the effect of metabolites of 2-naphthylamine and the mutagen

Nomenclature this paper

x-acetylaminofiuoreneN-hydroxy-2-acetylamino

fluorenex-hydroxy-2-acetylamino

fluorene2-hydroxylaminonaphthalene

Recently it has become apparent that anincreasing number of carcinogens are capable ofinteracting with nucleic acids (10, 14, 923). Theseobservations may be causally related to the mechanism of carcinogenesis as being a special case ofmutagenesis. The chemical reactions of somemutagenic agents such as nitrous acid, hydroxylamine, and alkylating agents with DNA have beenstudied with the view of explaining their mutagenic action.

A need exists, however, for a physical method,sensitive to small chemical alterations in DNA,

S A preliminary report was presented at the Meeting of the

Federation of American Societies for Experimental Biology,Atlantic City, April, 1969..

Thisinvestigationwasaidedby grantsfromAlliedChemicalCorporationandNationalInstitutesofHealth.

ChemicalAbstractnomenclaturewasusedthroughoutthispaper with the following exceptions:

which, according to this hypothesis, could providethe means to predict mutagenic action.

The use of the â€œmeltingâ€•behavior of DNAappeared promising from observations of modifications of this property as a result of ultravioletradiation (16) and reactions with nitrous acid ormustard gas (192).This approach is explored in thepresent paper. In a group of chemically relatedaromatic amine derivatives, only the two orthoaminophenols, known to be active carcinogens atthe tissue level, caused an irreversible alterationin the secondary structure of salmon sperm DNA.Inorganic hydroxylamine, a known phage mutagen(11),wasalsoshownto havea similareffectonthe melting behavior of this DNA.

These changes become apparent through a significantly altered thermal stability, measured byhyperchromicity as a function of temperature.This technic presumably measures the tenacity ofattachment between two strands of DNA, whichin turn is largely determined by the complimentariness of the bases guanine to cytosine andadenine to thymine. That this is indeed the mechanism for hyperchromicity was first clearly demonstrated by the experiment of Warner (927)showinga lowering of ultraviolet absorption when poly U'

1 The abbreviations used are: poly A, polyadenylic acid;

poly U, polyuridylic acid; poly I, polyinosinic acid; poly C,polycytidylic acid.

(Chemical Abstractnomenclature:)

(N-x-fluorenylacetamide)(N-hydroxy-N-9.-fiuorenylacetamide)

(N-(x-hydroxy-2-fiuorenyl)-acetamide)

(N-9.-naphthylhydroxylamine)

Receivedfor publication December9.4,1969..

841

The Effect of Metabolites of 2-Naphthylamine and theMutagen Hydroxylamine on the Thermal Stability

of DNA and Polyribonucleotides*

WALTER TROLL, SIDNEY BELMAN, AND ELLEN LEVINE

(Institute of Industrial Medicine, New York University Medical Center, New York, N.Y.)

SUMMARY

The melting properties of salmon sperm deoxyribonucleic acid (DNA) were used asa criterion for the study of the in vitro reactions of hydroxylamine, a phage mutagen,and aromatic amines related to the bladder carcinogen 92-naphthylamine. The mutagenhydroxylamine and the tissue carcinogens, 92-amino-1-naphthol and 1-amino-92-naphthol, lowered the transition midpoint (Tm) of salmon sperm DNA irreversibly and hadsimilar effects on synthetic polyribonucleotides. The reaction appeared to be specific,since a number of metabolically related noncarcinogens had no effect on the Tm. Thecarcinogen 92-amino-1-naphthol was bound to the DNA in proportion to its ability tolower Tm, whereas chemically similar compounds without action on the Tm were notbound. The parallel action of a mutagen and carcinogen on DNA is of interest to theunderstanding of the mechanism of carcinogenesis.

