J. Biol. Chem.-1980-Burger-11832-8

8/7/2019 J. Biol. Chem.-1980-Burger-11832-8

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T H E . JOURNAL OF BIO LO G ICALCH EM ISTRYVo l 255, No. 24, lssue of December 25, pp . 11R32-1183H. 19HoPrmted m U . S .A.

Origin of Malondialdehyde fromDNA Degraded by Fe(I1) *Bleomycin*(Received for publication, Febru ary 19,1980, and in revised form, May5 , 1980)

Richard M. Burger, Allen R. Berkowitz, Jack Peisach, and Susan Band H o h t z SFrom the Departmentsof Molecular Pharmacolo gy, Molecular Biology, an d Cell Biology, Albert Einstein CollegeofMedicine, Bronx, New York 10461

Ferrous bleomycinis known to break DNA efficientlyin vitro in the presence of 02,giving rise tooligonucle-otides, bases, and compoundsresembling malondialde-hyde in their chromogenic reaction with Z-thiobarbi-turic acid. Chromatography of radiolabeled DNA reac-tion mixtures resolves three kinds of malondialdehyde-like products, related by sequential conversions. Thefirst chromogenic product is linked to DNA, and itsformation does not entail the release of a base. It de-composes readily to the second product, a compoundcontaining the base and deoxyribose carbons 1-3. Hy-drolysis of either product yields the third, which isindistinguishable from authentic malondialdehyde.These findings suggest that the oxygen-dependentcleavage of DNA by Fe(1I). bleomycin can begin withthe rupture of the deoxyribose 3-4-carbon bond. Theinitiation of these events is concurrent with the initia-tion of another mode of DNA degradation, involvingthe release of free base alone, in a yield similar to thatof chromogen.

~~

The degradation of DNA by bleomycin, which is believedto underlie the antitu moractivity of this antib iotic( l ) ,followsthe formation of a bleomycin complex with both Fe(I1) andmolecular oxygen (2). The mechanismof ihe ensuing reactionwith DNA is obscure, and none yet proposed can account forall the DNA degradation products so fardetected.Thesereaction products include freenucleic bases (3-6) and baselikespecies containing the base label of their nucleoside precursors( 7 ) .The residual oligonucleotides bear few, if any terminal3-phosphate groups, but 5 termini are predominantly phospho-rylated (8).Material resembling malondialdehyde is also pro-duced (4 , 8 ) and is thoughtto derive from deoxyribosidecarbons 1-3 (6) as is a very similar product of X-irradiatedDNA (9, 10). Nucleosides, nucleotides, and free orthophos-phate are not liberated(3 ) .

Th e sequence specificity of susceptibility to Fe(I1) . bleo-mycin has been studied by measurements of base release (5-7) and by sequencing of oligonucleotide products (6, 11).Thebases released bybleomycin are enriched in pyrimidinesabout3-fold over their abundance in th e degraded DNA. An addi-tional strong preference is shown for att ack at nucleosidesHealth Service Grants CA 15714, CA 23187 (to S. B. H.), and HL* Thi s investigation was supported in partby United States Public13399 (to J. P.),and by American Cancer Society Gra nt CH-86 (toS.B. H.) , This paper is Communication 399 from the Joan and LesterAvnet Institute of Molecular Biology. TheNMR facility at th eAlbertEinst ein College of Medicine was supp orted by the BiotechnologyResources Prog ram, National Institutes of Health Division of Re-search Resources Grant RR 00636. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefo re he hereby mark ed adu ertisernent in accord-ance with 18 U.S.C. Section 1734 solely to in dicate this fa ct.$ Recipient of an Irma T . Hirschl Career Scientist award.

adjoining the guanydyl3-phosphate.Material resemblingmalondialdehyde,which is derived

from lipid oxidation, has long provided food chemists with asensitive assay of rancidity (12) by means of its chromogenicreact ion with 2-thiobarbituric acid, yielding an intensely col-ored adduct (EW = 1 .6 X 10 (13)) (Fig. 1 ) . Th e colored adductcharacteristic of malondialdehyde is also formed from prod-ucts of DNA, degraded by ionizing radiation (9,10, 14 , 15), orby aerobic Fe(I1) solutions (16),as well as by bleomycin. Th epossibility t ha t th e chromogen from DNA might not be ma-londialdehyde, but a precursor of malondialdehyde, was ap-preciated by Kapp and Smi th( l o ) ,who found that the chrom-ogen precipitated with X-irradiated DNA, while authenticmalondialdehyde did not.In an attempt to determine the chemistryof DNA breakageby ferrous bleomycin, we tr ied toisolate physicallythe malon-dialdehydereportedlyproduced.Ourrecovery of malondi-aldehyde indistillates of bleomycin- treated DNA reactionmixtures was so inferior to tha tfrom model mixtures contain-ing authenti c malondialdehyde th at we re-examined our re-action mixtures by chromatographic fractionation. Our anal-ysis indicates th at in bleomycin reaction mixtures, the chrom-ogen previouslyconsidered to bemalondialdehyde is notmalondialdehyde, but rather consists of two intermediates,each containing the deoxyribose carbons 1-3 derived from aninitial drug-induced cleavage of the 3-4 carbon bond of thesugar. These intermediates can react with 2-thiobarbi turicacid to give an adduc t identical with the one produced bymalondialdehyde, or they can undergoacid or basehydrolysisto produce malondialdehyde.

