Dioxyribosa Breakdown

8/3/2019 Dioxyribosa Breakdown

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Volume 14.5,number 1 FEBS LETTERS August 1982

Deoxyribose breakdown by the adriamycin semiquinone and HzOZ: evidencefor hydroxyl radical participationDiana A. Bates and Christine C. Winterbourn

Department of Clinical Biochemist ry, Christ church Hospit al, Christchurch, N ew ZealandReceived 22 June 1982

We report our finding that the reaction between the adriamycin semiquinone (produced by reduction ofthe drug by xanthine oxidase) and Hz02 in N2 causes deoxyribose degradation to a thiobarbituric acid-reactive chromogen. Deoxyribose breakdown was inhibited by scavengers of hydroxyl radicals, providingevidence for the participation of hydroxyl radicals. The reaction was detected in air, but was less efficientin air than in N2. Deoxyribose degradation did not require a metal catalyst, and was inhibited by super-oxide dismutase in air, but not N2. A similar reaction with deoxyribose in DNA may be of major impor-

tance in the antitumour action of adriamycin.Thiobarbituric acid

1. INTRODUCTIONThe antitumour effect bf adriamycin (doxorubi-

tin (HCl) is thought to involve intercalation of thedrug with the DNA molecule and free radical-in-duced scission of the DNA strands [l-3]. One pos-sible mechanism for this strand scission involvescleavage at deoxyribose residues. Breakdown ofdeoxyribose in DNA has been shown to occur withanother antitumour agent, bleomycin, when com-plexed with iron [4,5], and Halliwell and Gut-teridge [6] have found that other systems that gen-erate the hydroxyl radical (OH) also degradedeoxyribose to a thiobarbituric acid (TBA)-reac-tive substance (malondialdehyde). Adriamycin isknown to undergo redox cycling to give the semi-quinone (Adr), with subsequent formation of su-peroxide (02-) and possibly OH [3,7-l 11. OHcan be formed from Hz02 and 02- if an iron cata-lyst is present [121, but Hz02 has also been shownto react directly with Adr- to produce a specieswith the properties of OH which oxidizes methio-Abbreviations: Adr-, adriamycin semiquinone radical;TBA, thiobarbituric acid; OH, hydroxyl radical; 02-,superoxide; DTPA, diethylenetriamine penta-aceticacid.

Superoxide dismutase

nal or a-keto-y-methiol-butyric acid to ethylene(131.To further characterise this species, and to deter-mine whether it could be involved in adriamycin-induced DNA strand scission, we have examinedwhether it is capable of degrading deoxyribose.We report our finding that the reaction betweenAdr and Hz02 causes deoxyribose breakdown.The reaction does not require an iron catalyst, andin air is inhibited by superoxide dismutase. Thisprovides additional evidence that the reaction pro-

.duces OH and suggests that a similar reaction withdeoxyribose in DNA may be of major importancein the antitumour action of adriamycin.2. MATERIALS AND METHODS

The adriamycin semiquinone (Adr) was pro-duced by the xanthine oxidase-catalysed reactionof adriamycin with xanthine, in Nz-bubbled phos-phate-buffered saline (0.01 M sodium phosphate,0.14 M NaCl, pH 7.4) as previously reported [ 141.Reactions with deoxyribose were carried out for1.5 h at 22C, in 10 ml tubes sealed with rubberstoppers, previously bubbled with N2 for 2 min.The reaction rate was found to be approximatelylinear over this time period. Unless stated other-

Published by Elsevier Biomedical Press00145793/82/OOOl-oooO/$2.75 0 1982 Federation of European Biochemical Societies 137


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Volume 145, number I FEBS LETTERS August 1982wise, solutions contained 30 PM adriamycin, 180PM H202,0.38 mM xanthine, 75 PM diethylenetri-amine penta-acetic acid (DTPA), 3.2 mM deoxy-ribose and 8.6 x 10 -3 U xanthine oxidase/ml, in atotal volume of 1.3 ml. The radical generation rate,determined by measuring the rate of reduction of25 I_IM cytochrome c (aA550, c =2.1 x 104) in thepresence of catalase (30 pg/ml), was 0.91 pm01 .l-t . min-t . Similar reactions were carried out inair, or air/N2 mixtures prepared by replacing theappropriate volume of N2 in the tube with air,using a syringe. In the presence of 02, the xanthineoxidase reaction produced a mixture of 02- andAdr. All glassware was washed with 30% nitricacid and rinsed with deionised distilled water.Xanthine, deoxyribose and buffer solutions weretreated with chelex resin to remove contaminatingmetal ions.

adriamycin (Pharmitalia, Barnet, Herts), super-oxide dismutase (Diagnostic Reagents Ltd.,Thame, Oxon, UK), and Chelex-100 (Bio-RadLaboratories, St. Albans, Herts, UK).

