nuicleoli by histones anid nucleolar proteiins. Proc. Nat]. Acad. Sci. U. S. 53: 626-33. 13. LIlLY ... TELLER, D. C., J. M. KINKADE, JR., AND R. D. COLE. 1965.
Plant Physiol. (1967) 42, 731-735
Interactions of Aflatoxin with Histones and DNA' Homer S. Black2 and Bruno Jirgensons Department of Biochemistry, The University of Texas M. D. Anderson Hospital Tumor Institute, Texas Medical Center, Houston, Texas Received December 12, 1966.
Suimmtl7iarv. The interactions of aflatoxin B, with certain histone fractions and DNA were investigated by means of viscosity measurements and equiilibriuim dialysis. Two main histone fraction,s (F2b and Fl), both lysine-rich, were examined after treatment with the toxin. Fraction 2b and 1 differ in amino acid composition an(l behave differently, in regard to gross conformation, in the presence of electrolytes. Aflatoxin increased the viscosity of fraction 2b bu-t affected the viscosity of fraction 1 only slightly. Equtilibriuim d-ialysis experiments shoowed that aflatoxin was bound to both histone fractions. Aflatoxin also increased the viscosity of DNA and equilibrium dialysis showed that 1 molecule of the toxin -,vas boulnd to approximately every 5 nlucleotides of the nucleic acid. Binding constants for the aflatoxin complexes were calclulated as 1000 for F2b, 700 for Fl, and 5500 for DNA-. The biological implications of these data, in regard to the effect aflatoxin has on the information-transcription process, are discuissedl. Histones have been implicated in the control mechanism of the transcription process. They have been shown to inhibit DNA synthesis in vitro as well as DNA-dependent RNA synthesis (7, 12). Qtualitative changes in the base composition of newly synthesized RNA in the presence of histone have also been reported. Whereas interaction of carcinogens with nuicleic acids have been sttudied extensively, little is know-n of the effects these compouinds have on histone. Ts'o and Lti (22), althouigh tusing rather heterogeneous preparations o,f histone, reported binding of aromatic hydrocarbons and steroid hormones. In view of the possible role histones play, in regard to gene acttion, it seemed worthwhile to examine the interactions of histone and aflatoxin, the carcinogenic metabolite of Aspergilluls flazus. Aflatoxin inhibits amino acid incorporation by liver slices and amino acid activation by liver and Eschiericlhia coli (17, 18). Gabliks et al. (5), reported that in Chang liver cell cultures treated with aflatoxin, protein and RNA levels were lowered wvhereas DNA levels remained relatively constant. Others reported inhibition of DNA synthesis by aflatoxin in huiman lung cell ctultures (11). Binding of aflatoxin to DNA has now been reported (3, 19).
Aflatoxin also produlces a nuimber of toxic effects in plant tisstues. Chlorophyll development in seedlings of Lepidimn sativumin and cotyledons of cottonseedI is inhibited by the toxin (1, 16). Aflatoxin also inhibits mitosis and cauises fragmentation and bridging of chromosomes in roots of Vicia faba (13). Black and Alitschul (1) reported inhibition by aflatoxin of gibberellin-induticed lipase formation in cottonseed. Fturthermore it was observed that uinder certain conditions aflatoxin coulld stimuilate protein synthesis in cottonseed and exhibited similar activity to that of gibberellic acid (10). The present paper describes direct effects of aflatoxin uipoIn 2 histone fractions, comparative experiments with DNA, and discusses the implications of the experimental resuilts in regard to the effects of aflatoxin on living cells.