EXPERIMENTAL PROCEDURESPreparation, Assay, and Deriuatization of Malondialdehyde-

Malondialdehydewas generated (17) frommalonaldehydebis(dimethy1 acetal) (Aldrich) by treating a 5 mM solution with 0.1 NHC1a t 50C for 1h in a stoppered flask. The solution was then cooledand diluted5-fold with water. Stock solutions were stored a t10C forup to 2 months with no loss, based on colorimetric assay with 2-thiebarhituric acid (13 ) ,and samples elutedsimply from Sephadex G -10 columns (Fig. 2).We assayed malondialdehyde by heating a sampl e to 92C withexcess 2-thioharhituric acid a t pH 2 to 3 for 20 min, cooling, andmeasuring A331. Samples (50.3 m l ) were m ade up to 0.8 ml with asolution containing 42 mM 2-thiobarh ituri c acid and 1 mM EDTA.Assay mixtures containing 0.05 to 8.0 nmol of rnalondialdehydeobeyed Beers law. Th e published value of E M = 1.6 X lo5 M cm (13)agrees withour assay standardization,for which we assu med thatmalondialdehyde was obtained quantitatively from th e bis(dimethy1acetal).with a Cary model 118 recording spectrophotometer thermostated atTh e course of color development was monitored, whenappropriate,86C. Prehe ated cuvett es containing 3 ml of 35 mM Z-thiobarhituricacid and 1mM EDTA received 0.2-ml samples of authentic malondi-aldehyde or aerobic DNA reaction mixtures containing Fe(II).hleo-mycin. They were then tightly stoppered, and A5.v~was measured a t1-min intervals. First order rate constants were calculated by com-puter, using least squares criteria, asdescribed (161.

11832


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Malondialdehyde from DNA Degradedby Fe(I4.Bleomycin 11833Th e 2-thiobarbituric acid adduct of malondialdehyde was preparedaccording to Sinnhuber et al. (18) from th e bis(dimethy1 acetal) in 1

N HC1. A yield of 84% as washed crystals was obtained, with nospectrally detectable excess of 2-thiobarbituric acid. Aliquots weredissolved in H 20 for optical spectroscopy, and in perdeuterated di-methyl sulfoxide (299.5 atom B 'H; Aldrich) for 'H NMR. A similarpreparation of crystals was made from material distilled at 80Cunder a stream of N2 (18), from a 100-ml reaction mixture of 0.5 mMbleomycin, 2.5 mM DNA, and 1.0 mM Fe(I1) in 20 mM sodiumphosphate buffer, pH 7.0, subsequently adjusted with HC1 to pH 1.5prior to distillation. Optical spectra were obtained using a Cary model14R recording spectrophotometer with 1-cm pathlength cuvettes.Fourier transform 'H NMR spectra were obtained with a Briikermodel WH-360 spectrometer at th e University of Pennsylvania Re-gional NMR Center.Radiolab eled DNA-Variously thymidine-labeled DN As were ex-tracted from purified bacteriophage X, grown in a Thy- host, Esche-richia coli strain RS15, kindly provided by J. A. Wechsler and R. A.Scalafani, Department of Biology, University of Utah, SaltLake City(unpublished strain). Its genotype is tonA lam B str thyA deoC (ordeoB) (Xrr857 Sam7). This heat-inducible, lysis-defective bacterio-phage X lysogen efficiently utilized exogenous thymidine in DNAsynthesis.Amersham supplied thymidine, containing either U-I4C, methyl-I4C, 5'-'H, 6-'H, or 1',2',methyL3H labels. Bacteria were grown expo-nentially in Pennassay broth (Difco) at 30C to 2 X 10' cells/ml,heated to 42'C for 20 min, and then cooled to 37C. They thenreceived 85 pg/ml of uridine, followed at once by tracer amounts oflabeled thymidine, as recommended by O'Donovan (19). After 3 moreh of incubation with vigorous aeration, cells were harvested, lysed,and DNase-treated, and their bacteriophage were then purified byisopycnic banding in two successive CsCl gradients, all as described(20). DNA was extracted with formamide (21) and dialyzed four timeswith 200 volumes of 0.1 mM EDTA, 0.1 M Tris/Cl, pH 7.5, for 8 heach, then twice with 200 volumes of 20 mM sodium phosphate buffer,pH 7.0, for 8h each time. Doubly-labeled DNA solutions wereobtained by mixing portions of the appropriate preparations.Fe(ZI). Bleomycin/DNA Incubations-Bleomycin sulfa te (Blenox-ane) was the gdtof Bristol Laboratories and containedapproximately60% hleomycin A2, 30%bleomycin Bs,and 10%various other bleo-mycins. Solutions were prepared daily in 20 mM sodium phosphatebuffer, pH 7.0, and standardized optically, using 292 = 1.45 X lo4 M"cm" (22). Calf thymus DNA (Worthington) was dissolved in thesame buffer and standardized optically, using 2 ~ )= 6.6 X lo 3 M"nucleotide cm". Fe(I1) solutions, from reagent grade Fe(NH&(SO&.6H20 (Baker),were prepared in water 99%) before subsequent trea tments. Somecompleted reaction mixtures were then heated to 50C for 10 min;othe rs were exposed to 0.1 N HCI or NaOH at 92C for 10 min, thencooled and neutralized. For controls, unproductive reaction mixturesof similar composition were obtained by adding th e DNA las t, insteadof the Fe(I1) (16).Th e stability of the chromogenic products and their 2-thiobarbi-turic acid adducts to oxygen was demonstrated by addition of anaer-obic 2-thiobarbituric acid solution to a completed reaction mixturetha t had been equilibrated with argon. No difference in AIa2 wasobserved between this and the color-forming reaction under air.fractionated by precipitation of DNA with ethanol. received half the

