Base Propenals Research Articles

Bleomycin-induced unscheduled DNA synthesis in non-permeabilized human and rat hepatocytes is not paralleled by 8-oxo-7,8-dihydrodeoxyguanosine formation

The genetic toxicity of the antitumour antibiotic bleomycin (BLM) is thought to involve the formation of a reactive oxygen intermediate. 8-Oxo-7,8-dihydrodeoxyguanosine (oxo 8dG), an oxidation product of deoxyguanosine, is one of the major products formed when isolated DNA is exposed to oxygen radical generating systems. Gamma-irradiation (10–500 Gy 60Co; 10 Gy/min) or BLM and Fe 2+ (37.5–150 U/L and 0.5 mM, respectively) treatment of isolated DNA (0.25 mg/mL) increased oxo 8dG above background. In the latter case, the effect was greater than that with Fe 2+ (0.5 mM) alone and was dependent on the dose of BLM. When DNA was irradiated with 500 Gy 60Co, deoxyguanosine oxidation was inhibited by antioxidants (ethanol: 37.5 and 98% inhibition at 2 and 20 mM, respectively; mannitol: 20.5, 60 and 92% inhibition at 0.1, 1.0 and 10 mM, respectively). Similarly the BLM-induced production of oxo 8dG was inhibited (64%) by mannitol (10 mM). BLM also caused production of base propenals on interaction with isolated DNA. In contrast, oxo 8dG was not induced above background concentration (27 mol oxo 8dG/10 6 mol dG) in permeabilized (37°) and non-permeabilized (4° and 37°) rat hepatocytes treated with BLM (260 U/L). Despite this, there was extensive BLM-induced unscheduled DNA synthesis (10 and 100 U/L) in non-permeabilized rat and human hepatocytes in the absence of hydroxyurea. These findings, in accord with other observations, draw into question the role of OH in BLM-induced DNA damage and the mimicry of ionizing radiation in cellular systems.

New insight into the mechanism of base propenal formation during bleomycin-mediated DNA degradation

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNew insight into the mechanism of base propenal formation during bleomycin-mediated DNA degradationG. H. McGall, Lois E. Rabow, G. W. Ashley, S. H. Wu, J. W. Kozarich, and J. StubbeCite this: J. Am. Chem. Soc. 1992, 114, 13, 4958–4967Publication Date (Print):June 1, 1992Publication History Published online1 May 2002Published inissue 1 June 1992https://doi.org/10.1021/ja00039a002RIGHTS & PERMISSIONSArticle Views227Altmetric-Citations54LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Bleomycin pharmacology: mechanism of action and resistance, and clinical pharmacokinetics.

Bleomycin is a glycopeptide antibiotic with a unique mechanism of antitumor activity. The drug binds to guanosine-cytosine-rich portions of DNA via association of the "S" tripeptide and by partial intercalation of the bithiazole rings. A group of five nitrogen atoms arranged in a square-pyramidal conformation binds divalent metals including iron, the active ligand, and copper, an inactive ligand. Molecular oxygen, bound by the iron, can produce highly reactive free radicals and Fe(III). The free radicals produce DNA single-strand breaks at 3'-4' bonds in deoxyribose. This yields free base propenals, especially of thymine: cytotoxicity is cell-cycle-phase specific for G2 phase. In humans, bleomycin is rapidly eliminated primarily by renal excretion. This accounts for approximately half of a dose. In patients with renal compromise or extensive prior cisplatin therapy, the drug half-life can extend from 2 to 4 hours up to 21 hours. Thus, dose adjustments are needed when creatinine clearance is less than or equal to 3N mL/min. Finally, resistance to bleomycin in normal tissues can be correlated with the presence of a bleomycin hydrolase enzyme, which is in the cysteine proteinase family. The enzyme replaces a terminal amine with a hydroxyl, thereby inhibiting iron binding and cytotoxic activity. The low concentration of enzyme in the skin and lung may explain the unique sensitivity of these tissues to bleomycin toxicity. However, correlation of hydrolase levels with tumor cell sensitivity has thus far been negative.

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.

Bleomycin-dependent damage to the bases in DNA is a minor side reaction

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.