on April 9, 2018. © 1963 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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8492 Cancer Research Vol. 923, July 1963

and poly A were allowed to combine. The lostultraviolet absorption reappeared when the A+Upolymer was heated, and the strands of poly A andpoly U separated. The hyperchromic effect withheating has been observed with many differenttypes of DNA (13, 15, 17). The midpoint of thisrise of ultraviolet absorption has been called thetransition midpoint (Tm) or â€œmeltingpoint.â€• Aclear relationship of guanine-cytosine content toTm has confirmed the importance of the contribution of these bonds to the tenacity of the DNAhelix (13, 17). With an agent like hydroxylamine,the lowering of Tm can be readily rationalized,because the cytosine in the polymer has beenchemically modified, thus reducing the complimentariness of the two strands. The relative sensitivity of the melting point depression was mdicated by the fact that the ultra violet spectrum ofDNA treated with hydroxylamine was unchanged,whereas the Tm was greatly lowered. We havemade similar observations with synthetic polyribonucleotides which offered the additional opportunity of confirming that it was indeed cytosineand uracil that were attacked in the polymer inthe reaction leading to a lower Tm.

The carcinogen used in our study was 92-amino1-naphthol, the metabolic product of 92-naphthylamine, presumably responsible for bladder cancerin man and dog (5) . This carcinogen appears tointeract with nucleic acids through an unidentifiedoxidation product. When oxidation is preventedby addition of a reducing agent or by esterificationof the phenolic group by sulfate, both interactionwith DNA and tissue carcinogenicity are abolished(5).

MATERIALS AND METHODS

A sample of 92-naphthylamine, labeled in the8-position with C'4, was obtained commercially.This material was converted to 92-amino-1-naphthy! sulfate by the method of Boyland (8), andthis was converted to 92-amino-1-naphthol byhydrolysis at 100Â°C. for S hours with 6 N HC1containing stannous chloride at 92times the molarconcentration of 92-amino-1-naphthyl sulfate. The92-amino-1-naphthol crystallized from this mixtureand was washed with 6 N HC1 containing stannouschloride. Radioactivity was measured in a gas-flowcounter and corrected for self-absorption. Theconcentrations of 92-naphthylamine and 92-amino1-naphthyl sulfate were determined colorimetrically by converting them to naphthoquinone derivatives by reaction with 1, 92-naphthoquinonesulfonate (924). The concentration of 92-amino-i-naphthol was measured colorimetrically by conversion to the 1, 92-naphthoquinone with ferric

chloride and coupling with aniline to form the4-anilino-i, 92-naphthoquinone (925).

The oxidation products of 92-amino-i-naphtholwere prepared by shaking 925 mg. 92-amino-i-naphthol hydrochloride in 925ml. O.i M phosphatebuffer, pH 7.5, and allowing the mixture to oxidizefor 4 days at room temperature. A purple precipitate formed, and no free 92-amino-i-naphtholcould be demonstrated by the colorimetric testinvolving the conversion to the 1, 92-naphthoquinone (925).

The 92-amino-i-naphthylglucosiduronic acid (3)and 92-amino-i-naphthol were prepared as described previously (925), and the 92-amino-i-naphthyl sulfate (8) and 92-amino-i-naphthylphosphate were prepared according to Boylandand Manson (6). Salmon sperm DNA was obtamed commercially and had the ultraviolet absorption characteristics reported in the literature.The polynucleotides poly A, poly U, poly C, andpoly I were obtained commercially from Miles

Laboratories.Reaction of DNA with chemicak.â€”Salmon

sperm DNA, 9250 @ig/m1,was dissolved in 0.1 Mphosphate 0.001 citrate buffer, pH 7.5, and allowedto react with the chemicals tested. Hydroxylaminewas removed by 3 X repeated dialysis againstioox its volume of 0.01 M phosphate, O.OOiMcitrate, pH 7.5 (standard buffer). The aromaticamine compounds were removed by extractionwith an equal volume of water-saturated phenol,followed by three extractions with water-saturatedn-amyl alcohol. Alternatively the DNA was precipitated with indium chloride (1), and the precipitate was washed with ethanol until the washsolution was free of color or fluorescence. The precipitate was then dissolved in the standard bufferwith addition of 0.1 M ethylenediaminetetraaceticacid. After either procedure the salt concentrationwas adjusted to that of the standard buffer bydilution or dialysis. The solutions were diluted to10@ 40 j@gDNA/mi for the determination of theTm.

