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151

SHORT COMMUNICATIONS

Binding of radioactive Whydroxy-acetylaminofluorene to synthetic polyribonucleotides

Introduction Our previous studies l-3 have shown the binding of the chemical carcinogen

N-OH-AAF to liver RNA in vivo and in vitro. This binding has been confirmed by several groups * - ‘. Because of its possible importance to our understanding of the pathogenesis of liver cancer, some aspects of the interaction lietween N-OH-AAF and polyribonucleotides in vitro has been studied. The results of this investigation are reported here.

Material and methods Male Wistar rats of 100 g body weight starved over night were used. The

incubation conditions were as follows: 0.5 ml of soluble fraction (1600 rev./min; -25 mg of protein) from rat liver in trismaleate, 0.05 M, pH 7; 1.5 pmole of phos- phorus as polymer (Miles Laboratories, Inc.); 0.20 PCi of [9J4C)N-OH-AAF (Tracerlab, 7.7 mCi/mM) were incubated in a total volume of 2.5 ml for 20 min at 37”. At the termination of the incubation, 2.5 ml of cold-water-saturated phenol were added and extracted 2 times with phenol. The polymer was precipitated with 2 ~01s. of ethanol, redissolved in distilled water and dialyzed against cold distilled water for 24 h. The concentration of polymer in each sample was estimated from its absorption at 260 m,u. The 14C was determined in Bray’s solution and counted in a Nuclear Chicago scintillation counter.

Results Binding to copolymer G.-U (l/I). The data in Table I shows that this in vitro

system binds the radioactive carcinogen to the copolymer G:U. When the enzyme preparation was boiled before incubation, the reaction did not take glace.

TABLE I

BINDING OF [9-‘4CJN-OH-AAF To COWLYMER G:U

Liver extract counts/min per 20 units absorbance

(1) Unheated 682 (2) Heated 44

Conditions as described in methods.

Abbreviation: N-OH-AAF, N-hydroxy-acetylaminofluorene.

Chem.-Biof. Intemctiont. 2 (1910) ! Sl-1Sf

0.025 5s o.ow) 165 0.100 1014

0.m 1280

Conditions as dcscrkd ia methods.

Binding of (9-W]ZV-OH-AAF to t-i#erent polymers. To check for the binding to different bases, @molecular amounti of polymers were incubated with 0.5 pmok of [9-1~]N-GH-MF. The results in Tabk III show that the copolymer G:U binds 32 895 counts/min per 20 absorbance units, then follows po!_ymer G with 18 911. The rest of the polymers also did bind the carcinogen but In small amounts.

BINDING of [9-‘*C)N-OH-MF To DPftLLtNT ~0tYWfu

-- . .- Pdymrr counlr/mln per

Xl absortmnce units --------

G:U 32 893 G 18911 I S 216 A 3 259 C 2861 U 2 725

.- Conditions u described in methods, except that b.3 pmok of [9-‘*GIN-OH-AM (3.83 pCi) were used.

The data reported in this paper suggest that the binding of N-OH-AAF to certain bases in a polynuckotidc chain in vlrro is poaaibk. The binding seems to be specific for guaninc. This ir conoistent with the findings of MILLER et cl.“, of a N- (guanosin-8-yl) AAF.

The modScation of a specific site of a nucleic acid moltculc could alter the normal growth of the cell and allow it to grow with la rcutraint than a tumor cell.

Unpublirhed results from our laboratory indicate that the function of a poly- ribonuckotide as mWengcr in an in V&O protein 8yMbt5i8 8y8.tm, iS mdifd by the binding of the N-OH-AA?.

Chem.-Biol. In!ertxrlonr, 2 (1970) I=!-153

SHORT comwNtcATto~ :53

Supported by a grant from Research Corporation, a Fou~ti~ for Ad- vancement of Science. We wish to express our sincere apprcciatian to Dr. Jowe E. m of the University of Chile for his helpful technical advice.

I2abcmuory Of @Xhi??&t~JJ, Institute for Biology, FERNANDo NAttRoQuIN* University of Mexico City (Mexico) NEUEL COYOTE

I F. MAIUOQUM AND E. Flsraa*, The apparent binding of rndioactive 2-ac#ybminofluomne to rat&et ribonucleic acid k viw, Bloclirim. Bi@tys. Acta. 55 (1962) 403-405.

2 F. MARR~QUIN AND E. FARIKR, The binding of 2-acetylaminofluorenc to rat Liver ribonuckk: sid in tiw, Cunlcer Rcs., 2S (1%5) 1262-1269.

3 F. Mmlrtoqrm AND E F-R, Aparcnte union enzimatica de1 9-1*C-N-OH-acetilamino6~ al acid0 ribonuckko de1 higado de la rata In YIIIO, Communication preliminar, Bd. IIUI. Err& Med. Biol. Mex,, 24 (1966) 187-190.

4 E. C. MILLER, C. W. COOWF., P. D. L.oI~~rr;u AND 3. A. MILLER, The metabolism and car&u+ &city of N-hydroxy-2-acetylaminofluorene (N-HO-AAF) and some of its possibk metabolites, Proc. Am. Ass. Cuncer Bes., 5 (1964) 45.

S R. F. WILLURD AND C. C. IRVING, On the binding of the carcinogen, 2-acetylaminolluonoe, to rat liver RNA and DNA, Fedmrion Rot., 23 (1964) 167.

6 C. M. KIHG AND B. PHILLIPS, Enzyme-catalyzed reactions of the carciaogerz N-hydroxy-2-&m- rcnylacctar?udc with nucleic acids, &fence, 159 (1%8) 1351-1353.

7 J. R. DEBAUN, J. Y. ROWLEY, E. C. MILLF.R AND J. A. MILL@& Suifotmnsferase a&ration of N-hydroxy-Z-acetylaminofluorene in rodent livers sumptibk and resistant to this arci- Proc. Sot. E*p~i. Bioi. Med., 129 (1968) 268-273.

8 J. A. MILLER AND E. C. MILLER, The metabolic activation cJf carcinogenic aromatic %mines md amides, Prop. J!Zxprl. Tumor Ret., 11 (1969) 273-3G1.

Received April 24th. 1970 Accepted for publication May lst, 1970 -- .-- l present address: 2404 Pennsylvania Avenue, .\ciead Johns,ln Research Center, Evansvilk, tadiuu

47721 (U.S.A.).

C&m.-B&l. Interncrionr, 2 (1970) lfl-133