3. RESULTS

The method of Gutteridge [ 151 was used for themeasurement of TBA reactivity. 1 ml of TBA solu-tion (1% w/v in 0.05 M NaOH) and 1 ml glacialacetic acid were added to each tube, at the end ofthe reaction. The samples were heated for 30 minat 100C cooled, and A532 read against a reagentblank. The absorbance due to adriamycin was sub-tracted.

The reaction of xanthine oxidase with adria-mycin under N2, in the presence of H202 andDTPA, resulted in breakdown of deoxyribose to aTBA-reactive chromogen. The amount of TBA re-activity depended on the concentrations of adria-mycin, deoxyribose and xanthine oxidase (fig.1).Essentially no reaction occurred if any one of thesethree reagents or Hz02 was omitted (figs. 1 and 2).The reaction was inhibited by the OH scavengersmannitol, ethanol, formate and benzoate (table 1).The amount of inhibition by these scavengers wascomparable to their inhibition of deoxyribosebreakdown caused by Fe2+ (ETDA), xanthine andxanthine oxidase in air, a known OH--generatingsystem [ 161.

All biochemicals were obtained from the SigmaChemical Corp., St. Louis, MO, USA except for

Deoxyribose breakdown in the presence ofadriamycin was less in air than in N2, and de-creased as the 02 concentration increased (fig.2).In the absence of adriamycin, with DTPA present,virtually no reaction was detectable. When noH202 was added, deoxyribose breakdown in-

[Adriemycin] JAM

b

2.0 4.0peoxyribose] mM

0 10 20[Xanthine oxid ese] U/ k11 ~10~

F&l. Adriamycin-dependent breakdown of deoxyribose. Dependence on the concentration of (a) adriamycin, (b) de-oxyribose and (c) xanthine oxidase. Apart from the concentration of variable reactant, reaction conditions are given inthe Methods section. Each point on the graphs represents the mean and standard deviation from 6-10 experiments.

Absorbances were read after I .5 h reaction.

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Volume 145, number 1 FEBSLETTERS

Table 1The effect of radical scavengers on deoxyribose breakdown to a TBA-reactive

chromogen

August 1982

Inhibitor added

Mannitol (mM) 51020

Ethanol (mM) 51020

Formate (mM) 51020

Benzoate (mM) 5IO20

Superoxide 15 (N2)dismutase 15 (10% air)@g/ml) 15 (20% air)15 (air)CatalaseC 17 (10% air)(@ml)

Percent inhibition of deoxyribose breakdown byaAdr- and b02-, Hz02 and

Hz02 Fe2+ (EDTA)33r10 322 130+ 14 452 6412 13 482 1032+ 12 25+ 140? 8 4Ok 656& 10 48f 1226+ 6 242 335211 452 1053+ 12 582 1252? 5 402 2672 8 57* 7802 7 53& 7122 76622172211832 1282+11

a Reactions were carried out as described in the Methods section; in N2, unlessstated otherwise

b Reaction mixtures, in air, contained 15 PM FeS04. 100 PM EDTA, and 0~~generated using 0.38 mM xanthine and 8.6 X lop3 U/ml xanthine oxidasec In the absence of added Hz02

Means and standard deviations for 6-16 estimations are givenTable 2

The effect of Fe2+ and chelators on deoxyribose breakdown to a TBA-reactive chro-mogen

TBA-Reactive product (A532 , , , )N2 Air

Additional 30 /LM No 30 /JM Noreagent adriamycin adriamycin adriamycin adriamycinNo chelator 0.361 - 0.985 0.006 0.183+0.030 0.128+0.057DTPA (75 PM) 0.328 + 0.042 0.006 0.090+0.048 0.021&0.012EDTA (100 PM) 0.575 +0.113 0.043 0.283 2 0.028 0.226 ? 0.03 1Fe2+ (15 PM), 1.000+0.152 0.273+0.042 I.348 + 0.095 1.372 + 0.083EDTA (100 PM)Fe2+, EDTA, 0.191?0.008 0.180?0.006No xanthine oxidaseReactions were carried out as described in the Methods section. TBA reactivity isexpressed as means and standard deviations from 6-10 estimations