Materials and Methods Viscosity was measuired with Cannon-U'bbelohode type viscometers at 28.00 (±+0.030). The relationship (t-t. /to where t = flow time of the soluition and to =- flow time of the solvent, was ulsed to determine the specific viscosity. Solvent flow times were 192.67 and 191.18 seconds for D'NA and histone experimenits respectively. Binding of aflatoxin to histone and DNA (highly polymerized calf thymuls DNA from Worthington Biochemical Corp.) was determined directly bv equiilibriuim dialysis experiments. Aqueous soluitions (2.0 ml) of 2 main fractions of puirified calf
1 Supported in part by The Robert A. Welch Foundation Grant G-051, U.S.P.H.S. Institutional Grant FR 0551*1-03-IN-24, and NSF Institutional Grant GU-1009IN-8. 2 Present Address: Department of Biology, Sam Houston State College, Huntsville, Texas 77340.
731
732
PLANT PHYSIOLOGY
thy muis histoine ( F2b or FI) were placed inside dialysis tubing in small plastic screw-top tubes. These solutions were equilibrated with an eqtual volume of water contaiining various concentrations of aflatoxiin B1. Experiments with DNA were condutictedI in the same maniner as for histone uising distilled water or 0.2 \t NaCI as solvents. All mixtulres were equilibrated 112 hours at 5i. A rocking dialysis platform was uised to assture proper mixing. Concentrations of afl'atoxin were dletermine(l spectrophotometrically at 363 mpt (6, 15). A standard solution of aflatoxin was prepared in ethanol anidI aliquo,ts (liluite(l with 2.0 ml of solvent to give the desiredl conceintratioin (5-25 sg/ml). Although aflatoxiin is quite hydrophobic, once the toxin was dissolved in ethanol, dilution with water or salt soluitioin did Ilot significanitly lower the extinction coefficient except at high concentratiolIs. The toxinl solutions remained clear indicatiilg that no aggregatioll occuirred. Onily coIncentrations exhibiting a coeif ficient near that reportedl (EMI = 22,000) were uised. All controls coIltaine(1 a xoltume of ethainol equial to thait of the toxini treaLmelit. The association constailts (K) for the aflatoxin complexes were determine(d from the relationship
slope
rMrL\4ax. K} t P Concentrations of aflatoxin b)oth inside and outside the dialysis tubing were determined and reciprocal the ntumber of moles of plots of llr (where r toxin bound per mole of suibstrate) were plotted against 1/c (where c = molar concentration of free aflatoxin ). The maximuim niumber of moles of toxini botind per mole of histone or DNA (rM./ax.) was determined by extrapolation of this plot to the y axis. The slope of the plot was determined and the relationship solved for the association constaint (K). =
-Molecutlar weights of 14,000 and 21,000 were
used for histone fraction 21) an(l 1, respectively (9,20). The molectular weight of DNA as determined from viscosity measurements was 3.5 X 1-0; (4). Protein concentrations were determined by the biuiret-Folin reaction (14). The (liphenylamine reaction was uised to determiie DNA concenltrationis (2).
Results and Discussion In regard to the 2 histone fractions tested, the toxini inlcrease(l the viscosity of F2b strongly (fig lA) whereas it affected the viscosity of Fl very little (fig iB). From figuire 1A it can be seen that the viscosity cuirve of aflatoxindtreated histone had the same feattires as the cuirve for the putre aquleouts histone l)ut the viscosity valuies were mulch higher. At a concenitrationi of 0.5 % histone the redutcedl specific viscosity was 0.39 dl/g for the aqueous soluitioni whereas in the presenice of aflatoxin the valuie was increased to 0.80 dl/g. At concentrations below 0.5 %, the re(duiced specific viscosi,ty in the presence of the toxin increase(d Nvith (liluitioni exponenltially as did the uintreatedl solutions. Equilibriutm dlialysis experiments showed that the toxinl was bouind to both histone fractions (fig 2). Histone fraotionis F1 and F2b are both lysine-rich. However, F1 has a higher lysine an(d proline content than F2b. Contrary to F2b, the fractioni 1 remains in a disordere,d conformation even in the presenice of electrolytes (8). Approximately 35 molecuLles of the toxiIn were boutnd per molectule of F21). The association constaint for the aflatoxin-F21) ccmplex was 1000. AnI association constant of 700 was cal,cuilated for the aflatoxin-F1 complex with abouit 65 molectules of the toxiin botun1d per molectule of histone.
