fish MT and its possible biological functions.     

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.     

Inactivation of bleomycin by an N-acetyltransferase in the bleomycin-producing strain Streptomyces verticillus

Abstract Bleomycin-producing Streptomyces verticillus ATCC15003 possesses a bleomycin acetyltransferase which inactivates the drug in the presence of acetyl coenzyme A. The site of acylation in enzymically prepared acetylbleomycin A2 was determined by nuclear magnetic resonance analysis; the primary amino group of the β-aminoalanine moiety of bleomycin was acetylated. Acetylbleomycin A2 had no detectable antibacterial activity and did not induce in vitro DNA degradation.     

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 μM concentration of DMA and TBZ and irradiated at 5 Gy; both the ligands showed nuclei condensation suggesting a probable mechanism to protect DNA from radiation damage. The bisubstituted analogs of Hoechst 33342 are found to be better free radical scavengers and protect DNA against radiation-induced damage at a lower concentration than the parent molecule. Both the ligands also quenched free radicals in isolated free radical system suggesting their dual mode of action against radiation-induced damage to DNA. Molecules binding to the chromatin alter gene expression, whereas in this study both the ligands have not shown any profound effect on the nucleosome assembly and gene expression in vitro and in vivo. Both ligands afford a 2-fold protection by altering DNA structure as well as through direct free radical quenching in bulk solution in comparison to the parent ligand, which acts only through quenching of free radicals. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increase in free radical generation and lipid peroxidation following chemotherapy in patients with cancer

Several anti-cancer drugs are known to bring about their tumoricidal actions by a free radical dependent mechanism. Majority of the studies reported that adriamycin, mitomycin C, bleomycin, etc., augment free radical generation and lipid peroxidation process in vitro. Our results reported here suggest that following chemotherapy both stimulated and unstimulated human polymorphonuclear leukocytes generate increased amounts of superoxide anion and hydrogen peroxide. This was accompanied by increased formation of lipid peroxidation products as measured by thiobarbituric acid assay. These results confirm that many anti-cancer drugs augment free radical generation and lipid peroxidation even in an vivo situation.     

DNA damage induced by bleomycin in the presence of dibucaine is not predictive of cell growth inhibition

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.     

Protection of DNA damage by dietary restriction.

Dietary restriction is known to retard the aging processes and delay the onset of age-related neoplastic diseases. The mechanisms underlying these remarkable actions of nutritional intervention are not known in spite of recently intensified research efforts. However, the last couple of years' research on dietary restriction produced strong evidence indicating that its effective antiaging actions might be related to its ability to modulate free radical damage. In the present study, DNA damage and attenuation of the damage by dietary restriction were assessed by measuring 8-hydroxydeoxyguanosine 8-OH dG) in both nuclear DNA (nuDNA) and mitochondrial DNA (mitDNA) fractions. The data show that substantially more damage (approximately 15 times) occurred in mitDNA compared to nuDNA. More interestingly, the DNA damage was significantly attenuated in dietarily restricted rats.