[CANCER RESEARCH 35, 305-309, February 1975]

SUMMARY

The mean latent period for skin tumor production by thecarcinogen I 5, 16-dihydro- 11-methylcyclopenta [ajphenanthren-l7-one (Compound IVb) in the mouse was 30weeks for a dose of 60 zg/week and about 45 weeks for 60jzg/week, while at 0.6 zg/week, no tumors were observedduring 100 weeks. Simultaneous administration of theclosely related noncarcinogen (IVa) (54 @zg/week)togetherwith the carcinogen at 60 zg/week had no effect on themean latent period. Simultaneous administration of athreefold quantity of the microsomal enzyme inhibitor7,8-benzoflavone (I) with the carcinogen at the highest doseincreased the mean latent period to 38 weeks, while at theintermediate dose it completely suppressed tumor formation.

Neither ketone IVa nor IVb bound covalently to calfthymus DNA in vitro without prior metabolic activation.After incubation with rat liver microsomes and NADPH inthe presence of air, both ketones bound covalently to addedDNA in vitro, the noncarcinogen (IVa) about four timesmore extensively than the carcinogen (IVb), roughly inproportion to the overall extents to which these ketoneswere metabolized. In contrast, overall metabolism of thecarcinogen (IVb) was somewhat increased by the addition ofa threefold quantity of the inhibitor (I) to the incubationmixture, but binding to added DNA was almost completelyprevented.

These results are discussed in connection with the hypothesis that cellular DNA is the target of the carcinogen (IVb)for tumor initiation.

INTRODUCTION

The ability of 7,8-benzoflavone (I) to inhibit the production of skin tumors by simultaneous application of thepotent carcinogen DMBA2 (II) has recently been demon

1 Recipient of a research bursary from the Imperial Cancer Research

Fund. Presentaddress: MetabolicStudies Laboratory,G. D. Searle& Co.Ltd., Lane End Road, High Wycombe,Bucks HPI2 4HL, England.

2 The abbreviation used is: DMBA, 7,l2-dimethylbenz(a)anti@racene.

Received June 17, 1974; accepted October 14, 1974.

strated by Kinoshita and Gelboin (8, 9). These authorsalso discovered that tumor production by the relatedhydrocarbon benzo(a)pyrene (III) was not inhibited, butrather was somewhat enhanced by this flavone. Theyshowed that metabolism of DMBA was required beforecovalent binding of the latter to DNA could be observed invitro and that the flavone (I) prevented this binding whenadded to the incubation mixture. In addition, binding ofDMBA to the DNA ofmouse skin in vivo was inhibited 60%by this flavone.

The carcinogen 15,l6-dihydro-ll-methylcyclopenta[a]phenanthren-l7-one (IVb) (2, 4) has, in common withDMBA, a phenanthrene ring system bearing a methyl groupin the same relative ring position. Although this compoundis not purely a hydrocarbon, it was of interest to investigate

whether its capacity to induce skin tumors in mice would beinhibited by 7,8-benzoflavone. This paper reports the outcome ofthis experiment and ofexperiments on the influenceof the flavone on the in vitro metabolism and binding of thiscaltinogen to DNA in the presence of microsomes preparedfrom the livers of untreated rats (Chart 1).

MATERIALS AND METHODS

Animal Experiments

The compoundsand mixtures weretestedby twice-weeklydorsal painting of toluene solutions (6 z1) for 50 weeks,followed by observation of the animals for a 2nd year.Groups of formally randomized TO albino mice, 10 maleand 10 female, were used for each solution. The conditionswere generally as previously described (2, 4).

C@A@oo4

w wI II

0R..

a , R:H or3H

b. RaCH3 orC3H3

Chart 1. Chemicalcompoundsmentionedin the text.