0.9
0.8
I
1.6
0 0
0.7 0
u
40
1.4
0.6
0
0.5 0
0.4
1.2
o--------
0~~~~~~~
0.3 0
0.5
on
1.0
0
0.5
1.0
Concentration in X FIG. 1A. (left) Effect of aflatoxin on the viscosity of histonie fraction 2b. FIG. lB. (right) Effect of aflatoxin the viscositv of histone fraction 1. One ml of a 1 % aqueous solution of histone was incubated for 2 hours at 5° Concentration
with 100,g of aflatoxini. C(
-
in
%
Control:
-
Aflatoxin-treated.
733
BLACK AND JIRGENSONS-AFLATOXIN, HISTONES, AND DNA
These stuidies indicate that aflatoxin affects in some specific way the gross conformation of certain histone fractions. The increase in viscosity of F2b couild be explained by a stretching effect on the protein or, alternatively, by aggregation. Because the number of molecuiles of aflatoxin bouind
25
20 re) 0
15
x _|o.
10 5
-1'-
2 0
120 x
3
25
75 100 10 FIG. 2. Equilibrium dialysis of histone fractions 2b and 1 Nvith aflatoxin. Two ml of an aqueous solution of fractioni 2b or 1 (500 ,ug/ml) were dialy zed against at) equtal volume of water containing aflatoxin. Concentrationis of the toxin inside and outside of the tubing were used in determination of actual free and bound quantities. About 10 % of the toxin was absorbed to the dialysis tubing. Not more than 4 % of the histone was dial! zed through the tubing. 0 - Fl; - F2b.
0
50
C
40;
0
[
'0~
0
30 .
0
I.0
4.0 3.0 * 2.0 Concentration in % x 103
FIG. 3. Effect of aflatoxin on the viscosity of DNA. A 0.003 % DNA solution was prepared in 10 mNi phosphate buffer (pH 7.0). This solution was incubated at 50 for 2 hours with aflatoxin. Final concentration of aflatoxin was 200,gg/ml of DNA solution. 0 ConAflatoxin-treated. trol; 0 -
-
180
240
Ic50
FIG. 4. Equilibriumi dialysis of DNA with aflatoxin. Two ml of DNA solution (500 lAg/mil) was placed in,side dialysis tubing and dialyzed against 2.0 ml of the solvent (0.2 At NaGI) containing aflatoxin. Controls and determination of free and bound quantities of aflatoxin were the same as described under figure 2.
x
0~~~0
06 N0 35
60
to F2b is relatively small, i.e. 35 (fig 2), aggregation could possibly occur by staggere(l jlnctioln of the fibrotus macromolecules aligning them in a coplanar arrangement. Optical rotatory dispersion measurements indicated that the secondary structure of the histone fraction 21) was not significantly altered by the toxin. In the presence of aflatoxin the viscosity of DNA was greatly increased (fig 3). This indicated that the asymmetry of the DNA molecule was possibly increased due to bindiing of the toxin. Since stretching of the double-stranded DNA molecule seems highly unlikely, a staggered linear aggregation similar to that postulated for histone wotuld seem to be more probable. Equtilibrium dialysis experiments showed that 1800 molecules of the toxin were bound to each molectule of t)NA (fig 4). An association constanit (K) of 5500 was calculated for the DNA-aflatoxin complex. When expressed in terms of ntucleotide residtues, 1 molectule of aflatoxin was bound per each 5 nutcleotides. Sttudies of the melting profiles of DNA in the presence of aflatoxin revealed no differences from utntreated DNA. Both DNA preparations, in 0.2 M NaCl, exhibited a Tm of 890. However, the toxin showed an 8- to 9-fold greater affinity for denatured DNA than for the native form. Also when experiments were conducted in distilled water, approximately 6 times as mtuch aflatoxin was boutnd per mole of native DNA as when 0.2 M NaCl was tused as solvent, although the association constaant was lowered to 1500. The affinity of aflatoxin to DNA appears to be related to the conformation of the DNA molecule.