Fractionation of Reaction Products-Reaction mixtures to beusual concentrations of bleomycin and Fe(I I), in order to diminishth e formation of small, ethanol-soluble oligonucleotides. One equiv-alent of chromogen was formed per 56 eq of oligonucleotide phosphateprovided. Using an ethanol ice bath, reaction constituentswere equ&ibrated to -5"C, which was measured with a YSI model 42 ScTelathermometer and a Teflon-sheathed, solution-type thermistorprobe. Those few reaction mixtures that froze were discarded. Reac-tion mixtures (100 pl) contained 18 mM sodium phosphate buffer , pH7.0, 1 mM DNA containing [6-3H]thymidine (1.6 Ci/mol), 0.1 mMbleomycin, and 0.12 mM Fe(I1). Th e last was added to initiate the

reaction, which was run for 5 min at -5C before addition of 1M LiCland 0.25 mM undigested nonradioactive DNA, followed at once bythe addition of 3 volumes of cold (-5'C) ethanol. The mixtures werecentrifuged at 18,000 X g for 2 h a t -25"C, and the precipitates wereredissolved in 50 p1 of 20 mM sodium phosphate buffer, pH 7. Super-natants and precipitateswere assayed for radioactivity and P-thiobar-bituric acid-reactive material. Each assay mixture was supplementedto contain the sameamount ofLiCl andethanolpresent in thesupernatants.

Sephadex G-10 columns served to fractionate some products by gelfiltration and others by adsorption chromatography (23). Columns(18X 1cm diameter) were equilibrated and eluted with 20 mM sodiumphosphate buffer, pH 7.0, at 6C. DNA eluted at 6.0 ml, and 'Hz0eluted at 11.5 ml, which were taken to indicate void and includedvolumes, respectively. The reaction mixtures analyzed were incubatedat 0C. Reaction aliquots (0.6 ml) were applied to th e column, eitherat once, after heating to 50"C, or after base hydrolysis with 0.1 NNaOH at 92'C for 10 min followed by neutralization. Columns wereeluted at a rate of -4 ml/h with a hydrostatic pressure head of 12 to14 cm. Thirty-nine 0.66-ml fractions were collected, followed by 3.3-ml fractions. Of each fraction, 0.3 ml was assayed with 2-thiobarbituricacid, and the remainder was mixed with 5.0 ml of TT-21 scintillant(Yorktown) and assayed for radioactivity by scintillation spectrome-try. Recovery of radioactivity was always complete (95 to 105'%),butrecovery of chromogenic activity gradually decreased (from 90%) asthe column was re-used, except tha t the chromogen in hydrolyzedsamples was always completely recovered.Reversed-phase thin layer chromatography of radioactive DNAreaction products was carried out on Analtech RPS 0.25-mm plates,developed with ascending 4 M ethanol, 15 mM sodium phosphatebuffer, pH 7, at 6C for 4.5h. The solvent front moved 15 cm.Reaction mixtures contained 0.2 mM [6-'H]thymidine-labeled DNA(30 Ci/mol of nucleotide), 50 ELMbleomycin, 50 p~ Fe(II), and 18 mMsodium phosphate buffer, pH 7.0, plus 5p~authentic [methyl-Clthymine (55 Ci/mol) used as an internal standard. They wereincubated at 0C and analyzed either before or after base hydrolysisat 92C in a sealed glass capillary. Chromatography was startedpromptly after applying a 1-p1 aliquot to thethin layer a t 6C withoutdrying. Fractions (0.5 cm) were scraped from the support plate forscintillation counting in Aquasol (New England Nuclear).

14

RESULTS2-Thiobarbituric Acid Reaction Products and Kinetics-When Fe(I1) is added to aerobic mixtures of bleomycin and

DNA, the ensuing reaction produces material which can reactwith 2-thiobarbituric acidto form an adduct having the samespectral characteristicsas the 2-thiobarbituric acid adductofmalondialdehyde. The optical absorption spectrum (Fig. 1 ) isidentical with that of the authentic malondialdehyde adduct,as reported previously (5, 8, 24), and the 'H NMR spectra(Fig. 1) are consistent with the knownstructure (17) . Thefluorescence spectra have also been reported to be identical(25).Although these 2-thiobarbituric acid adducts appear iden-tical, several observationslead us to conclude that they arisefrom differentprecursors. The most evident difference be-tween authentic malondialdehyde and the chromogen fromDNA is in the kinetics of their reactions with 2-thiobarbituricacid. At 86"C, the adduct from malondialdehyde forms ho-mogeneously with tlp = 2.2 min, while the reaction with theDNA products is >90% complete in 2 min, the time of ourearliest observations. If the Fe(1I).bleomycin/DNA reactionmixtures are exposed to 0.1 N HC1 or 0.1 N NaOH for 10minat 92OC before the 2-thiobarbituric acid reaction, theirratesof subsequent color development are equal to the rate withauthentic malondialdehyde.