FEBRUARY 1975 305

The Relationship between Metabolism, DNA Binding, andCarcinogenicity of 15, 16-Dihydro- 11-methylcyclopenta[a]phenanthren-17-one in the Presence of a MicrosomalEnzyme Inhibitor

Maurice M. Coombs, Tarlochan S. Bhatt, and Cohn W. Vose'

Chemistry Department, Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London WC2A 3PX, England

No. ofmicewithSqua

Squa

GroupTreatmentNo.

oftumorless survivors atmous

papillomamous

carcinoma6mo. 12 mo. 18 mo. 24mo.1lVb,6O@ig/wk14

4 10410@b2IVb,6gig/wk(lst2013 727Iaexperiment)3IVb,6pg/wk(2nd19

11 8262experiment)4IVb,0.6pg/wk20

19 1765IVb,6@ig/wk+ IVa,20 10 2073c54@ig/wk6IVb,60@g/wk+I,19

9 3039@l80@ig/wk-7IVb,6@g/wk+I,20

18 13518@g/wk8I,

l80pg/wk19 18 1259Toluenecontrol2019 17410Clipping

control20 18 18 8

M. M. Coombs et a!.

The carcinogen (IVb) was applied at 3 dose levels: 0.5%w/v, the dose arbitrarily chosen for all previous work(Group 1); 0.05% w/v in 2 separate experiments (Groups 2and 3); and 0.005% w/v (Group 4). This corresponds to 60,6, and 0.6 zg of IVb weekly; the highest dose level is knownto produce a high tumor incidence with a mean latent periodof about 30 weeks (2, 4). Four other solutions, namely, 0.5%IVb + 1.5% w/v of 7,8-benzoflavone (I) (Group 6), 0.05%IVb + 0.15% I (Group 7), 0.5% I alone (Group 8), and0.05% IVI, + 0.45% w/v ofthe noncarcinogen (IVa) (Group5), were also used. Groups painted with toluene (Group 9),or clipped but otherwise untreated (Group 10), served asnegative controls. The dates of first appearance of skintumors were recorded, and the tumors were generallyallowed to grow to 8 to 10 mm in diameter before theanimals were killed. A few sick animals were also killed toavoid loss of skin tumors. Autopsies were performedwhenever possible, but histological examination was confined to the skin tumors; no other tumors were observed byvisual inspection. In previous work with this carcinogen (2,4), skin tumors of this size usually involved the panniculuscarnosus muscle and were, therefore, classified as carcinomas rather than papillomas. With this criterion, the pathology ofthe tumors induced with IVb in Groups 1, 2, 3, 5, and6 is presented (Table I).

Preparation of Tritiated Compounds

The [I l-3H3]methyl ketone (IVb) (specific activity, 24.6mCi/mmole) was prepared as recently described (3). The

[1 l-3H]ketone (IVa) was synthesized as follows: 17,17-ethylenedioxy - 11,12,13,14,15,16 - hexahydrocyclopenta[a]phenanthren-ll-one (100 mg, 0.34 mmole) (I) was reducedwith sodium borotritide (54.6 mg, 1.44 mmoles, 100 mCi!mmole) in dry tetrahydrofuran, under reflux. Exchangeable tritium was removed from the product dissolved indiethyl ether, by washing with N NaOH, and the crude11-hydroxyketal (1 11.4 mg, 33.8 mCi/mmole) was dehydrated and aromatized by heating with glacial aceticacid (4 ml), nitrobenzene (1 ml), and concentrated HC1 (1ml), as previously described (I). The product was purifiedby chromatography on Wöelm Grade II alumina (10 g) toyield pale yellow needles of the [1 l-3H]ketone (IVa) (88mg; specific activity, 20.4 mCi/mmole), m.p. l99_20l0[literature, 202—203°(1)], UV and infrared spectra identicalwith those of an authentic, unlabeled specimen.

Metabolism and DNA Binding

Calf thymus DNA and NADPH were purchased fromSigma Chemical Co., London, England. A Spinco preparative ultracentrifuge was used for high-speed sedimentations. Radioactivity was counted in a Nuclear-ChicagoModel 2 liquid scintillation counter using Triton phosphor(Triton X-lOO, 500 ml; PPO, 4 g; toluene, 1 liter); counting times were adjusted to give l0@ counts and were corrected for quenching. Thin-layer chromatography wascarried out on glass plates (20 x 20 cm) coated to a thickness of 0.25 mm with Silica Gel G (E. Merck AG, Darmstadt, Germany) and dried for at least 20 hr at ambient

Table INumber of mice surviving without tumors and histology of induced tumors at site of application

Each group initially consisted of 10 male and 10 female animals.

a The number of tumors unavailable for histology were in Group 1, 2; in Group 2, 1; and inGroup 6,2.

b One additional animal had a tumor (spindle cell sarcoma) at the site of application.