Deterininalion of Tm.â€”A Beckman spectrophotometer was modified by inserting a heating tapebelow the cell carriage. Tap water was circulatedthrough the thermo spacers. One cuvette was fittedwith a thermometer or glass-tipped thermistor,and absorbancy was determined in i-cm. cuvetteswith a capacity of 3 ml. at i@C. temperature intervals. The temperature rise was kept at approximately i@C/mm. Identical curves were obtained whenheating was carried out at either twice or half thisrate. The Tm was the temperature of the medianabsorbancy increment. The slope was arbitrarilytaken as the average absorbancy rise per degree



TROLL et al.â€”Thermal Stability of DNA 843

centrigrade for the 3Â° preceding and the 3Â°following the Tm.

RESULTSHydroxylamine.â€”Hydroxylamine HC1, brought

to pH 7.5 and allowed to react with DNA over an8-day period at 37Â°,lowered both the Tm andslope. At 4Â°the effect on the melting profile wasmuch smaller (Chart 1). This temperature effectwas observed with all the active agents used andmay represent the consequences of a slightlymodified secondary structure of the DNA athigher temperatures. When cytosine was allowedto react with hydroxylamine following the procedare of Freese (ii), the ultraviolet changes whichhe described were observed to appear slowly overan 8-day period. The absorption spectrum of theDNA solutions after 8 days of reaction withhydroxylamine was identical, however, with that

CHART 1.â€”Salmon sperm DNA (250 pg/ml) in 0.1 as

sodium phosphate, 0.001 as sodium citrate buffer was treatedwith 1 as hydroxylamine adjusted to pH 7.5 at 37Â°C. Sampleswere removed at different time periods and dialyzed for 48hours against 0.01 as sodium phosphate, 0.001 as sodium citrate,pH 7.5. Optical density measurements were performed from9.50 C. to 95Â° C. at 1Â° intervals.

of the control. The relatively lower reactivity ofthe cytosine in the DNA as compared with thefree compound may be the basis for this finding.The lowering of the Tm is apparently a moresensitive indicator of nucleic acid alteration thanthe ultraviolet absorption spectrum.

Similar findings were made with the syntheticpolyribonucleotides. When poly U was exposed to

hydroxylamine and then allowed to interact withpoly A, the melting point was strikingly loweredcompared with the control A + U polymer. Onthe other hand, when poiy A was allowed toreact with hydroxylamine and then treated withpoly U, the Tm was identical to that of thecontrol (Chart 92). This was the result to beexpected from the known reaction of hydroxylamine with uridine and lack of reaction withadenosine (ii) (Table 1). Similar experiments with

I

CHART 2.â€”The reaction of hydroxylamine with poly A and

poly U. Conditions as described in Table 1.

polyinosinic acid and polycytidylic acid revealedthat both reactions resulted in a lowered Tm in thedouble-stranded I + C polymer. Chemical modification of the cytosine-containing compounds hasbeen described (ii). The reaction of hydroxylamine with inosine, however, will require investigation.

Naphthylamine and relaied compounds.â€”Theeffect of interaction of 92-naphthylamine and several related compounds with DNA is summarizedin Table 92.All compounds were allowed to react

E@20

C

a,

C

TemperatureC 50 60Temperature â€˜C



A+U polymer.Tmdegrees

centigradeI+CpolymersTin

degreescentigradePoly

A+UPoly Ah+UtPolyA+UhPolyAh+UhPoly A..+U@Poly A+U,,Poly A.,+U,,,69

.569.553.053 .068 .068.066 .5Poly

I+CPoly Ih+CPolyI+ChPolyIh+ChPoly I.,+CPoly I+C.@Poly @73

655851697165

.MetaboliteConc.

(mg/mi)TmSlope.Cancer inszIuReferences10

Controls (no metabolite)Q-Naphthylamine2-Amino-1-naphthol@2-Amino-1-naphthol+sodium dithionite5

59. *9. @71â€”74

7460733.3

3 .30 .62 .9â€”

+â€”(5)

(9., 5)(+SnCl2,5)1,2.Naphthoquinone

1-Amino-2-naphthol2-Amino-1-naphthyl phosphate

2-Amino-1-naphthyl glucosiduronide9.-Amino-1-naphthyl sulfate2-Ilydroxylamino naphthalene5

55 55

222 @71

6073

7172702.7

0 .83 .0

2 .82.72 .0+

+(bis(2-amino-1.naph.thyl phosphate)

+â€”(5)