Another difference, which is similarly nullified upontreat-ment of reaction products with acid or base, is seen in thestability of chromogenic activity. The DNA-derived chromo-gen is lost from reaction mixtures at 6C with t,,P = 70 h,while the chromogenic activity of authentic malondialdehydeadded to unproductive control incubations is stable, like our1 mM malondialdehyde stock solutions.


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11834 Malondialdehyde from D N A Degraded by Fe(I4 - Bleomycin

WAVELENGTH (nm) CHEMICAL SHIFT (ppm)FIG. 1. Structure and spectra of the 2-thiobarbitu ric acidadduct with malondialdehyde. The left panel depicts the opticalspectra, and the right panel depicts the 'H NMR spectra of the 2-thiobarbituric acid adducts with:A, authentic malondialdehyde,or B,the malondialdehyde-like productof DNA degradation by oxygenatedFe(II).bleomycin. The baselines have been arbitrarily offset. Theoptical spectrawere obtained from:A, an unproductive200-pl reactionmixture (0.1 mM Fe(I1) added to 0.1mM bleomycin,followed by 1 mMDNA), supplemented with2.2 nmol of authentic malondialdehyde, orB, a productive reaction mixture. Eachwas treated with 2-thiobarbi-turic acid. The shoulder at 453 nm is due to a reaction of 2-thiobar-bituric acid with the iron present. The NMR spectra were obtainedfrom the crystallized products of 2-tbiobarbituric acid refluxed withmalondialdehyde bis(dimethy1 acetal) (A) ,or with the distillate of ableomycin/DNA reaction mixture ( B ) ,as described under "Experi-mental Procedures." NMR sample A contained 10 mg of adduct/ml,and sample E contained 0.1 mg/ml. The indicated chemical shifts

were determined with respect to tetramethyl silane. The structureproposedfor the malondialdehyde adduct (18) is shown with ourN M R assignments. The exchangeable protons givea broad resonanceat 9.06 ppm (not shown). Doubletand triplet coupling constants are14Hz.

A minor but consistent difference is also seen in th e effectof ethanol on theyield of adduct. Addition of 1 to 5 M ethanolto the 2-thiobarb ituric acid assay mixture has no effect oncolor development with malondialdehyde, but enhance s theyield from the DNA products by 8%.

These results are consistent with the hypoth esis that reac-tion mixtures contain a produc t that may be converted tomalondialdehyde by hydrolysis. T he prod uct is less stable,and reacts faster than does malondialdehyde in forming th e2-thiobarbitu ric acid adduct.

Fractionation of 2-Thiobarbituric AcidChromogens-Ma-londialdehydedoes notco-precipitate with DNA in coldethanol, but when bleomycin-treated DNAis ethanol-precip-itated, as described in the legend to Table I, as much as 88%of its chromogenic product is recoverable in the prec ipita te.Such completeness of precipitation is lost if react ions areperformed at higher temperatures, for longertimes, or containa smaller ratio of DNA to bleomycin.

A more detailed analysis of the DNA degradation productswas obtained by fractionating reaction mixtures ona Sepha-dex G-10 column (Fig. 2). Four chromogenic fractions wereresolved, each present in an am ount dependingon the condi-tions of the reaction and of postreaction treatment.

Whena0Creaction mixtureis applieddirectly to thecolumn (Fig. 2b), most of the chromogenic material elutes intwo fractions: the first with thevoid volume (P eak I ) , and thesecond subsequen t to t he included volume near the positionof the pyrimidine bases (Peak 3).A minor amoun t of chrom-ogen elutes at about four times theincluded volume (Peak 4)near the position of the purine bases.

A rechromatography experiment suggests th at the chrom-

TABLEIEthanol precipitation of Fe(II). bleomycin-degradedD N A products

Reactionmixtures (100-pl) contained 18 mM sodium phosphatebuffer, pH 7.0, 1 mM UN A containing [6-3H]thymidine(1.6 Ci/mol),0.1 mM bleomycin, and 0.12 mM Fe(I1).Reactions were run for 5 minat -5C before addition of 1M LiCI, 0.25 mM carrier DNA, and then3 volumes of cold ethanol. Both supernatant and precipitate wereassayed for radioactivity and 2-thiobarbituric acid chromogens asdescribed under "Experimental Procedures." Supernatants of other-wise identical mixtures that had received Fe(I1) before the additionof DNA to bleomycin had one-fifthof the radioactivity and one-tenthof the chromogen; these have been subtracted incalculating thetabulated values.__

Material Assayed in ethanolSupernatantPrecipitatenmol

[6-"H]Thymine radioactivity 0.29 282-Thiobarbituricacid addend 0.21 1.6ogen appear ing in theregion between peak s 1 and 3 (Fig. 2, b,e, and h)is due to therelease of chromogen from DNA duringchromatography.WhenthecentralPeak 1 fractions of areaction mixture were applied within 1 h of collection to anidentical column, the chromogenic material eluted mainly asPeak 1 , but 15% of it eluted as Peak 3.