C One additional skin tumor of latent period 67 weeks regressed when it had reached 2 mm in

diameter.

306 CANCER RESEARCH VOL. 35

DNAbindingindex(jzmolcsketone/moleMicrosomalof

DNASubstratepreparationphosphorus)IVbPreparation

IActive34.4 ±3.OaIVbPreparation

2Active18.1 ±1.2IVbPreparation

3Active46.5@ 2.5IVbHeat

inactivated (2mm at100°)0IVb

+ 3 moleequivalentsActive07,8-benzoflavone(I)IVbPreparation

4Active18.1 ±2.0IVbAbsent0IVb

+ 3 mole equivalentsIActive0.87IVaActive77.3±5.0IVaAbsent0a

Mean ± SE.

Effect of 7,8-Benzoflavone on Carcinogenicity

temperature. Before use, the plates were washed by ascending elution with chloroform:methanol (3:1, w/v), andmaterial washed to the solvent front was isolated by drawing a line across the silica gel layer 1 cm from the top of theplate. After the plates were redried in air, the metabolitemixture, dissolved in ethanol, was applied as a thin line Icm from the base of the plate, which was then developedby ascending elution with a mixture of toluene:ethylacetate:methanol ( 15:5: 1, by volume) to the line drawn 1cm from the top. The plate was divided into bands corresponding to fluorescent zones seen in UV at 254 nm, andeach band was carefully scraped off. The metabolites wereeluted from the recovered silica gel with ethanol and theirradioactivity was determined by scintillation counting.When possible, the UV absorption of the ethanolic solutionwas determined with a Perkin-Elmer Model 402 UV spectrophotometer. In each case, the unmetabolized ketone ranas the least polar band, and was identified on the basis ofits RF value, and its fluorescent and UV spectra.

Preparation of the Microsomal Fraction of Rat Liver.All work was carried out at 4°,following the method ofGelboin and Sokoboff (6). Liver from male 75-g SpragueDawley rats, freshly killed by stunning, followed by cervicaldislocation, was homogenized in ice-cold 0.25 M sucrose(10 ml/2 g liver). After removal of cell debris at 9000 x gfor 10 mm, the supernatant was centrifuged at 16,000 x gfor 10 mm to remove mitochondria, and the microsomeswere aggregated at 90,000 x g for 40 mm. The microsomeswere washed by resuspension in 0. 1 M sodium phosphatebuffer (pH 7.4) (5 ml/2 g liver), centrifuged at 90,000 x gfor 40 mm, and finally resuspended in fresh buffer (1.4 ml!1 g of liver) for the binding experiments.

DNA Binding in Vitro. Incubations were carried out in0. 1 Mphosphate buffer at 37°for 30 mm with gentle shakingin air. Each tube contained DNA (2 mg), NADPH (2 mg),microsome suspension (0.2 ml), and 3H-labeled ketone (IVaor b) (30 ;zg) in pure dimethyl sulfoxide (20 jul) in a totalvolume of 3 ml. Metabolism was quenched by cooling in ice,the microsomes were removed by centrifugation at 80,000 xg for 1 hr at 4°, and the supernatant was extracted withwater-saturated phenol (3 ml). The upper, aqueous layerwas separated, treated with ethoxy-ethanol (3 ml), and theprecipitated DNA was sedimented at 3,000 x g. The DNAwas washed with 1:1 v/v ethoxyethanol:water (3 ml), withabsolute ethanol (3 x 3 ml), and with hot absolute ethanol(3 x 3 ml). The residue was finally dissolved in l0@ Msodium chloride solution (1 ml), and DNA phosphorus wasdetermined colorimetrically (7). Aliquots were each madeup to 1 ml with water, and radioactivity was counted in 10ml of Triton scintillant. Each value quoted in Table 2 is amean of 5 replicate determinations.