Footnote 3(2)(2, 5)Footnote3Oxidized

products of 9..amino.1.naphthol0.5 @631.2

844 Cancer Research

for 8 days at 37Â°C. Significant lowering of Tmwas observed only with 92-amino-i-naphthol andi-amino-92-naphthol. The effect of reaction of92-amino-i-naphthol over a 192-day period withDNA is shown in Chart 3. These results may becorrelated with the biological activity of thesecompounds (Table 92). The aminonaphthols (1-amino-92-naphthol and 92-amino-i-naphthol) areactive carcinogens in the mouse bladder test,whereas 92-naphthylamine and 92-amino-i-naphthylsulfate are inactive (92,5). The carcinogenicity of

the glucosiduronate and phosphate conjugates of92-amino-i-naphthol is probably due to the aminonaphthol formed by enzymatic hydrolysis. Urineand bladder mucosa contain very active phosphatases which have been shown to hydrolyze 92-amino-i-naphthyl phosphate.2 Much weaker glucuronidase activity has also been observed.@Sulfatases, however, although present in urine and

2 S. Belman and W. Troll, unpublished observations.

S G. M. Bonser and D. B. Clayson, personal communica

tion.

TABLE 1

THE EFFECTOFTHEREACTIONOFHYDROXYLAMINEAND 2-AMINO-i-NAPRTHOL ON THE TM OF POLYRIBONUCLEOTIDES

Stock solutions of poly A, poly U, poly I, and poly C (0.5 mg polyribonucleicacid/ml 0.1 M phosphate, 0.001 M citrate buffer, pH 7.5) were treated for S daysat 87Â°C. with 9.mg 9.-amino.i-naphthol/mlpolyribonucleicacid or for 5 days at370 C. with 1 M hydroxylamine adjusted to pH 7.5. The 9.-amino-i.naphthol

treated samples were extracted S times with water-saturated n-amyl alcohol andadjusted to 0.45 M sodium chloride, 0.01 M sodium phosphate, 0.001 M sodiumcitrate. The hydrox@rlamine-treated samples were adjusted to 0.45 M sodiumchloride, 0.01 M sodium phosphate, 0.001 M sodium citrate with a G-9.5 Sephadex column. The reacted sampleswere treated with unreacted polyribonucleicacids in the same buffer solution.

S The samples were placed on a Sephadex.G25 column equilibrated with the

0.45 M sodium chloride buffer and eluted in the outside volume with the samebuffer.

t Subscripth-polymerreacted with hydroxylamineat 37Â°C. 5 days.@ Subscript an-polymer reacted with 9.-amino-1-naphthol at 37Â°C. S days.

TABLE 2

THE EFFECT OF NAPHTHYLAMINE METABOLITES ON THE MELTING OF SALMON SPERM DNA

Salmon sperm DNA, 250 @g/mlof 0.1 M phosphate, 0.OOi M citrate, pH 7.5, was treated with metabolites ofnaphthylamine for 8 days. The solutions were freed of the metabolite by thorough extraction with phenol, n-amylalcohol, and dialysis against 0.01 M sodium phosphate, 0.OOi M sodium citrate, pH 7.5.

S These compounds are not soluble to this extent and were used in suspension.

Vol. 923, July i963



TROLL etal.â€”Thermal Stabilityof DNA 845

tissue, are unable to hydrolyze 92-amino-i-naphthyl sulfate (92). The conversion of 92-naphthylhydroxylamine, which is also carcinogenic,8 to92-amino-i-naphthol by tissue enzymes is a possibility, since the analogous N-hydroxy-92-acetylaminofluorene is converted to ortho-hydroxymetabolites (18).

The presence of a reducing agent such as sodiumdithionite prevents the reaction with the DNA.This parallels the observation that the presence ofstannous ion in 92-amino-i-naphthol abolished thecarcinogenicity of this compound (5). The majoroxidation product of 92-amino-i-naphthol in tissuesis 1, 92-naphthoquinone, which reacts with theamino group of lysine in proteins (4). The 1,92-naphthoquinone is, however, inactive withDNA. Other oxidation products which are possiblecandidates for reaction with DNA are the intermediate-free radical, the iminoquinone, or polymers formed between oxidized products. The

CHART 3.â€”Salmon sperm DNA (250 @g/mlof 0.1 as sodiumphosphate, 0.001 as sodium citrate, pH 7.5) was treated at370 C. for different time intervals with 9.-amino-1-naphthol

(2 mg/mI). Solutions were dialyzed for 48 hours against 0.01as sodium phosphate, 0.001 as sodium citrate. Optical densitymeasurements were performed from 9.5@to 95Â°at 1Â°intervals.

latter view was supported by the observation thataminonaphthol oxidation products did lower theTm of the DNA (Table 92).

