Bmchim~ta ea BiopRyswe Acta, 1008 ~lggq) 339- M5 339 ~s~wier

BBAEXP 9]976

DNA damage and cytotoxicity of nilracrine in cultured HeLa cells

Leszek Szmigiero and Kaz]mierz Smdzian Delmrtmem of General Cl, em~st~: Institute of Phvsud~Kv and Ba~cltemz~t~'o Sch~d o/ Med:c~ne ~n ~dL / . /~ c P~and~

~Recc~ved 5 Janua~/ 1989~ (~¢vl~ m~nuscnpl ¢e,~vcd ~ April 1989}

K¢~" '~-o~ds: ~,NA d~m,~; DNA tV|oh~xieety. DNA,pro|~n crc~s|mk; N~tmc~nc: fHum~n c¢|L~: ,(HcL~ c¢~| c~*hu~

DNA ~ breakage ~ | n ~ ~ | | ~ i ~ g as wtq| ~s DNA-~'o~e~n c r o g s q ~ n g were ~ r e ~ by

~ d e t ~ in the com:~Wafio~ ~ that h~h~t¢~ ce~ ~ h up |o ~ . DNA .~i~ge-sm~u~ b~e:~ ~e~e

~ n e were D N A - ~ e l n ~ which ~ e d fog 24 h ~ ter drug ~ x , ~ a ~ L | | is concluded th~ DNA b f e ~

|naeductk~

An anticancer drug, nitracrine is known to achieve its DNA ~.~ pro,.6n bi~ding propensity upon reductive activation [I-4]. |n ce|l-frce systems, reduction of the drug by thiols ~eads to the formal/on of irreversib|e complexes with polynucleotides [2.3]. Severn| mam- malian and bactefiM systems were shown to transform nitracfine to ~ c t i v e intemediates which bound cova- lendy to macromolecules [4].

it was reported that treatment of HeLa cell with the drug resulted in DNA interstrand cross-linking [5] and DNA breakage [7]. The ability of nitracfine and some other 1-nitro-9-aminoacridine derivatives to produce in- re,strand cross-links as well as DNA breaks was torte° ia~ed with the cytotoxic activity of these compounds [5.6]. Although it is believed that DNA damage plays an important role in cell killing by nitracfine~ which par- ~icular type of DNA lesion is :esponsib|e for the drug's eytotoxicity remains unclear. In previous studies dealing

Abbreviations: Hepes, dM2-hydroxycthy|b|.piper~neet~nosulfo.'fic acid; EGTA, eth,ylcne glycol bis[2-~ninoeahyl ¢therFN, N, N', N'-te- lraacetic add; DUg. dith/othrcizol: ISC. in|erstrand crossqink: DPC. DNA.protein cross-Enk; SSB, stogie-stand break: ED~, drug dose inhibiting cell proliferation by 50~.

Correspondence: L. Sunigiero. Department of General Chemistry. Institute of Physiology and Biochemistry, School of Medicine in L6dL IAndleya 6. L6d~ 90-131. Poland.

wilh DNA damage c a u ~ by nitr~crine, a ve~, high d~es of the drug were u.~d 15.6L Thus. it is difficuh to distinguish whether a particular type of DNA |e~sion occarred in 1he pharmacologicM|y ,~sonable r~nge of drug dosage, or whether it represents g~me arlehc~s due to DNA from dyirg ceBs.

|n order Io overcome this dffficuhy, we u.~d DNA alkMine elution tec|miquc 1o study the reIalio~ship be- tween DNA damage and nitracfine cytotoxicity. "J~,: high sensitivity o[ this technique enables Io measure° merit of DNA lesions induced by sublethM do~s of 11~e drug. We have focused on ~he cross-linking activity of nitracrine, because DNA interstrand cross-links were postulated as iLs crucial effect [5]. An attempt was ai:~ made to explain the mechanism of DNA |¢sion forma° tion in the isolated nuclei sys|em.

We report here that DNA-protein crogs-Iinks ~ m to be the sole lesion produced in the cel~ by low d o ~ of nitracrine. Slow ~.pair of DNA-prozein crossqinks and correlation between ils frequency and the cytoloxicily of nitracrine suggests that this type ~ff DNA damage may play an important role in cyloto~t~c action of thins compound.