When DNAcleavage reactio ns are run atroom tempe ratureor warmed after their completion, the transfer of chromogenfrom Peak 1 to Peaks 3 and 4 is enhanced. It is almostcomplete in mixtures heated to50C (at pH7) for 10 min (Fig.2c). Chromogenic material elutinglike authentic malondialde-hyde is now manifest in a minor shoulder preceding Pe ak 3;no trace of color is obtained between the vestige of Peak 1and this shoulder.

When reaction mixtures are treated with 0.1 N NaOH orHC1 at 92C for 10 min, the n chilled, neutralized, and chro-matographed (Fig. 2d), all chromogenic material elutes likemalondialdehyde, in Pe ak 2. The ra teof color development inthe 2-thiobarbitu ric acid assay of these fractions is also char-acteristic of authen tic malondialdehyde and is unlike that ofthe chromogens eluting elsewhere (Peaks 1,3, and 4 ) .Fate of Thymine Radioactivity -The origin of the mater ialseluting in Peaks2, 3, and 4 was investigated by fractionatingreaction produc ts of radioactive DNA. A react ion runat -5"C,stopped at


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Malond ialdehyde from D N A Degraded by Fe(II) - Bleomycin 11835I 1 I I I 1

MDA

0.61 L. 1 2 3 40.4 Cu* 0.20 . 4 p0.2 r\ 92CpH12.

pH 12

EFFLUENT(ml)FIG. 2. Sephadex G-10column fractionation of Fe(I1)-bleo-mycin-degraded DNA products. The DNA cleavage reactions tobe fractionated were incubated at OC, and the reaction mixtureswere applied to the column directly or afterthe indicated treatments.Columns were run at 6C and pH 7.0, as described under Experi-mental Procedures. a, the elution of the indicated reference com-pounds (arbitrary ordinate); b to d, the elution of chromogenicincubation products; e to g, the elution of radioactivity from DNAwith [U-4C]thymidine;h toj , the elution of radioactivity from DNAwith [6-3H]thymidine. The ordinates indicate the radioactivity perfraction and the A 6 3 2 developed in assaying a 0.3-ml aliquot with 2-thiobarbituric acid. The arrows indicate the elution position of ma-londialdehyde (MDA).The peaks are numbered for reference to thetext. Reference compounds tested but not shown here are: formate,thymidine, and cytosine, which elute with peaks centered at 10,13,and 15.5ml, respectively.

chromogenic Peak 3 product isnot demonstrableusing Seph-adex G-10 columns, since thymine itself elutes inPeak 3.

Thin layer chromatography of Fe(I1). bleomycin/DNA re-action mixtures reveals that muchof the thymine label first

released from DNA partitions not as free thymine, but as aseparable species that is susceptible to hydrolysis, and thenyields a product with the mobility of the free base. When adigest of [6-3H]thymidine-labeled DNA is fractionated byreversed-phase thin layer chromatography (Fig. 3 ) , most ofthe radioactivity remains associatedwitholigonucleotidesnear the origin, but the remainder is found in two mobilefractions. One ( R F= 0.73) co-migrates with anauthentic[14C]thymine interna l marker ; the otheris less mobile ( R F=0.56). Unlike thymidine ( R F = 0.75; notshown), this lessmobile fraction is susceptible t o hydrolysis in 0.1 N NaOH at92C for 10min. When a reaction aliquot is hydrolyzed beforechromatography, the fractionhaving R F= 0.56 is absent, butthe radioactivityfound to co-migrate with thymine is en-hanced bythe amountotherwise found inth e missing fraction.The r ati o of these two products is variable and depends onthe separation system used. We obtained results similar tothose of Povirk et al. ( 7 )when we used a cellulose thin layer,but found that therecovery of the non-thymine ( R F= 0.56)product is much enhanced with reversed-phase chromatog-raphy.Fate of Deoxyribose Radioactivity-On Sephadex G-10fractionation, all t he radioactivity in digests of [5-3H]thymi-dine-labeled DNA elutedwith the oligonucleotide fraction, asin Fig. 2, Peak 1, unless the completed reaction had beenhydrolyzed prior t o fractionation. Th e radioactivity then re-leased (5%)eluted as3H20and probablyderives from tritiumexchange w ith the solvent, No radioactive formate was de-tected in anyof our react ion mixtures.

When DNA containing[U-4C]thymidine is incubated withbleomycin but nothydrolyzed (Fig. 2, e and f ), the distributionof label inthe SephadexG-10 effluent is qualitativelylike tha tof thymine-labeled DNA (Fig. 2, h to j ) .When the reactionmixture was first hydrolyzed, radioactivity was also found inthe Peak 2 eluant (Fig, 2g); otherwise, no new peaksorshoulders in the distr ibutio n of radioactive produc ts are dis-cerned. The distr ibutio n of radioactivity in Peak 2 eluants

5 6\ pH12.392CX10 0.2 0.4 0.6 0.8 1.0

RfFIG.3. Thin layer chromatography of Fe(II) bleomycin-de-graded DNA products. The DNA cleavage reactions t o be fraction-ated were incubated at OC, and 1-pl aliquots were applied to thereversed-phase chromatography plate either directly or after basehydrolysis and neutralization, as indicated. The reaction mixturescontained 0.2 m~ DNA labeled with [6-3H]thymidine, 50 PM bleo-mycin, 18 mM sodium phosphate buffer, pH 7.0, and 50 PM Fe(I1).plus 5 p~ authentic [rnet/~yZ-~C]thymine. Chromatograms were de-veloped for 4.5 h at 6C with ascending 4 M ethanol, 15 mM sodiumphosphate buffer, pH 7.0, and assayed for radioactivity as describedunder Experimental Procedures.The amount of tritium ( R F= 0.56,0.73) released from DNA is comparable to that in Fig. 2, but thedetectionof DNA tritium at the sample origin is impairedby quench-ing.