In Vitro Metabolism. Incubations were carried out withgentle shaking at 37°in 250-mi conical flasks open to air.Flasks contained microsome suspension (5.6 ml), 0. 1 Mphosphate buffer, pH 7.4 (56 ml), NADPH (56 mg, 60.7zmoles), and either labeled ketone (IVa) (1.0 mg, 4.3

jzmoles), or the latter plus 7,8-benzoflavone (3.3 mg, 12.1jsmoles) in dimethyl sulfoxide (0.3 ml). Incubations were

Table 2Extent ofcovalent binding of the ketones, IVa and IVb, to calf thymus

DNA in vitroEach value rcpresents the mean of 5 replicate determinations, and each

section contains results from 1 batch of microsomes. For further details seethe text.

terminated by cooling in ice, followed by extraction withethyl acetate (5 x 25 ml). Emulsions were separated bylow-speed centrifugation, the organic solutions were cornbined, dried over anhydrous Na2SO4, and evaporated todryness under reduced pressure below 40°. The residueswere dissolved in ethanol (10 ml) for chromatographic andspectral examination. The percentages of the total radioactivity recovered in the ethyl acetate extracts were as follows:IVa, 73%; IVb, 67%; and IVb + 7,8-benzoflavone, 69%.

RESULTS

Table 1 and Chart 2 summarize the skin-painting experiments. At the highest dose, the carcinogen (IVb) inducedtumors at the site of application in 17 of 20 mice alive at theappearance ofthe 1st tumor, with a mean latent period of 29weeks in agreement with previous results (2, 4). Reductionof the dose to one-tenth prolonged the mean latent period byabout 50%, to 43 weeks in the 1st experiment, and to 46weeks in the 2nd, and reduced the tumor incidence to 9 of 20and 8 of 19, respectively. At this dose, simultaneouspainting ofa 9-fold amount ofthe inactive ketone (IVa) hadlittle effect on the latent period, although the tumorincidence was slightly higher ( 11 of 20). No tumors wereobserved in the animals painted with one-hundredth of thehighest dose, and their survival was similar to the toluenetreated and untreated controls.

The ratio of carcinomas to papillomas amongst the tu

FEBRUARY 1975 307

Jbflb

IVa

,;,o b p?71

M. M. Coombs et a!.

respectively, and each gave rise to a similar band of metabolites at RF 0.7 to 0.75. In addition, both ketones gavesmaller quantities of a number of more polar metabolites,several of which, by comparison of patterns of R@valuesand fluorescence colors in UV, appeared to possess homologous structures. Incubation of IVb in the presence of a 3-fold amount of the inhibitor 7,S-benzoflavone (I) did notappreciably alter the pattern of these metabolites, but rather more of IVb was metabolized (35%).

No covalent binding ofeither IVa or IVb was observed invitro (Table 2) without metabolism, for the radioactivityinitially associated with added DNA was readily extractedwith organic solvents. After incubation of both ketones withrat liver microsomes and NADPH, nonextractabbe radioactivity remained associated with the DNA. This is interpreted as covalent binding of the ketones, since there is noevidence to suggest loss of tritium from the molecules underthe mild conditions of these experiments. Although thebinding index of IVb varied appreciably with differentbatches of microsomes, as did the extent of its metabolism,there was fairly close agreement among replicate assaysusing the same microsome preparation. Under theseconditions (Table 2, Preparation 4), the noncarcinogen (IVa)bound to DNA about 4 times more than the carcinogen(IVb), while binding of the latter was almost completelyprevented in the presence of a 3-fold quantity of 7,8-benzoflavone in the incubation mixture.

DISCUSSION

A 10-fold reduction in the highest dose (60 pg/week) ofthe carcinogen increased the latent period by 50%, cornpared with an increase of 30% when 7,8-benzoflavone wasapplied together with the carcinogen at this high dose. Theinhibitor therefore substantially reduces the “effectivedose―of the carcinogen in vivo. The complete suppression oftumor formation by the inhibitor with the carcinogen at 6

IVb

pyb bbb b py@-@-@----

RF 1P 0.9 0.8 0.7 0.6 0.5 04 03 0.2

Chart 3. Profiles of metabolites produced by in vitro incubation withrat liver microsomes of the ketones, IVa and IVb, as disclosed by thin-layerchromatography. For details, see “Materialsand Methods.―The followingletters refer to fluorescence colors seen in UV of wavelength 254 mm: b,blue; g, greenish yellow; o, orange; p, purple; y, yellow. In each case, theunmetabolized substrate formed the least polar band (RF 0.9 to 0.95).