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Volume 145,number 1 FEBS LETTERS August 1982

c .1 I I

25 50 75 100Percent air

Fig.2. Effect of 02 concentration on deoxyribose break-down to TBA-reactive products. (0) adriamycin, H202,added. (m) adriamycin, no Hz02 added. (A) No adria-mycin, with or without H202. Reaction conditions aregiven in the Methods section. Radical generation rates(from xanthine oxidase), in pmol . 1-l . min-I, were:~0.2 (no adriamycin) and 0.91 (30 PM adriamycin) inN2, and 2.14 (no adriamycin) and 2.43 (30 PM adria-mycin) in air. Results are expressed as A532 relative tothe value obtained with adriamycin and H202 in N2,

and represent means from 4- 12 experiments.

xsnthlne+xs;thine ox idru\

2superoxidedlsmutase

1lsmutetion OH/ deoxyrlboseJTEA-reactiveFig.3. Sequence of reactions leading to

breakdown.

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chromogen

deoxyribose

creased to a maximum in lo-20% air as the 02concentration increased, and then decreasedslightly (fig.2). This reaction was also H202-depen-dent, and was inhibited by catalase (table 1).Superoxide dismutase inhibited deoxyribosebreakdown in air and air/N2 mixtures (table 1).There was very little inhibition in N2 and this wasprobably due to the presence of traces of &.

The reactions described above were carried outin the presence of DTPA, which complexes iron sothat it cannot catalyse OH formation from Hz02and 02- [ 171. When DTPA was replaced byEDTA the amount of deoxyribose breakdown wasabout 80% higher (table 2). The increase caused byEDTA in the absence of adriamycin was insuffi-cient to account for this difference. In the absenceof a chelator deoxyribose breakdown was ex-tremely variable, and was almost undetectable un-less the reaction was carried out in tubes that hadbeen washed in nitric acid. This was most likely tobe due to the presence of an impurity that inhibitsthe reaction, but which has no effect in the pres-ence of a chelating agent. The reason why EDTAand DTPA affected the reaction differently is un-clear, but no requirement for an iron catalyst wasevident. Although addition of 15 PM Fez+(EDTA) to the reaction mixture under N2 in-creased the amount of deoxyribose breakdown, itwas less than double that observed with EDTAalone (table 2). Most of this increase was account-able for by the reaction between Fe2+ and Hz02(Fenton reaction), with a small additional contri-bution from xanthine and xanthine oxidase, prob-ably via 02- produced from any 02 still present.In air, Fez+(EDTA) caused a much greater in-crease in the rate of deoxyribose breakdown, bothin the presence and absence of adriamycin. Hence,under these conditions, OH production via theiron-catalysed Haber-Weiss reaction predomi-nated over the adriamycin-dependent reaction.EDTA alone also increased deoxyribose break-down in air, presumably by complexing con-taminant iron and catalysing the Haber-Weiss re-action.

4. DISCUSSIONDeoxyribose breakdown to TBA-reactive sub-stances has been shown to occur when Adr is

generated from adriamycin in the presence H202.


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Volume 145, number 1 FEBS LETTERS August 1982The reaction was most efficient under N2, whereadriamycin can accept an electron from xanthineoxidase to generate Adr- [14]. In air or air/N2mixtures, where deoxyribose breakdown was lessefficient, xanthine oxidase produces predominate-ly 02- [13], but because of the equilibrium be-tween Adr and 02- (fig.3), subsequent reactionscould be due to either radical. OH has been shownto cause degradation of deoxyribose to the TBA-reactive substance malondialdehyde [6]. Our pres-ent findings that the reaction between Adr- andHz02 degrades deoxyribose, and that this reactionis inhibited by OH scavengers strongly supportearlier evidence [ 131 hat OH is produced. The al-ternative, that it produces a related species withvery similar reactivity (e.g., crypto-OK, as sug-gested by Youngman and Elstner [ 18]), cannot beentirely excluded. But biologically this is of littleconsequence, since the radical is energetic enoughto undergo reactions typical of OH, and is there-fore capable of causing extensive damage to cellconstituents.