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11836 MalondialdehydefromDNA Degraded by Fe(II).Bleomycin(Fig. 2g) appears identic al with t ha t of th e chromogen (Fig.2d).

The release of deoxyribose fragments was studied quanti-tatively using DNAdoublylabeled with [U-'4C]thymidineand [6-3H]thymine. Fe(I1)ebleomycin digests of this DNAwhich were otherwise unt reated (O'C), warmed to 50"C, orbase-hydrolyzed were fractionatedonSephadex G-10 col-umns, and the ratiosof isotopes releasedpermi tted calculationof the fraction of thymidine carbons appearingin Peak 2 and3 eluants. Thus,for example, if only the thyminebase moietywere releasedina particularreaction,the fraction of [6-"Hlthymine label released would be twice th at of th e [U -l4C]t,hyrnidinelabel released, since only half of the thymidinecarbon atoms are in th e base. Such calculations a re inter -preted cautiously: they express average s tha treflect apossiblemixture of products.

The results of these experiments are summarized in Table11. When a 0C reaction mixture is applied directly to thecolumn, the material eluting after Pea k 1 contains 8 (of 10)thymidine I4C carbons forevery[6-3H]thymine equivalentreleased. Th e 5'-3H (and, presumably, th e 5"carbon) was seento remain associated with t he oligonucleotide fract ion (PeakI ) , and no thymidinewas detected.When reactionsrun at 0Careheatedto 50C beforecolumn chromatography, our calculations indicate that 92%of the increaseinreleased 14C radioactivityderives fromthymine and8%derives fromdeoxyribose products. The num-ber of thymidinecarbons found per eq of [6-3H]thymine,either in Peak 3 or in all fractions subsequent to Peak 1, isnow 6. This would result if, a t 50"C, for every new fragmentcontaining 8 thymidine carbons, fourfragments now appeared,containing only 5 carbons. Although th e release of thyminelabel more than doubles on heating to 50"C, no significantincrease in total chromogen is seen.

When the same reaction produc ts are hydrolyzed beforecolumn chromatography, littl e additional radioactivity is re-leased, but the products are altered so tha t only Peak 2 ischromogenic. It now includes some deoxyribose radioactivitythat would otherwise have appeared in Peak 3 (Fig. 2, f andg).Th e overall I4C:'H fractional release ratio remains 0.6, butwhen Peak 3 alone is considered, the ratio is 0.5, indicatingthat Peak 3 now contains only thymine. The deoxyribosecarbons now elute as malondialdehyde i n Peak2.

A parallel experiment was done using DNA labeled with[1',2',n~ethyZ-~H]-and [methyZ-'4C]thymidine (Fig. 4 ) . DNAcleaved a t 0C released equal fractions of both labels, whichis consistent with a continuing association of base with deox-yribose carbons 1'-3'. However, in this experiment, notall th e

TARLEI1Release of base and deoxyribose moieties from D N AThe double label incubations of Fig. 2, e to j , were analyzed by

comparing the fraction of [U-"Clthymidine label released to thefraction of [6-3H]thymidine label released. A ratio of 1 signifies thestoichiometric release of base label and nucleoside label. Details aregiven under "Experimental Procedures."

Label eluting %/"H elutingTreatment Thymidine after: after:label 1 O m l 14ml 10ml 14 ml

70 '% "C/% 3 HO T , pH 7 U"4C6-'H9.6 6.67' 4 5' 3 0.77 0.8050C. pH 7 U-14C6-3H 22.1 20.113'4 12" 0.61 0.6092OC,pH 12.3 u-"C6-3H 23.822.5 14.' 0.62 0.50

"L O? ?0 0x xl-1-Ea0I0

3t n i03:b0

EFFLUENT (ml)FIG.4. Fractionation of deoxyribose-l',2'-tritiated DNA re-action products. Reactions were run and fractionated as describedin the legend to Fig. 2, except for the isotopic labels used. DNA

contained [n~e thyl-'~C ]-and [1',2',methyl-3H]thymidine.The arrowsindicate the elution positions of 3H20 and of malondialdehyde ( M D A ) .

released 3H appears in Peak 3 about 20% of th e released "Helutes as3H20. An exchange of 2'-3H with solvent could resultfrom enolizationof a 3'-aldehyde, whichis a possible structurefor th e base-sugar fragment. As expected, heating the reactionmixture to50C at pH7 releases additional radioactivity, with

C predominating, but no addition al 3Hz0 ap pears. The'H20detected exceeds by 5-fold that foun d insimilarly treatedcontrols containing undigested DNA.Base hydrolysis releases9%of DNA tritium as 'H20 after incubationwith bleomycin,but releases very little from untreated DNA. These experi-ments are refractory to more complete interpretation, sincethe quantitative distribution of tritium in this thymidine isnot precisely known.