Materials and Methods

Drugs. Nitracrine (Ledaknn) was kindly donated by Professor Jetty Konopa from the Department of Pharmaceutical Technology and Biochemistry, Techni- cal University of Gdahsk, Poland. Stock drug (2.5 pM)

0167-4781/89/$O3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division~

340

was prepared in water and stored frozen, lVlitomycin C (Calbiochem-Behring, U.S,A.) was dissolved in water immedi,'ately before use.

Cells and radioactive labeling. HeLa cells (Flow Laboratories, U.S.A.) were cultured in minimum essen- tial medium (Wytwbrnia Surowic i Szczepionek, Po- land) supplemented with I0% fetal calf serum and 0.02 M Hepes btfff~r ( ~ a , F.R.G.) in monolayer cultures. Cells (5-I0 s) were seeded in 50-cm 2 tissue culture flasks in medium containing [14C]thymidine (0.02 ttCi/ml, Chemapol, Czechoslovakia) and grown for 24 h. Labeled thymidine was removed at least 24 h prior to the DNA damage measurement.

Cytotoxicity studies. Replicating HeLa cells were pre- pared at 200000 cells per 2~-cm 2 flask and 24 h later the drug was introduced. Cells were exposed to the drug for 1 h at 37°C. After 72 h of incubation in fresh growth medium, the cells were trypsinized and counted,

DNA 3~mage measurements. DNA single-strand breaks (SSB), DNA interstrand cross-links (ISC), and DNA-protein cross-links (DPC) were assayed according to the alkaline elution procedure of Kohn et al~ [I0] except that the pumping rate was 0.1 ml/min. Fractions of 6 ml were collected at 1 h intervals for 5 h. Poly- carbonate filters, pore size 1 Itm (Nucleopore, Pleasan- ton, CA, U.S.A.) were used in the SSB and ISC assay. For DPC as ~ l l as for total cross-linking (combined effect of DPC and ISC) measurement, type BSWP poly(vinyl chloride) filters, pore size 2 /zm (Milllpore, Bedford, MA, U.S.A.) were used. The SSB frequency (PEP) expressed in rad equivalents, was calculated by the formula

P log(r I/r o ) . ^ ~ - -

B D ---" ~ ' .4UU r a o

where r l, r o and Ro are relative retentions of [t4C]DNA from drug-treated, untreated and 300 rad-irradiated cells, respectively. The retention end points were taken at,the point of elution when 20 ml of e l u ~ g solution passed through the filter [10]. DNA-protein cross-links f requency ~PcD) in rad equiTalents was calculated by the formula:

P e n = ( 1 1 ).3000tad jr: Ro

where Rt and R o are the fractions of DNA in the slow eluting component extrapolated back to the ordinate, r e f e ~ g to drug-treated and control cells, respectively (see Ref. 10 for details).

Nuclei isolation and treatment with nitracrine, t4C- labeled cells w~e gently scraped from the flask surface, washed and resuspended in buffer B containing 0.15 M NaCI, 5 mM MgC[2, .~ mM KH2PO 4 and I mM EGTA (Serva, F.R.G.) pH 7.0. Cells were lysed and nuclei were

isolated as described by Filipski a~ad Kohn [8]. For treatment of nuclei, 0.2 ml of nuclei suspension ((0.5-1) • 106 nuclei/nd) were combined with buffer B and incubated with 1/~M nitracrine for 1 h at 37°C. Final volume of the sample was 1 nd. To terminate the nuclei treatment 5 nd of cold buffer B was added to each sample, the nuclei was centrifuged fot~ 5 ~ at ~3~ x g, and resuspended in 5 nd of cold buffer B. The micro- somal proteins preparations were obtained according to the procedure described in Ref. 9.

Results

Cytotoxicity The cytotoxic effect of nitracrine was assayed by

measuring the drug inhibitory effect on HeLa cell pro- liferation (Fig. 1). Nitracrine was found to be a potent antiproliferative agent. Its EDs0 calculated by linear regression was 0.12/xM. Mitomycin C, used as a refer- ence compound in this study, exhibited EDs0- 0.39 ~tM. A total inhibition of cells proliferation occurred when cells were treated with 1/~M nitracrine for 1 h. The higher drug concentrations were highly toxic and caused detachment of cells from the flasks bottorf~, several hours after drug treatment•

, i

I00

50

10

5

0,5

Fig. 1, The effect of different nitracrine concentrations on the pro- fiferation of HeLa cells. Cells were treated with drug for 1 h at 37°C in growth medium. Cell number relative to control was determined after 72 h. The points are means + S.D. of three independent determi-

nations,

0.1 i • t ! • • I , |

O 0.5 I,O flitracrine (~JM)

1.0 0.9 0.6

03

_0.E ¢ u l

- - 0.5

== 0.4

as , I E

L----1

- 0.2

._~

$.