A definite relationship was observed betweenthe lowering of Tm and the quantity of radioactivity bound to the DNA when radioactive92-amino-i-naphthol was used as a reactant (Chart4). Reaction of 0.4 p@192-amino-i-naphthol/mmoleDNA phosphorus lowered the Tm by 1Â°.Theradioactivity bound to the DNA was not removed

by amyl alcohol extraction or dialysis. The quantity bound was a direct function of time during

the first 8 hours, probably representing the rateof formation of the active oxidized intermediate.The melting point lowering follows the samepattern with time as the increase in bound material. No significant binding of radioactivity wasobserved when 92-naphthylamine or 92-ammo-i-naphthol in the presence of dithionite was usedin the reaction with DNA. This was ascertainedby incubating 92 mg. of C'4-labeled aromaticamines, containing 4.5 X iO@ counts/min/mmole

CHART 4.â€”Salmon sperm DNA (250 ,@g/ml) in 0.1 as sodiurn phosphate, 0.001 as sodium citrate buffer, pH 7.5, was treated with C'4-labeled 2-amino-1-naphthol (9. mg/mi) at 87Â°C.for various times. The solutions were extracted S times withwater-saturated n-amyl alcohol and dialyzed for 48 hoursagainst 0.01 as phosphate, 0.001 as citrate buffer, pH 7.5. Tmwas obtained as described in the experimental part, and aliquots of each solution were used for measurement of radioactivity in a gas-flow counter.

92-amino-i-naphthol, in the presence of 10 mg.sodium dithionite, with 9250jig. DNA in standardbuffer for 924 hours at 37Â°C. and observing nosignificant radioactivity remaining in the DNAafter solvent extraction and dialysis as describedabove. The lack of effect on the Tm under thesame reaction conditions supports a relationshipbetween binding and effect on Tm shown inChart 4 for 92-amino-i-naphthol.

After the material is bound to the DNA, additional changes occur in the melting characteristicswithout any direct reaction of 92-amino-i-naphthol.When samples were freed of 92-amino-i-naphtholwith n-amyl alcohol after 5 and iO minutes andsubsequently incubated at 37Â°C., lowering of themelting point occurred over a 7-day period, whereas

12Timein hours

Temperatureâ€˜C



846 Cancer Research Vol. 923, July 1963

no significant Tm changes were observed beforethe 7-day incubation (Chart 5). The lowering ofTm observed after 7 days of incubation may bedue to the interaction of purines or pyrimidineschemically modified by 92-amino-i-naphthol.

No decisions as to which purine or pyrimidineis most decisively affected by 92-amino-i-naphtholcould be made by studying the interaction withsynthetic polyribonucleotides. It was apparent,however, that interaction with the carcinogen hada somewhat greater effect on the Tm if either

C

CHART 5.â€”Salmon sperm DNA (250 pg/ml of 0.1 as sodiumphosphate, 0.001 assodium citrate) was treated at 37Â°for5- and10-minute intervals with 9.-amino-i-naphthol (2 mg/ml). Thesamples were extracted 3 times with water-saturated n-amylalcoholand then kept at 37Â°for7 days. When the Tm was determined immediately after the extraction, it was identical to thecontrol curve. The samples were adjusted to 0.01 as phosphate,0.001 ascitrate for melting. The curves labeled 5 minutes and10 minutes were obtained when the extracted samples wereincubated for 7 days.

poly I or poly C, rather than poly U or poly A,was reacted (Table i). Further work is clearlyindicated to learn the precise nature of the reaction of the carcinogen with DNA.