The release at 50C of chromogen fromDNA (Fig. 2c)without therelease of equivalent thymidinedeoxyribose label(Table 11) requires comment. It a ppea rs t hat thechromogenformed at 0C but released from DNA at 50C must derivemainly from nucleosides other than thymidine. Conversely,the chromogen released at 6C might be expected to derivemainly fromthymidine, and the ratioof chromogen recovered(Fig. 221) to the thymine and deoxyriboseradioactivity re-leased(Fig. 2, e and h) indicatesthatthis is so. Thus,itappears that release from DNA of different nucleoside deg-radation productsis differentially affected byincubation tem-perature.

14

DISCUSSIONThe fist observed effects of bleomycin on DNA were a

reduction in meltingtemperature and sedimentationvelocity,reflecting DNA polymer cleavage in vivo and in vitro (26).Muller et al. (4 ) observed the format ion of aldehyde groupsand proposed thi s to be a probable consequence of t he liber-ation of free thymine. They titrat ed 0.58 aldehyde eq perthymine released. Th e aldehydic species wascharacterized byKuo and Haide (8) as malondialdehyde-like in forming thecharacteristic 2-thiobarbituric acid adduct. They noted thesimilarity of products of DNA damaged by x-rays and by


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Malondialdehyd e from D N A Degraded by Fe(Il ) .Bleomycin 11837

Bleomycin+FeIIIl I+0 2

OPO00

[x ] 2%

[VIb'c.

SCHEME1.Steps in DNA degradation following ferrous bleo-mycin treatment. Twomodes of DNA disintegration have beenresolved, one releasing a chromogeniccompoundcomprising thecarbon atoms of the base and a %carbon deoxyribose fragment, andthe other releasing free base. The early DNA products, [XI and [yl ,are ethanol-insoluble, but upon warming, release the fragments de-picted. The products enclosed in brackets have not been definitivelycharacterized, but the carbon linkages depicted in the 8-carbon nu-cleoside fragment seem probable in lightof its precursor and products.

bleomycin. Free bases, 5"phosphate termini, and a malondi-aldehyde-like chromogen were formed.

Haidle et al. (3) and subsequent workers (5-7) observedliberation of all four bases. Haidle et al. (3) proposed that thebleomycin acted primarily by removing bases,as analkylatingagent.Such lesions would then rend er the DNA polymerunstable. Closer examination of the produc ts of the drug-degraded DNA by Povirk et al. (7,27) revealed fur ther com-plications: the DNA contained alkali-labile sites in addition tobreaks, and thebase-like products included species th at weredistinguishablefrom authenticfree base. Th e alkali-labilesites were attributed to base-free deoxyribose residues (27).

Th e possibility tha t the bre aks occurring without alkalitreatment give rise to a derivatized base species is suggestedby the discovery (14) among the produc ts of irradiated de-oxynucleotides, of compounds comprising base and deoxyri-bose fragments, aswell as the malondialdehyde-like chromo-gen. Products originating in DNA degradation and in lipidoxidation resemble malondialdehyde in their product with 2-thiobarbituric acid but appearby other criteria tobe differentfrom, though possibly precursors of, malondialdehyde (10,28).

Scheme 1 summarizes our interpretation of the reactionsinitiated by bleomycin. The early productsof DNA degrada-tion, X and Y, are relatively stable at low temperatures andare macromolecular. Theymustcontain lesions, however,which predispose them to two modes of disintegration. Evena t low temperatures, Y slowly releases acompound containingthe carbon atomsof th e nucleic base and3 of th e 5 deoxyribosecarbons. This compoundis relatively stable in its chromogenicproperties, and a hydrolysis procedure is necessary to cleavethe deoxyribose fragment, as malondialdehyde, from the nu-cleic base moiety. (At room temperature and pH 7, the stoi-chiometry of chromogen formed per DNA cleavage is about1.)'Following cleavage of the deoxyribose 3'-4' bond, carbons5' and, probably, 4' remain associated withthedegradedoligomer. A mechanism forsuch a cleavage has been proposed(29).The othermode of disintegration takeseffect when reaction' R. M. Burger and S. B. Horwitz, manuscript in preparation.

I3PO3

o=c c-N '9Z'COH-.03c--c' c=o

The 8-carbon compound is released at low temperatures ( -6C); thefree base isreleased at higher temperatures. The former may behydrolyzed, yielding base and malondialdehyde as shown. The C-3'-aldehyde and C-4'-hydroxyl functions shown are conjectured, to ac-count for patterns of tritium exchange from carbon atoms 2' and 5'.Hydrogens bonded to carbon or nitrogen have been omitted fromthese drawings. The released bases are shown as thymine, the onemost often released; the polymer cleavage event is shown occurringadjacent toa 3"guanidylphosphate, the highly preferred site.

mixtures arewarmed, releasing freethymine and,presumably,other bases. This lesion yields no malondialdehyde-likechromogen, but theremoval of nucleic base renders theresid-ual phosphodeoxyribose oligomer susceptible to cleavage (27)in moderate alkali. Thus, under appropriateconditions, DNAincubated withoxygenated ferrous bleomycin may releasemalondialdehyde and free nucleic bases, a compound com-prising the nucleic base and three deoxyribose carbon atoms,a mixture of the latt er two, or nothing. The hypothes is thatDNA cleavage results as a consequence of free base release isonly partly true,since the released compound combining baseand deoxyribose carbons 1'-3' preserves the glycosidic bond.The hypothesis that the free base detected is a breakdownproduct of this compound is, likewise, only partly true, sincebase is also released independently.