0.1 | !

10 Eluling volume

20 30 (ml)

Fig. 2. Effect of nitracrine on DNA in HeLa ce:ls. Cells were exposed to drug for 1 h at 37°C and harvested. EDNA from control cells (®); nitracrine concentrations: 0.5/tM (~ m)~ 5/~M ( ~ A). Assays were performed on poly(vinyl chloride) filters without proteinase K (open symbols) or on polycarbonate filters with proteinase K (filled sym- bols). DNA from cells was treated with 5 FM nitracaine and post-in-

cubated in drug-free medium for 6 h. Assay with proteinase K (A).

DNA breakage The presence of SSB in DNA from drug-treated

HeLa cells was as~yed by means of alkaline elution, The rate of elution depends on the molecular size of polynudeotide chains. Undamaged DNA elutes slowly. Whe, n SSBs are introduced into DNA elution, kinetics increase [10]. Treatment of cells for 1 h with nitracrine concentrations lower than 0.5/zM did not change the elution patterns (data not show~). At a concentration of 0.5 /tM, nitraerine produced a very low number of DNA breaks (Fig. 2). Elution rates increased signifi- cantly at highly toxic drug doses. Elution curves pro- duced by 5 / tM nitraerine were not linear which suggest a different break frequency induced among different cells in the population. Such elution profilos may be due to the presence of dead or dying cells. As shown in Fig. 2, DNA breaks were detected in elution assays which include deproteiniTation. When lysates were not treated with proteinase K, DNA elution curves were very close to that of the controls.

DNA breaks caused by nitracrine were found to be irreversible. The break frequency produced by 5 ttM nitracrine increased sharply during 6 h post-d.~g in- cubation of cells (Fig. 2). A s,.'milar tendency was ob-

341

100

A

w

e o

' ' I I I I

12 20, 36

Time Iollowieg drug renloval (h) Fig 3. DNA single-strand breaks as a function of time after treatment of HeLa cells with 0.5 FM nhracrine. Cells were exposed to drug for 1 h at 37 °C and then incubated in drug-free medium. The points are

means :1: S.D. of three independent experiments.

served when cells were treated with 0.5 ~tM nitracrine and then incubated in drug-free medium (Fig. 3). A very low DNA break frequency found immediately after

!.0 0,9 0,8

0.7

0,6

0,1 ,,, , ' " 0 I0 20 3 0

Elulion vol~Jme (nd } Fig. 4. Alkaline elution assays for total DNA cross-finking and DNA interstrand cross-linking. Control cells and cells treated with the drug for 1 h at 37°C were harvested and irradiated on ice with 300 tad of y-radiation. Total cross-linking assays were performed without pro- teinase K on poly(vinyl chloride) filters (open symbols). ISC ~',ssay~ (filled symbols). DNA from control cells (o, ®). Nitracrine concentra- tions: 0.5 ~M (o), I FM (~, A). Assay for ISC induced by 2.5/tM

mitomycin C (il).

.E

"-'0.5

ia3

~-=0.2

342

drug treatment increased about 5-times during 36 h of incubation.

DNA cross-linking by nitracrine The rate of alkaline elution is reduced both by ISC

and by DPC. Both types of DNA lesion can be dis- tinguished on the basis of their susceptibility to pro- te]nase K digestion. The effect of ISC on elution rates does not depend on proteinase K digestion, while the DPC-induced reduction of the elution rate is reversed after deproteinization [10]. Fig. 4 shows elution patterns for control cells and cells treated with different nitracrine concentrations. Prior to the lysis, cells were irradiated with 300 rad of "l-radiation in order to obtain a suitable D~A elution rate [10]. When cross-link assay was per- formed without proteinase K, the elution rates were reduced showing total DNA cross-linking (combined effect of ISC and DPC). Deproteinization of cells lysates prior to the elution, reversed the effect of the drug (Fig. 4.) Hence, the observed decrease of the elution rate is due exclusively to DPC. The ability of nitracrine to [n<~uce ISC was compared with that ¢.f mitomycin C. At

concentration of 2.5 pM, which inhibited cells pro- h~eration to 6% of control, this antitumor antibiotic induced a high frequency of ISC (Fig. 4).