0E

E

0z

Chart 2. Date of first appearance of skin tumors and number of mice

with tumors produced by the carcinogen (IVb) at 60, 6, and 0.6 @tg/week,and also on simultaneous administration ofthe noncarcinogen (IVa) and ofthe inhibitor 7,8-benzoflavone (BF, I) at the doses indicated. Experimentaldetails are given in “Materials and Methods.― Neither the differences intumor incidence (a) with IVb at 60 ag/week in the presence and absence ofinhibitor, or (b) in the 3 experiments with IVb at 6 ag/week are statisticallysignificant (p > 0.05). For the difference in tumor incidence between thegroups treated with IVb at 60 and 6@zg/week,p < 0.01. expt., experiment.

mors produced appeared to be dose dependent. At 60 zg/week (Group 1), carcinomas predominated, whereas at 6ag/week (Groups 2, 3, and 5), the reverse was true. Amarked tendency was also noticed for the rate of growthof the skin tumors, as determined by the time taken forthem to grow to 8 to 10 mm from their 1st appearance, toincrease with increasing latent period. Thus for Group Ithis time was 10 to 30 weeks for tumors appearing beforethe 25th week, but was more than 40 weeks for those appearing after the 40th week. Also, histology demonstratedthat tumors of this size were usually carcinomas, whereassmaller tumors were often papillomas. Most of the tumorsinduced with the carcinogen at the lower dose (Groups 2,3, and 5) grew slowly, and more than one-half of them didnot reach 10 mm. This may at least partly account for theobserved differences in the carcinoma:papilloma ratio.

7,8-Benzoflavone was not found to be carcinogenic in thistest system, as was previously established also for mice of adifferent strain (9). When 7,8-benzoflavone was painted,together with the carcinogen, at 60 ag/week (Group 6) at 3times the dose of the carcinogen, it prolonged the meanlatent period by almost 30% (8 weeks) and reduced thetumor incidence to 14 to 20. The carcinoma:papillomaratio and rate of growth of the tumors were similar to thoseobserved with the carcinogen above (Group I). Tumorproduction was totally inhibited by this flavone in the samerelative excess with the carcinogen at 6 @tg/week (Group 7).The general survival of these animals was similar to that ofthe controls.

In a number of experiments, it has been observed consistently that the noncarcinogen (IVa) is metabolized about3 times faster than the related carcinogen (IVb) understandard conditions. In the present experiment (Table 2,Preparation 4), summarized diagrammatically in Chart 3,IVa and IVb were metabolized to the extent of64 and 24%,

6pg +l8pg BF@0 tumorsorO6pg alone

y py

308 CANCER RESEARCH VOL. 35

40

0

@20

S

V

A@•@@

Effect of 7,8-BenzoJlavone on Carcinogenicity

jzg/week, and the failure of the latter to produce tumorswhen administered alone at 0.6 zg/week seem to be in linewith this interpretation.

The manner in which 7,8-benzoflavone reduces the effective dose can be rationalized if we accept that chemicalattack leading to covalent binding with a crucial, cellular,informational macromolecule (DNA, RNA, or protein) isrequired for initiation of the carcinogenic process (10). It isthen apparent from Table 2 that the reactive form of thecarcinogen is generated by metabolism, for no binding toadded DNA occurs in vitro in the absence of active microsomes. If the inhibitor functions by diminishing theamount of proximate carcinogen formed in this way andavailable for covalent binding, it does not do so by generallyinhibiting metabolism, for enhancement of the overallmetabolism of (IVb)@is observed.

In the absenceofthe inhibitor, the in vitro binding of boththe carcinogen (IVb) and the noncarcinogen (IVa) to DNAis roughly proportional to the extent to which these ketonesare metabolized. Thus in both casesmetabolism appearstoproduce chemically reactive derivatives capable of covalentmacromolecular binding. Consequently, the amount ofbinding to DNA in vitro is not positively correlated withcarcinogenicity, for the noncarcinogen (IVa) binds moreextensively. This result is similar to that of Rayman andDipple (12) who were unable to find a positive correlationbetween carcinogenicity and the extent of in vitro binding tosalmon sperm DNA of the strong carcinogen 7-bromomethyl- l2-methyl-benz[a]anthracene in comparison with itsweaker homolog, 7-bromomethylbenz[a]anthracene. Theseauthors likewise failed to establish a correlation withbinding to mouse skin DNA in vivo, after treatment of theanimals with these compounds under conditions used forskin tumor induction (13). They enumerate reasons whylack of correlation between extent of DNA binding andcarcinogenecity does not necessarily exclude DNA fromconsideration as the initial target for cancer induction bychemicals, and this topic has recently been discussed in amore general way be Miller and Miller (I 1).