734
PLANT PHYSIOLOGY
No effect of aflatoxin on the viscosi,ty of RNA was observed. Equilibrium clialysis experiments showed that the toxin was reaclily boulnd to RNA althouigh the conformation of this molecule apparently remains unaffected. Although the gross conformation of DNA may be affected, the melting profile of aflatoxin-treated DNA indicated that the secondary struictulre is not altered by the toxin. Aflatoxin showed a muich greater affinity for the coil form of D\NA than for the helix form, however. Ts'o (23) on the basis of binding studies, concluded that when carcinogens enter a living cell the com,pouinds are more likely to interact wvith DN-A than with anythung else, that interactions of these compouinds will leadl to uinicoiling of DN`A: and(I that interaction wvith uincoile(d DNA is most probable. Aflatoxin, tinder oulr experimental condlitionis, appears not to act entirely in accordance wvith Ts'o's predlictions. The notalble exceptioins are that aflatoxin appears to have no effect on the secondary struictuire of DNA and(I that binding forces for the histone, when the molecullar weights are considered, are as great or greater than those for native DN-A. It reimaiins specuilative as to whether these findings are relate(l to the modle of acition of aflatoxin. However, in view of ouir present knowle(dge on the general affects of this toxin oIn cells, it is coniceivable that aflatoxin exerts its influieince at the information-transcription level. Indeed, others have reporte(l the effects of afla,tox.n on RNA metabolism in vivo aind it has beeii suLggeste(l that these effects couldk occur by (lirect interaction with DNA thereby interfering with such processes as DNA replication anid( DNA-dependent RNXA synithesis
(3, 19). Alternatively aflatoxin could(I affect the informatioin-trainscriptioil process in(lirectly by affecting the stability of the nutcleoproteini complex. It has already been pointed ouit that a conformational change of either DNA or histone could redutce their affini ty in the nutcleohistone complex (21). Aflatuoin cauises conformational changes in both
DNA and specific histones. It is conceiv,able that at low concentrations aflatoxin (loes affect the stability of the nucleohistone complex, that consequently previously repressed DNA template is derepressed, andl finally, increased concentrations of the toxinl again inhibit DNA acti-vity by d(irect interaction. A mechanism of this sort cotuld explain the concentration-depen(lent effects of afla-
t0xin oIn protein sxyinthesis (10).
Acknowledgments We gratefully ackinowledge the technical assistance of Mrs. M. L. King. Generous gifts of aflatoxin B1 were donated by Dr. G. Wogan of the Massachusetts Institute of Technology and Dr. D. A. van Dorp of the Unilever Research Laboratorium, The Netherlanids. The purified calf thvmus histone fractions +-ere generously provided by Dr. L. S. Hiuilica, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas.