It is known that some cross-linking agents produce the highest ISC frequencies several hours after drug removal [13]. In the case of nitracrine, this possibility was also tested. Cells were exposed to 0.5/tM and 1.0 ttM nitracrine for 1 n and then incubated in drug-free medium. At 3 h and 6 h ISC had not appeared (data not shown). Longer post-treatment times were not tested because DNA breaks increase during cell incubation. An additional attempt to detect nitracnne induced ISC was made. The method of D'Incalci et al. [14], enabling measurement of the labile DNA lesion was used. Also in this assay, ISC were not detectable (data not shown).

DNA-protein cross-linking by nitracrine In order to measure the concentration dependence of

DPC frequencies a specific assay including T-irradiation with 300 rad was used [10]. The DPC frequency was not saturable and exhibited a linear dependence on drug concentration up to 1 ~tM (Fig. 5). DPC formation occurred at very low doses of nitracrine, also below 0.5 ttM when DNA breakage was not detectable (compare Fig. 2). The highest DPC level was observed im- mediately after drug treatment. During 3 h of post-drug incubation, breaks persisted without significant change. Slow repair occurred later, and after 24 h about 30~ of the initial DPC frequency w~.s found (Fig. 6).

To determine whether the growth inhibition effect of nitracrine was correlated with its DNA-protei~3 cross- linking activity, the cytotoxicity data were plotted against DPC frequency at a given drug concentration (Fig. 7). The regression line was log F = -1 .225.10-2

2OO

lOg

+

- 015 1.0 Nitracrine I~M)

Fig. 5. Dependence of DN'A-protein cross-links frequency on nitracrine concentration. Cells were exposed to drug for l h at 37 o C. DPC were assayed immediately after drug treatment. The points

represent data from independent determinations.

[DPC] + ( - 0.237) where F and DPC are surviving frac- tion and yield of DPC expressed in rad equivalents, respectively. These data suggest a linear relationship between the surviving fraction and DPC frequency (cor- relation coefficient, r - - -0.95).

Induction of DPC wag also examined in an isolated nuclei system. As nitracrine is believed to be activated in the cell by a reductive metabolism, nuclei were treated with the drug in the absence or in the presence of some reductants (Table I). When reductants were omitted from the incubation mixture, no DPC was formed. NaBH4, which is known to be an activator of quinone- containing anticancer drugs [15] failed to activate r.ltracrine. A somewhat surprising result is that also DTI" did not activate nitracrine for DPC formation. This is because DTT is an efficient activator of 1-nitro- 9-aminoacridines, causing their irreversible binding to

IOC

• @

+®

I I I I ~..I.-- 0 6 t~ 18 ~

Time following drug r~maval {h )

Fig, 6. DPC frequency as a function of time after treatment of HeLa cell with nitracrine, Cells were exposed to 0.5 mM nitracrine for | h 37 °C and then incubated in drug-free medium. The points represent

data from independent experiments,

=_=

i m

i

50 I00 t5O 200 0PC hequenc~ (tad equivalenls)

Fig. 7, The relationship between the inhibitory effect of nitracrine on HeLa cells growth and its DNA-protein ¢ross-finking activity. The

bars shows S.D. of at least three independent determinations.

TABLE I

Induction of DPC by nitracrine in isolated HeLa nuclei in the presence of different activating systems

Isolated nuclei were treated w~th 1 pM nitracrine as described in Materials and Methods. The concentration of microsomal proteins was 120-360 t~g/ml. DPC frequency expressed in tad.equivalent.