Nevertheless, the parallelism between, on the one hand,inhibition by 7,8-benzoflavone of skin tumor productionwith either the carcinogen (IVb) or DMBA (8), and on theother hand, inhibition of in vitro macromolecular binding ofthese compounds is noteworthy. The inhibitor also protectsce!ls in culture from the cytotoxic effects of polycyclichydrocarbons (5) and suppresses the microsomal enzymesystem (aryl hydrocarbon hydroxylase) responsible for theirmetabolic activation. It seems reasonable to expect that acloser study of the way in which this inhibitor diminishesthecarcinogenecity of IVb and DM BA will lead to a better

understanding of the nature of proximate forms of these 2carcinogens of related chemical structure.

ACKNOWLEDGMENTS

We are indebted to Dr. L. Pang for confirming the pathology of theinduced skin tumors. We also thank M. Hall for valuable technicalassistance.

REFERENCES

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2. Coombs, M. M., Bhatt, T. S., and Croft, C. J. Correlation betweenCarcinogenicity and Chemical Structure in Cyclopenta[a]phenanthrenes. Cancer Res., 33: 832—837,1973.

3. Coombs, M. M., and Crawley, F. E. H. Characterisation of an

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4. Coombs, M. M., and Croft, J. C. Carcinogenic Cyclopenta[aJphenanthrenes. Progr. Exptl. Tumor Res. II: 69-85, 1969.

5. Diamond, L., and Gelboin, H. V. Alpha-Naphthoflavone: an Inhibitorof Hydrocarbon Cytotoxicity and Microsomal Hydroxylase. Science,166:1023—1025,1969.

6. Gelboin, H. V., and Sokoloff, L. Effects of Methylcholanthrene andPhenobarbital on Amino Acid Incorporation into Proteins. Science,134:611—612,1961.

7. King, E. J., and Wootton, I. D. P. Microanalysis in MedicalBiochemistry, Ed. 3, p. 77. London: J. & A. Churchill Ltd., 1956.

8. Kinoshita, N., and Gelboin, H. V. Aryl Hydrocarbon Hydroxylase andPolycyclic Hydrocarbon Tumorigenesis: Effect of the Enzyme Inhibitor 7,8-Benzoflavone on Tumorigenesis and Macromolecule Binding.Proc. NatI. Acad. Sci. U. S., 69: 824-829, 1972.

9. Kinoshita, N., and Gelboin, H. V. The Role of Aryl HydrocarbonHydroxylase in 7, l2-Dimethylbenz[ajanthracene Skin Tumorigenesis:On the Mechanisms of 7,8-Benzoflavone Inhibition of Tumorigenesis.Cancer Res., 32: 1329—1339,1972.

10. Miller, J. A. Carcinogenesis by Chemicals: An Overview—G.H. A.Clowes Memorial Lecture. Cancer Res., 30: 559—576, 1970.

11. Miller, C. E., and Miller, J. A. Biochemical Mechanisms of ChemicalCarcinogenesis. In: H. Busch (ed), The Molecular Biology of Cancer,pp. 377—402. London: Academic Press, Inc., 1974.

12. Rayman, M. P., and Dipple, A. Structure and Activity in ChemicalCarcinogenesis. Comparison of the Reactions of 7-Bromomethylbenz[a]anthracene and 7-Bromomethyl-12-methylbenz[a]anthracene withDeoxyribonucleic Acid in vitro. Biochemistry, 12: 1202—1207, 1973.

13. Rayman, M. P., and Dipple, A. Structure and Activity in ChemicalCarcinogenesis. Comparison of the Reactions of 7-Bromomethylbenz[a]anthracene and 7-Bromomethyl-l2-methylbenz[a]anthracene withMouse Skin Deoxyribonucleic Acid in vivo. Biochemistry, 12:1538—1543,1973.

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