DISCUSSIONAlthough there appears little doubt that com

bination with a tissue constituent must precedethe modification of normal cells to malignant ones,the nature of this constituent has remained ohscure. Many investigators have stressed the importance of protein combination with carcinogens,pointing in some cases to a relationship of protein

binding to carcinogenesis (92i). Indeed, in workreported from our laboratory concerning the combination of 92-amino-i-naphthol with bladdertissue, protein combination with the oxidationproduct 1, 92-naphthoquinone was observed to bequantitatively the most extensive binding (4).

On the other hand, progress in the understanding of the biochemistry of genetics, as well as theclear demonstration that viruses containing RNAand DNA are causative agents of certain cancers,have made nucleic acids an attractive object forcarcinogen reactions. The main difficulty with thehypothesis that nucleic acids are modified bycarcinogens has been a failure of clear demonstration of reaction (921). Yet recent observations ofincorporation of radioactive N-hydroxy-N-92-acetylaminofluorene, after feeding, into liver DNA,as well as the demonstration that DNA bindsdimethylnitrosamine, have given encouragementto the somatic mutation theory of carcinogenesis(10, 14). The present paper, concerning the invitro DNA reaction with carcinogens, demonstrates a high selectivity among compounds forreaction. The carcinogens used in this study arerelated to a group of bladder carcinogensâ€”i.e.,aromatic amines with the para position blocked,such as 92-naphthylamine, benzidine, and 4-aminobiphenyl. The view that these compounds causebladder cancer in man by virtue of their ortho-hydroxy metabolites has long been expounded, andonly recently has this view found a competitorin the hydroxylamine metabolite described byCramer, Miller, and Miller (9) from the analogousacetylaminofluorene. The corresponding 92-hydroxyaminonaphthalene has been observed as ametabolite of 92-naphthylamine in man and dog(7, 926), and this compound has recently beenshown to be carcinogenic in the mouse bladdertest.3 Our in vitro work with DNA demonstratesthat 92-amino-i-naphthol does react with DNAand that 92-hydroxylaminonaphthalene does not.Although the final decision as to the precise natureof the proximal carcinogen cannot be made fromthe available data, the ortho-aminophenol, or moreprecisely an unidentified oxidation product of thiscompound, appears to be a strong candidate forthe following reasons : (a) It serves to explain theimportance of the blocked para position of thesecarcinogens; the block making orthohydroxylationimperative. This is also true for the aminofluorenecompounds since the 4-acetylaminofluorene is inactive as a carcinogen (928). (b) The rearrangementof hydroxylamino compounds is a known chemicalreaction (920). Indeed, the in vivo biological conversion of N-hydroxy-92-acetylaminofluorene toi-hydroxy-92-acetylaminofluorene has been demon

Temperature.C



TROLL et al.â€”Therinal Stability of DNA 847

strated (i8). The mechanism proposed for thisconversion involves deacetylation, which wouldlead to the ortho-aminophenol as an intermediate(920). Although the lack of carcinogenic activity ofthe i-hydroxy-92-acetylaminofluorene on producingmammary cancer on intraperitoneal injections(19) and the failure of 1- and 3-hydroxy-92-acetylaminofluorene on feeding to produce liver cancer(9292)point out the unusual position of the acetylN-hydroxy compound in these experimental cancers, it does not exclude the possible final role ofan ortho-hydroxy amine in the production of thesetumors. No evidence that the acetylated amineswere hydrolyzed before conjugation of the hydroxyl group to sulfate and glucuronide wasobtained, and it is difficult to speculate about theproximal carcinogen from experiments involvingmany metabolic steps such as feeding or intraperitoneal injections at a site removed from theoccurrence of the tumor (i9, 9292). The greatercarcinogenicity of N-hydroxy-92-acetylaminofluorene may in fact be due to its capacity to passcell membranes and then rearrange to an orthohydroxylamine. The reverse reaction, the conversion of the ortho-aminophenol to a hydroxylaminocompound, is highly improbable. This would tendto put the ortho-aminophenol at a later stage inmetabolism than the 92-hydroxylaminonaphthalene.

ACKNOWLEDGMENTS

The continued interest and advice of Dr. Norton Nelson aregratefully acknowledged.

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1963;23:841-847. Cancer Res Walter Troll, Sidney Belman and Ellen Levine PolyribonucleotidesHydroxylamine on the Thermal Stability of DNA and The Effect of Metabolites of 2-Naphthylamine and the Mutagen

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