Activation system DPC frequency

None 0 Dithiothreitol 2 mM 0 LiBH41 mM 0 NADPH 0.2 mM 0 NADPH + micro~omes 104 + 12 a

a Means + S.D. of four independent determinations.

polynucleotides in ce!l-frec systems as has been demon- strated [1-3]. The only activating system that was able to stimulate DPC formation by nitracrine was micro- somal fraction of HeLa cells in the presence of 0.2 mM NADPH. The DPC frequency measured in isolated nuclei was abot~t 2-times lower than that observed in intact cells. It is worth mentioning here tha~t DNA sh~81e-strand breaks as well as interstrand cross-finks were not detected in isolated nuclei whatever activation system was used (data not shown).

Discussion

Extensive studies on the mode of nltracrine action have been carrie~ e,ut in ~everal laboratories [l-7,16]. The recent inter~:st ;in n;tracrine is due to the fact ~,hat this drug bearing a re~dil~ ret3ucible nitro group was shown to be ~ei#~tivety to~,; towards i~yp~ :~ac ce!!~ [17]. Konopa et al. [3-.7] .ev,.deno~d ~hal rutracrine is a potent DNA cross-linking and DNA breaking ag:~nt. In these experiments, however~ DNA damage was observed at high toxic drug doses. We made an attempt to examine whether the same types of DNA lesion arc induced by

343

low drug doses. In the present work, DNA damage was measured by the widely used, sensitive method of al- kaline elution [10]. As regards the DNA breakage by nitracfine, this lesion was not observed when drug doses close to EDs0 were used (Fig. 2). Significant DNA degradation occurred after exposure of the cells to 5 ~M rfitracfine for 1 h. Our results confirm previous data from Woynarowski et al. [7], who found substantial DNA breakage in HeLa $3 cells exposed to the drug for 2.5 h at concentrations above 1.25 ~M. Besides the known capability of nitracrine to form covalent com- plexes with DNA, this drug is ~t~o an intercalator [1]. A common feature of many intercalating agents appears to be their ability to produce protein-associated DNA breaks in the cell [11,12]. It was evidenced that this type of cellular lesion is mediated by topoisomerase II (see Ref. 21 for a review). In the case of nitracrine, DNA breaks were apparently masked by proteins (Fig. 2), which raises the questions of whether DNA damage occurs in the manner typical for topoisonlerase ll-medi- ated drugs or whether rdtracrine induces such a lfigh frequency of DPC that breaks are difficult to detect. Although we did not address this question in detail, the latter possibility is favored. This is because, in contrast to the other acridine derivative m-AMSA, which was demonstrated to induce approximately eqaal frequen- cies of DNA breaks and DPC [13], nitracrine induced severalfold higher DPC frequency than that of DNA breaks (Figs. 3 and 5). Both of these lesions exhibited a different time course during post-drug incubation. DPCs were repaired slowly, whereas the frequency of DNA single-strand breaks increased (Figs. 3 and 6). The above suggests that DNA breakage and DPC formation by nitracrine are two independent processes caused by different mechanisms. The lack of IS(?. in DNA from nitracr~ne-treated cells was an unexpected result (Fig. 4). "[~e drug was reported previously as a potent cross- linking agent in bacterial an~t mammalian cells [5]. Its interstrand cross-linking activity was evidenced by several experimental approaches [5]. Activation of nitracrine by thiols also leads to ISC formation in cell-free systems [2]. Contrary to those data we did not fin~ ISC in cells treated with nitracrine (Fig. 4). This discrepancy may be due to the low drug concentrations and relatively short duration of drug treatment in our study. It is possible that I$C formation by nitracr'ne is too slow to produce detectable amounts of ISC, when the concentration of the drug does not exceed 1 ,~M during ~ h treatment. However, mitomycin C which was shown to be a less active cross-linking agent then ni~tacrine [5], in our s~udy induced a large amount ,'~f ~$t" a~ the ~c.sc ~uitoxic to 0.75 ,~M nitrac~ine ~::ig 4). Th~is last result may indicate a different stab,iJgy of mhomycin C and nitracrine induced iSC ia alkaline solutions. This seems likely, because most ISC assays used previously were performed in neutral solutions or

344

cross-linked by nitracrine DNA which was exposed to alkali for short time (5 rain at 45 ° C) [5]. We used the alkaline elution method in which DNA was exposed to alkali for 5 h. If nitracrine forms alkali-labile DNA cross-links, these adducts could be disrupted by eluting solutions of pH 12.1.