The proposed mechanism of deoxyribose break-down in the presence of adriamycin (shown infig.3) is essentially the same as that proposed formethional oxidation by the same system [13]. Thedecrease in deoxyribose breakdown with increas-ing 02 concentration is due to the fast reaction ofAdr with 02 competing with the reaction ofAdr with H202. The inhibitory effect of super-oxide dismutase is explained by its displacing theequilibrium between Adr and a- to the left,and lowering the Adr- concentration. Hz02 wasessential for the reaction, but only needed to beadded in N2. In the presence of 02 the reactioncould proceed, albeit more slowly, with Hz02 pro-duced either through dismutation of 02-, or di-rectly from the xanthine oxidase.An alternative explanation, that 02- was re-quired for deoxyribose breakdown in the absenceof an iron catalyst, must be excluded because thereaction was much more eflicient in N2 than air,and it did not occur when 02- was generated inthe absence of adriamycin. In the presence of ironcomplexes, OH can be produced from 02- viathe Haber-Weiss reaction. Little evidence for thisor any other iron-catalysed reaction was seen inNz% but in air with 15 PM Fe2+(EDTA), theHaber-Weiss reaction predominated over adria-mycin-dependent OH production. OH production

by either mechanism could potentially occur inbiological situations and both would be inhibitedby superoxide dismutase. The Haber-Weiss reac-tion can take place only if a suitable catalyst ispresent. Whether metal catalysts are generallyavailable has not yet been established, but if so, itis likely to be favoured at high 02 concentrations[19,20]. The Adr--dependent reaction is morelikely to be important if little or no metal catalystis available. It will be more efficient at lower 02concentrations, although some 02 is essential as asource of H202. These conditions could be met inmost tissues.

If deoxyribose breakdown occurs within theDNA molecule, the likely outcome would be scis-sion of the DNA strands. The cleavage of DNA bybleomycin has been shown to involve OH-induceddeoxyribose degradation [4,5]. The adriamycinmechanism must differ from that for bleomycin,which requires co-ordination of both Fe2+ and Q,but the production of OH may be common toboth mechanisms. The adriamycin-dependent de-oxyribose breakdown we have observed requiresthe semiquinone and H202, but not a metal cata-lyst, and is inhibited by superoxide dismutase. Ifthis reaction occurs with deoxyribose in DNA, itcould be the cause of the DNA cleavage thought tobe involved in the antitumour action of adria-mycin.ACKNOWLEDGEMENTS

The help and interest of Professor R.W. Carrel1is gratefully acknowledged. This work was sup-ported by a grant from the Medical ResearchCouncil of New Zealand.REFERENCES111

121131I41151

Lown, J.W., Sim, S.K., Majumdar, K.C. andChang, R.Y. (1977) Biochem. Biophys. Res. Com-mun. 76,705-710.Lown, J.W. and Chen, H.H. (1981) Can J. Chem.59, 390-395.Berlin, V. and Haseltine, W.A. (1981) J. Biol.Chem. 256,4747-4156.Gutteridge, J.M.C. (1979) FEBS Lett. 105, 278-282.Giloni, L., Takeshita, M., Johnson, F., Iden, C. andGrollman, A.P. (1981) J. Biol. Chem. 256, 8608-8615.

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Volume 145, number I FEBS LETTERS August 1982(61 Halliwell, B. and Gutteridge, J.M.C. (1981) FEBS [ 141 Bates, D. and Winterbourn, CC. (1982) Biochem. J.

Lett. 128, 347-352. 203, 155-160.(7) Handa, K. and Sato, S. (1975) Gann 66,43-47.[8] Thayer, W.S. (1977) Chem. Biol. Interac. 19, 265-

278.1151 Gutteridge, J.M.C. (1981) FEBS Lett. 128, 343-

346.[9] Bachur, N.R., Gordon, S.L. and Gee, M.V. (1977)

Mol. Pharmacol. 13,901-910.[lo] Bachur, N.R., Gordon, S.L., Gee, M.W. and Kon,

H. (1979) Proc. Natl. Acad. Sci. USA 76,954-957.[ 111 Goodman, J. and Hochstein, P. (1977) Biochem.Biophys. Res. Commun. 77,797-803.

[ 121 McCord, J.M. and Day, E.D. (1978) FEBS Lett. 86,139-142.

[161 Diguiseppi, J. and Fridovich, I. (1980) Arch. Bio-them. Biophys. 205,323-329.

[I71 Halliwell, B. (1978) FEBS Lett. 92,321-326.(181 Youngman, R.J. and Elstner, E.F. (1981) FEBS

Lett. 129, 265-268.[ 19) Halliwell, B. (1981) Bull. Europ. Physiopath. Resp.17 (suppl), 21-28.

1201 Winterbourn, C.C. (1981) Biochem. J. 198, 125-131.[ 131 Winterbourn, C.C. (1981) FEBS Lett. 136,89-94.

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