Acyl migration in the production of thymine. Propenal from 3'-O-benzoyl-5'-deoxy-4'-(hydroperoxy)thymidine: a reinterpretation of a putative model for bleomycin-mediated DNA degradation

Studies of Saito et al. (Saito, I.; Morii, T.; Matsuura, T. J. Org. Chem. 1987, 52, 1008) analyzing the decomposition of 3'-O-benzoyl-5'-deoxy-4'-hydroperoxythymidine (7) claimed to model the decomposition of the putative 4'-hydroperoxynucleotide intermediate in the bleomycin (BLM) mediated production of base propenal, 3'-phosphoglycolate, and 5'-phosphate termini from double-stranded DNA. A number of puzzling observations reported in this paper prompted a reinvestigation of this model system in detail. [4'-HY 18 O 2 ]-7 and its 4' epimer 8 were prepared and their fate in aqueous solution as a function of pH was examined

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeoxyguanosine (8OHdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 8OHdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O2-) and hydrogen peroxide (H2O2). Thus, TBA reactivity is destroyed in the formation of O2- and H2O2 which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.

A Comparative Study of the Interactions of Bleomycin with Nuclei and Purified DNA

Fe(III)-bleomycin associates strongly with rat liver nuclei and binds to nuclear DNA. Metal-free and Cu(II)-bleomycin, however, do not bind to nuclei. The treatment of nuclei with activated iron-bleomycin results in nucleic base and base propenal release from the DNA, and also gives membrane peroxidation. Isolation and quantitation of the base propenals and free bases released subsequent to activated bleomycin treatment reveal an alteration in the stoichiometry of these products compared to those released from purified DNA. With nuclei, significantly less propenal is formed, although the yield of free base is equivalent to that from purified DNA. The membrane peroxidation products from nuclei are the same as those obtained from microsomal membranes treated with activated bleomycin. Superoxide dismutase inhibits the membrane peroxidation but has no effect on the DNA breakage reactions. The results implicate a role for iron in mediating the in vivo action of bleomycin and also reveal a potentially toxic effect, membrane peroxidation, separate from DNA damage.

Deoxyribonucleic acid (DNA) damage induced by bleomycin-Fe(II) in vitro: Formation of 8-hydroxyguanine residues in DNA.

Treatment of calf thymus deoxyribonucleic acid (DNA) with bleomycin-Fe(II) at 0 degree C for 5 min resulted in the formation of 8-hydroxyguanine (8-OH-Gua) residues in DNA in a dose dependent manner, in addition to the formation of base propenal, a DNA-degradation product. The amount of 8-OH-Gua was about one-hundredth of that of base propenal. Treatment of cellular DNA with bleomycin did not result in any increase of 8-OH-Gua even under conditions of 100% cell killing.

DNA cleavage activity of liblomycin (NK313), a novel analog of bleomycin.

Liblomycin (NK313), a novel analog of bleomycin and peplomycin (PEP), produced acid soluble DNA, base propenals and nucleo bases from isolated DNA. This was similar to the action of PEP. However, the DNA cleavage activity of NK313 was 1/2-1/10 of that of PEP in the absence of reducing agents. In the presence of reducing agents such as 2-mercaptoethanol and ascorbic acid, the activity of NK313 was stimulated more strongly than PEP. NK313 was also different from PEP in the formation and decomposition of active intermediates. This result suggested that differences in DNA cleavage activity between NK313 and PEP may be due to the different properties of their active intermediates. NK313 released preferentially pyrimidine bases from DNA, and the molar ratio of the released pyrimidine bases to the total of the released bases was little affected by the concentration of NK313 relative to DNA. In contrast, the ratio of the released purine bases by PEP increased with the concentration of PEP relative to DNA. NK313 induced double strand cleavage on pBR322 DNA as efficiently as did PEP. The major cleavage sites of NK313 on pBR322 DNA were similar to those of PEP. However, certain minor cleavage sites of NK313 were specific for NK313. Increase of PEP concentration led to increased degradation of DNA fragments; this was not the case with NK313. These results indicate that the cleavage sites of NK313 were similar to, but more limited than those for PEP.

Chemistry of the alkali-labile lesion formed from iron(II) bleomycin and d(CGCTTTAAAGCG)

Two sets of products are formed from DNA upon treatment with Fe(II).bleomycin + O2. One set, which is believed to derive from a C-4' hydroperoxy derivative of the DNA deoxyribose moiety, includes the four possible base propenals, as well as DNA oligomers having deoxynucleoside 3'-(phosphoro-2"-O-glycolates) at their 3'-termini. The other set of products consists of free bases and alkali-labile lesions, the latter of which had not previously been characterized structurally. By use of the self-complementary dodecanucleotide d(CGCTTTAAAGCG) having a site modified by Fe-bleomycin three nucleotides from the 5'-end, it has been possible to characterize the alkali-labile product as a C-4' hydroxyapurinic acid. When the bleomycin-treated dodecanucleotide was treated with agents that effected decomposition of the alkali-labile lesion, products of the form CpGpx were obtained, and these proved useful for structural characterization of the alkali-labile lesion. Treatment with alkali produced CpGpx, where x was 2,4-dihydroxycyclopentenone. Alternatively, treatment with hydrazine provided a pyridazine derivative, and aqueous alkylamines led to formation of CpGp itself. The structures of all dinucleotides produced from the alkali-labile lesion were verified by direct comparison with authentic synthetic samples.