The eventspreceding the cleavage reactions were elucidatedby Sausville et al. (30, 31), who demonstrated the necessaryparticipation of both Fe(I1) and 0 2 in the bleomycin-catalyzedreaction. They appreciated theradiomimetic aspects of bleo-mycin activity and proposed that such free radicals as .OHand - O n- might be formed as a consequence of Fe(I1) .bleo-mycin oxidation, and that these might attack DNA. Indeed,the detectionof free radicalsusing spin traps in aerobicFe(I1).bleomycin mixtures (32, 33) has been interpreted asc o n f i i -ing t he proposal th at .OH or . 0 2 - accumulate and damageDNA in a way analogous to th atresulting from irradiation orfrom treatment withaerobicFe(I1)solutions (34-36). Al-though this proposal is attractive, there is no compulsion toassume tha t radicals formed from 02 -Fe(II ).bleomycin au-tooxidation a re the species responsible for the specific DNAcleavage outlined inthis paper. Theoxygenated complex itself(2) may be the a ctiv e species or may give rise to one tha t isnot necessarily a free radical.

Oxidation reactions are well known for the heterogeneityoftheir pathways and products,so the observed release of base,both with and without thechromogenic deoxyribosefragment,does not in itself requ ire tha t bleomycin inflict upon DNAmore than onekind of primary lesion. However, it seemslikelythatthe early intermediates in Scheme 1, X and Y, are


7/7

11838 Malondialdehyde from DNADegraded by Fe(II) - Bleomycindifferent, since the prior formation of one final product doesnot seem to prejudice the yield of the other.

Acknowledgments-We are grateful for the advice and help of Drs.C. Fred Brewer, Felicia A. Gaskin, Pradip Bandyopadhyay, James A.Wechsler, and Robert A. Sclafani. We thank Drs. S. T. Crooke andW.T. Bradner of Bristol Laboratories for supplying bleomycin sulfate.Addendum-After submission of this paper, an abstract appeared

which reported the characterization, by mass spectrometry, of aderivative of adenine plus a 3-carbon deoxyribose fragment obtainedfrom bleomycin-degraded DNA (37) .REFERENCES

1. Umezawa, H. (1979) in Bleomycin: Chemical, Biochemical andBiological Aspects (Hecht, S. M., ed) pp. 24-36, Springer-Verlag, New York2. Burger, R. M., Horwitz, S . B., Peisach, J., and Wittenberg, J. B.(1979) J. Biol. Chem. 254, 12299-123023. Haidle, C. W., Weiss, K. K., and Kuo, M. T. (1972) Mol. Phar-macol. 8,531-5374. Miiller, W. E. G. , Yamazaki, Z., Breter, H.-J., and Zahn, R. K.(1972) Eur. J. Biochem. 31,518-5255. Sausville. E. A., Stein, R. W., Peisach, J ., and Horwitz, S. B.6.7.8 .9.

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21 . Thomas, M., and Davis, R. W. (1975) J. Mol. Biol. 91 ,314-32822 . Dabrowiak, J. C., Greenaway, F. T., Longo, W. E., Van Husen,M., and Crooke, S. T. (1978) Biochim. Biophys. Acta 5 1 7 ,5 1 7 -52 623 . Sweetman, L.,and Nyhan, W. L. (1968) J. Chromatogr. 3 2 ,6 6 2 -67 524 . Honvitz, S. B., Sausville, E. A., and Peisach, J. (1979) in Bleo-mycin: Chemical,Biochemical and Biological Aspects (Hecht,25 . Gutteridge, J. M. C. (1979) FEBS Lett. 105 ,278-282S. M., ed) pp. 207-221, Springer-Verlag, New York26 . Suzuki,H., Nagai, K., Yamaki, H., Tanaka, N., and Umezawa, H.(1969) J. Antibiot. (Tokyo)22,446-44827 . Povirk, L. F.,Wiibker, W., Kohnlein, W., and Hutchinson, F.(1977) Nucleic Acids Res.4,3573-358028 . Pryor, W. A., and Stanley, J . P. (1975) J. Org. Chem. 40, 3615-361729 . Takeshita, M., and Grollman, A. P. (1979) in Bleomycin: Chemi-cal, Biochemical and BiologicalAspects (Hecht,S.M., ed) pp.207-221, Springer-Verlag, New York30. Sausville, E. A. , Peisach, J., and Honvitz, S. B. (1976) Biochem.Biophys. Res. Commun. 73.814-82231 . Sausville, E. A., Peisach, J., and Horwitz,S. B. (1978) Biochem-istry 17,2740-274632 . Sugiura, Y., and Kikuchi, T. (1978) J. Antibiot. (Tokyo)31 ,1310-131233 . Oberley, L. W., and Buettner, G. R.(1979) FEBS Lett. 9 7 ,4 7 - 4 934 . Haber, F., and Weiss, J. (1934) Proc. R. SOC.Lond. Ser. A 147 ,35. Zamenhof, S., Griboff, G., and Marullo, N. (1954) Biochim. Bio-36. Czapski, G., and Ilan, Y. A. (1978) Photochem.Photobiol. 28 ,37 . Takeshita, M., and Grollman, A. P. (1980) Fed. Proc. 39, 1880

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