The only DNA lesion detected by the alkaline elu- tion technique in cells treated by nitracrine at doses lower then 0.5/~M remains the DNA-protein cross-link (Fig. 5). The mechanism by which the drug forms DPC is not clear. Topoisomerase II mediation in this process was excluded by Woynarowski et al. [25].

The results obtained with isolated nuclei showed that only microsomal activation in the presence NADPH led to DPC formation by nitracrine (Table I). This is con- sistent with earlier data showing the neecessity of metabolic activation in the binding of nitracrine to DNA [1-4]. Interestingly, thiol activation by D T r was not efficient and no DPCs were found (Table I). This is difficult to explldn, b~ ' a se ~?~:!" has been d~r.:x~n- strated as a nitracrine activator iin cell-free systems [1-3,16] and DNA addtt,~ts form~.~ ~, ~he presence of DTT appear to have a structure similar to that of adducts produced in the cell [22]. It may be inferred from this result that only certain active species gener- ated during reductive activation are able to form DPC and these are not present when D T r is ~:~sed. Although no direct evidence exists, it is probable the.t DPC is caused by covalent binding of active nitracrine metabo- lite to DNA and proteins.

It is known that covalent binding of a chemical agent to polynucleotides, often leads to SSBs, wl~ch are pre- sent in DNA along with ISC and DPC. SSB may be caused by direct interaction of a drug with bases or phosphate groups, or they may be formed as a results of action of DNA-repair enzymes. Such mechanisms of induction of DNA lesions are typical for alkylating agents, e.g., nitrosoureas [23]. There are also some com- pounds which form DNA cross-links by covalent bind- ing and introduce breaks into DNA by a free radical m~hanJ~m as observed with aziridinvlbenzoquinones [15] and 'second generation' platinum compounds [24]. As was mentioned earlier, two independent processes may cause DPC and SSB in DNA treated with nitracrine cells. The lack of SSB at low nitracrine doses which ;reduced DPC (Figs. 2 and 5) seems to be consistent with this assumption. It is possible that the active metabolite of the drug binds to DNA and proteins, but has no DNA breaking a,~tivity. SSBs which appeared at highly toxic nitracrine doses do not seem to be a specific cellular lesion induced by the drug, but they probably represent degraded DNA from killed cells.

Little is known about the biological significance of nitracrine-induced DPC. Its possible involvement in the drug cytotoxicity as well as mutagenicity should be considered. From our findings, the following may sug-

gest a contribution of DPC in the cytotoxic action of nitracrine: (i) slow DPC repair (Fig. 6), (ii) close corre- lation between nitracrine-induced DPC frequency and the antiproliferative potency of drugs (Fig. 7). These results agree with data obtained by Woynarowski eta!. (unpublished data), who have demonstrated a correla- tion between the ability of several 1-nitro-9-aminoacri- dines to form DPC and their growth inhibitor potency against L-1210 cells. On the other hand, DPC induced by a variety of DNA-interacting compounds are not commonly considered as highly lethal to cells (see Ref. 13 for a review). In the case of nitracrine, it is not clear why relatively low DPC frequency may cause cell death. It may be assumed that the drug exhibits high cross-lin- king ~pecificity against some proteins critical for cell survival. As a!kafiv.e elution does not distinguish be- tween different proteins, an alternative interpretation that nitracrine-induced DPC is not necessarily toxic, but this lesion simply reflects reactivity of the drug in ce!ls, is also possi~!e. Further research with nitracrine- resistant c,e|i iines should clarify this question.

The current work may have interesting implications with re, spect to mutagenicity of 1-nitroaeridines. It is know.~ that nitracrine is a much higher mutagenic than its 2,3- and 4-nitroisomers [18-20]. The reason for this disadvantageous feature is not known. It was suggested by Fergus~o6 an~ -i-umer [20] that the DNA lesion responsible for nitracrine mutagenicity is not ISC but an unidentified lesion induced when low drug doses are used. Although there is no direct proof, we suspect that DPC may be this mutagenic lesion. If this assumption is right, the reu'a~tion of DPC-forming ability and (or) enhancement of ISC-fotming ability may be a rationale for synthesis of new potentially anticancer 1-nitroacri- dines.

Acknowledgments

The authors thank Professor M, Gniazdowsld for critically reading the manuscript. We also thank Miss M. Affeltowicz for technical assistance.

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