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

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

The DNA cleavage mechanism of iron-bleomycin. Kinetic resolution of strand scission from base propenal release.

The action of iron-bleomycin and O2 in cleaving DNA has been resolved into two kinetic events following the initial attack on DNA by the kinetically competent drug species, "activated bleomycin." At 4 degrees C, DNA strand scission, monitored both viscometrically and fluorimetrically (t1/2 = 2.5-5 min), precedes the release from DNA of nucleic base propenals, which is half complete in about 40 min. Therefore, a moderately stable intermediate consisting of cleaved DNA bearing a base propenal precursor is formed. The release of tritium from deoxyribose carbon-2 occurs at the time of DNA scission, which is consistent with the base propenal precursor retaining the deoxyribose-3'-phosphate bond. Specific mechanistic proposals are discussed.

DNA degradation by bleomycin: evidence for 2'R-proton abstraction and for carbon-oxygen bond cleavage accompanying base propenal formation

Reaction of poly(dA-[2'S-3H]dU) with activated bleomycin yields [3H]uracil propenal that completely retains the tritium label. In contrast, we have previously shown that reaction of poly(dA-[2'R-3H]dU) with activated bleomycin affords unlabeled uracil propenal [Wu, J. C., Kozarich, J. W., & Stubbe, J. (1983) J. Biol. Chem. 258, 4694-4697]. We have also prepared both cis- and trans-thymine propenals by chemical synthesis and have observed that the trans isomer is the exclusive product of the bleomycin reaction. Moreover, the cis isomer was found to be stable to the conditions of bleomycin-induced DNA degradation. Taken together, these results establish that the formation of trans-uracil propenal occurs via an anti-elimination mechanism with the stereospecific abstraction of the 2'R proton. The question of phosphodiester bond cleavage during base propenal formation has also been addressed by the analysis of the fate of oxygen-18 in poly(dA-[3'-18O]dT) upon reaction with activated bleomycin. The 5'-monophosphate oligonucleotide ends produced from thymine propenal formation have been converted to inorganic phosphate by the action of alkaline phosphatase, and the phosphate has been analyzed for 18O content by 31P NMR spectroscopy. The oxygen-18 is retained in the inorganic phosphate, establishing that the formation of thymine propenal by activated bleomycin proceeds with C-O bond cleavage at the 3'-position.

Deoxyribonucleic acid cleavage specificity of a series of acridine- and acodazole-iron porphyrins as functional bleomycin models

A series of metalloporphyrins linked through basic chains to certain DNA interactive groups has been synthesized. Several of these agents reproduce the characteristic properties of the antitumor glycopeptide bleomycin, including the oxygen-mediated scission of DNA in the presence of thiols, antibiobic activity under aerobic conditions, and activity against human and animal tumor models. Initial screening by scission of PM2-CCC-DNA identified six of the compounds, including those bearing acridine and acodazole intercalating groups, as the most active. The specificity of the oxygen-mediated scission of a 139 base pair HindIII/NciI restriction fragment of pBR322 by these six selected agents was then determined and compared with the action of pancreatic DNase by densitometric scans. All six of these compounds produce uniform base and sequence neutral cleavage of the restriction fragment at each base site. The six active compounds bear either of two types of intercalators, 6-chloro-2-methoxyacridine or acodazole, and with linkages to the ferric binding domain of -NH(CH2)2-, -NH(CH2)3-, -NH(CH2)4-, or -NH(CH2)3NH(CH2)3- and either porphyrin or deuteroporphyrin moieties. Comparison of the Kassoc values for binding to calf thymus DNA suggests that the enhanced binding observed with the linker -NH(CH2)3NH(CH2)3- contributes to the efficiency of sequence neutral DNA scission and may be a factor in the relative anticancer activities of these agents. The iron porphyrins give no evidence of the production of base propenals in DNA degradation, and the autoradiograms clearly indicate that a phosphate group is attached to the 5' end of the oligomer. The scission is partially suppressible by catalase and superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of bleomycin: evidence for a rate-determining 4'-hydrogen abstraction from poly(dA-dU) associated with the formation of both free base and base propenal