Literature Cited 1. BLACK, H. S. AND A. 'M. ALTSCHUL. 1965. Gibberellic acid-induced lipase and a-amylase formation and their inhibition by aflatoxin. Biocheimi. Biophys. Res. Communi. 19: 661464. 2. BURTON-, K. 1956. A study of the conditions and mechan-ism of the diphenvlamine reaction for the colorimetric estimation of deoxv rihonucleic aci(l. Biochemn. J. 62: 315-23. 3. CLIFFORD, J. I. AND K. R. REES. 1966. Aflatoxinl: A site of action in the rat liver cell. Natuire 209: 312-13. 4. DOTY, P., B. B. MCGILL. .N-D S. A. RICE. 1938. The properties of sonic fragmnenits of deoxy ribose nu1icleic acid. Proc. Nattl. Acad. Sci. 'U. S. 44: 432-38. 5. GABLIKS, J.. W. SCIHAEFFER, L. FRIEWMANX, AND G. WOGAN. 1965. Effect of aflatoxin B , on cell cultiures. J. Bacteriol. 90: 720-23. 6). HARTI.E, R. D, B. F. NESBITT, AN-N) . O'KELI-Y. 1963. Toxic metabo)lites of Jspr/f/nli\ls flar"ls. Nature 198: 1056-58. 7. HNILICA, L. S. AND D. BILLEN. 1964. The effect of DNA-l1istone interactions on1 the iosyi-nthesis of DNA in x-itro. Biochlimii. Bioph\ys. Acta 91:
271-80. 8. JIRGE.NSONs. B. AND L. S. HNILTCA. 1965. The conformiational changes of calf-thlimus hiistolle fracitionis as determined by the optical rotari dispersion. Biochimii. Biophys. Acta' 109: 241-49. 9. JIRGENSONs, B., L. S. HNILIC.\, AND S. C. CAPE TILLO. 1966. Viscosity and(l coniformiiationi of calf thlvnmus histones. NIakromol. Clhem. 97: 216-24. 10. JONES, H. C.. H. S. BLACK, AND A. 'M. ALTSCHUL. A comparisoni of the effects of gibberellic aicid andl aflatoxini in germinating seeds. Nature (In press). 11. LEGATOR, M. S. AND A. \WITHROW. 1964. Aflatoxin : Effect on mitotic division in cuiltuire(d emlbryonic luing cells. J. Assoc. Offic. A-r. Chelmiists 47: 1007-09. 12. LJAU, 'M. C., L. S. HNILICA. AND R. B. HURLBERT. 1965. Regutlation0 of RNA Synthesis in isolated nuicleoli by histones anid nucleolar proteiins. Proc. Nat]. Acad. Sci. U. S. 53: 626-33. 13. LIlLY, L. J. 1965. Induction of chromlosomie aberrations bv aflatoxini. Nature 207: 433-34. 14. LoNVRY, 0. H., N. J. ROSEBROUGIT. A. L. FARR, AN-D R. J. RAN-DALL. 1951. Proteini measuremenit ith the folin pheniol reagent J. Biol. CiheImi. 193: 265-
7. 15. NABNEY, J. AND B. F. NESBITr. 1964. Determin1iatioIn of the aflatoxins. Nature 203: 862. 16. SCHOENTAL, R. AND A. F. WNHITE. 1965. Aflatoxins and 'albinism' in plants. Natuire 205: 57-58. 17. S-MITH, R. H. 1963. The influience of toxins of .Aspcrgillus flavus on the incorporationi of 14C leucine into proteins. Biochem. J. 88: 50-51 p. 18. S'MITH, R. H. 1965. The inhlibitioni of amtlino acid activation in liver and Eschcrichia co/i preparations by aflatoxin in vitro. Biochlemtl. J. 95: 43-44 p.. 19. SPORN, M. B.. C. W. DING-MAN, H. L. PHELPS, AND G. N. WOGAN. 1966. Aflatoxini B1: Binding to DNA in vitro and alteration of RNA metabolismi in vivo. Science 151: 1539-41.
BLACK AND JIRGENSONS-AFLATOXIN, HISTONES, AND DNA
20. TELLER, D. C., J. M. KINKADE, JR., AND R. D. COLE. 1965. The molecular weight of ly3sine-rich histone. Biochem. Biophys. Res. Commun. 20: 73944. 21. Ts'o, P. 0. P. AND J. BONNER. 1964. Biology a-nd Chemistry of the histones. In: The Nucleohistones. J. Bonner and P. Ts'o, eds. Holden-Day Inc., San Francisco, California. p 367-79.
735
22. Ts'o, P. 0. P. AND P. Lu. 1964. Interactions of nucleic acids. I. Physical binding of thymine, adenine, steroids, and aromatic hydrocarbons to nucleic acids. Proc. Natl. Acad. Sci. U. S. 51: 17-24. 23. Ts'o, P. 0. P. 1964. The interactions of nucleic acid. In: The Nucleohistones. J. Bonner and P. Ts'o, eds. Holden-Day Inc., San Francisco, California. p 149-62.