When poly(dA-[4'-3H]dU) was degraded by activated bleomycin under a variety of conditions, 50 +/- 10% of the deoxyuridine residues were converted to uracil and uracil propenal, paralleling observations made with DNA. By manipulation of the concentration of O2 in solution, the relative ratio of uracil propenal to uracil could be varied between 0.03 for anaerobic activation and 7.0 for activation at 3 atm of O2. Tritium selection effects on 4'-hydrogen abstraction were also measured under these conditions and found to range from 7.2 to 12.5. These results strongly suggest that the formation of both uracil and uracil propenal is the consequence of a rate-determining 4'-carbon-hydrogen bond cleavage and of an O2-dependent partitioning of the intermediate produced by this cleavage.

Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release

Incubation of poly(dA-[3'-3H]dU), poly(dA-[5'-3H]dU), or poly(dA-[5'-3H]dT) under a variety of conditions with activated bleomycin resulted in the production of free nucleic acid base, base propenal, and a small amount of 3H2O. Adjustment of the terminated reaction mixture to pH 10 and incubation at 95 degrees C resulted in a time-dependent increase in 3H2O to an amount equal to the amount of free base. If the terminated reaction mixture was incubated with NaBH4 prior to the heat and alkaline treatment, the release of 3H2O was significantly inhibited. These results are consistent with the generation by activated bleomycin of a 4'-ketone yielding free base, with the exchange of the 3'- and 5'-hydrogens by enolization and with the alkaline-induced strand scission occurring from this intermediate.

Analysis of products formed during bleomycin-mediated DNA degradation

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2''-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.

Stimulation of iron(II) bleomycin activity by phosphate-containing compounds

Orthophosphate and phosphate derivatives including pyrophosphate, hexametaphosphate, ATP, ADP, and inositol hexaphosphate enhance the extent of DNA degradation by iron(II) bleomycin. These phosphate-containing compounds increase both the release of free nucleic base and that of base propenals which are DNA cleavage products, probably by enhancing the efficiency with which Fe(II) is recruited into the drug. Phosphate action occurs during drug activation prior to the attack on DNA. In addition, phosphates affect the stability of the activated drug complex, overcome the inhibition observed with high concentrations of DNA, and reduce the size of the DNA fragment necessary for reacting with the drug. Phosphate derivatives bind to iron(II) bleomycin and alter its optical spectrum. An analysis of titration data for pyrophosphate and inositol hexaphosphate indicates that each phosphate compound binds to more than one iron(II) bleomycin molecule. With ATP, ADP, and 2,3-diphosphoglycerate, only a single phosphate-containing compound binds to the ferrous drug complex. The affinity for ATP is sufficiently high as to suggest that the ternary complex formed in vitro may exist physiologically.

Cobalt-bleomycins and deoxyribonucleic acid: sequence-dependent interactions, action spectrum for nicking, and indifference to oxygen.

Light-induced strand scission of DNA by cobalt-bleomycins is more likely to occur at certain base sequences than others. By use of 32P-end-labeled DNA restriction fragments as the substrates for cleavage, products have been analyzed on high-resolution polyacrylamide gels and compared to those produced by iron-bleomycin. The results indicate that the sites of damage to DNA are similar in both cases: pyrimidine residues located at the 3' side of a guanine are preferentially attacked. Consistent with the observed nicking specificity, interactions between cobalt-bleomycin and guanine residues in the trinucleotide sequence GGT are revealed in a dimethyl sulfate methylation experiment. The action spectrum for the light-induced DNA cleavage reaction correlates with the absorption spectrum of cobalt-bleomycin in the wavelength range between 330 and 450 nm. In contrast to iron-bleomycin, the extent of DNA degradation by light-activated cobalt-bleomycins appears to be indifferent to the concentration of dissolved oxygen in the reaction medium, and little or no base propenal is produced. Bases (e.g., thymine) are released by both agents. Fluorescence quenching experiments show that apparent binding constants of cobalt-bleomycin complexes with DNA are in the 10(7) M-1 range in 25 mM tris(hydroxymethyl)aminomethane, pH 8, 1 mM NaCl, and 1 mM ethylenediaminetetraacetic acid at 25 degrees C.