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[CANCER RESEARCH 41, 2168-21 0008-5472/81 /0041 -OOOOS02.00 74, June 1981] Structural Consequences of Modification of the Oxygen Atom of Guanine in DMA by the Carcinogen N-Hydroxy-1-naphthylamine1 F. F. Kadlubar,2 W. B. Melchior, Jr., T. J. Flammang, A. G. Cagliano, H. Yoshida, and N. E. Geacintov National Center for Toxicological Research, Food and Drug Administration, Sac/ay, 91190 Gif sur Yvette, France [A. G. G.]; Institut Universitaire University. New York, New York 10003 [H. Y., N. E. G.] Jefferson, Arkansas de Technologie, ABSTRACT Since the ultimate carcinogen W-hydroxy-1-naphthylamine (N-HO-1-NA) reacts selectively with DMA at the O6 atom of the guanine base, an investigation of the consequences of this potentially mispairing lesion upon DMA structure was under taken. Fluorescence spectroscopic studies, which detected only the major N-HO-1-NA-O6-guanine adduct, showed that the fluorescence decay rate for the naphthyl residue in DMA was similar to that for N-HO-1-NA in solution. Furthermore, the naphthyl fluorescence was efficiently quenched by O2 and was relatively unaffected by Ag+, indicating the free accessibility of the bound naphthyl moiety to the surrounding solution. Electric linear dichroism studies revealed that the transition moment of the 1-naphthylamine adducts, which are aligned along the short axis of the naphthyl ring, tended to be parallel (within 20°) to the transition moment of the DNA bases and thus perpendicular to the DNA helical axis. From these data, space filling molecular models of DNA containing the major (^-sub stituted guanine-naphthylamine adduct were constructed. A model is shown in which the naphthyl residue resides in the major groove of the DNA with complete freedom of rotation about the naphthyl-1-NH bond without causing major conformational changes in the DNA helical structure. Quite unexpectedly, N-HO-1 -NA decreased the thermal sta bility of the DNA in proportion to the degree of reaction. However, derivative melting curves suggested that a major part of this effect is due to preferential reaction with high-melting satellite components (guanine:cytosine-rich regions) of the DNA and that the effect on the stability of the main component is considerably less. In contrast, reaction of DNA with N-acetoxy-2-acetylaminofluorene had qualitatively different effects on thermal stability, indicating that binding of the fluorene residues to DNA occurred randomly. The role of N-HO-1-NADNA adducts as promutagenic, site-selective lesions leading to the initiation of N-HO-1-NA carcinogenesis is proposed. INTRODUCTION N-HO-1-NA3 is an ultimate carcinogen and induces tumors primarily at sites of application (3, 27).' In bacterial systems, this W-hydroxy arylamine is also highly mutagenic (3, 20, 24). 'Supported in part by NCI PHS Grant CA 20851 and by Department of Energy (EP-78-02-4959 E(11-1)2836). 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: N-HO-1-NA, N-hydroxy-1 -naphthylamine; NA, 1-naphthylamine; NA-DNA, DNA containing covalently bound 1-naphthylamine; N-AcO-2-AAF, W-acetoxy-2-acetylaminofluorene; AAF-DNA, DNA containing co valently bound 2-acetylaminofluorene; G:C, guanine:cytosine. 4 E. C. Miller, F. F. Kadlubar, J. D. Scribner, and J. A. Miller, unpublished studies. Received December 9, 1980; accepted February 17, 1981. 2168 72079 [F. F. K., W. B M., T. J. F.¡;Centre d'Etudes Nucléaires de Paris XI, Cachan, France [A. G. G.]: and Chemistry Department, New York Under slightly acidic conditions (pH 5 to 7), N-HO-1-NA is converted to an electrophile, with nitrenium ion and carbocation resonance forms, that binds covalently to DNA, RNA, and protein (16, 17). Previously, we reported that the O6 atom of guanine in DNA was selectively substituted by this carcinogen in vitro (17). The major adduct, which accounted for up to 60% of the NA bound to DNA, was identified as W-(deoxyguanosinO6-yl)-1-naphthylamine. Minor adducts, identified as 2-(deoxyguanosin-O6-yl)-1-naphthylamine and its decomposition prod uct, accounted for at least 30% of the covalently bound carcin ogen (Chart 1). Further analyses of the NA-DNA hydrolysate by high-pressure liquid chromatography have not detected additional adducts, and the limit of detection was judged to be 1% of the total binding.5 Thus, the high selectivity of this reaction offered a unique opportunity to study the structural consequences of carcinogen binding to DNA at the O6 position of guanine. Although this is a minor site of substitution by many carcinogenic nitrosamines and nitrosamides, reaction at this position represents a mispairing lesion that is believed to play a critical role in the initiation of the neoplastic process (1, 19, 21). In this study, the thermal stability (7"mand hyperchromicity) of N-HO-1-NAreacted DNA was determined, and its fluorescence and electric linear dichroism properties were investigated. From these data and from space-filling molecular models, the conformation of A/-(deoxyguanosin-O6-yl)-1 -naphthylamine in the major groove of the DNA helix is proposed. Similar conformational studies have been carried out on DNA reacted with the ultimate carcin ogens, benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide, which binds primarily to the N2 atom of guanine in the minor groove of DNA and causes relatively 25, 26), and N-AcO-2-AAF, the C-8 position of guanine destabilization and unwinding MATERIALS little perturbation of the helix (9, which reacts predominantly with and causes considerable local of the DNA (4, 7, 8, 11). AND METHODS Materials. DNA (calf thymus, type I) and deoxyguanosine were purchased from the Sigma Chemical Co. (St. Louis, Mo.). Calf thymus satellite DNA (density, 1.705 g/cu cm) was pre pared by Ag+-Cs2SO4 centrifugation (6) and subsequently was dialyzed against 0.10 mw citrate buffer (pH 5) prior to reaction with carcinogens. [3H]-N-HO-1-NA (14.4 mCi/mmol) and [93H]-N-AcO-2-AAF (4.29 mCi/mmol) were obtained from Mid west Research Institute (Kansas City, Mo.), and N-HO-1-NA was synthesized according to the method of Willstatter and Kubli (33). All other chemicals were of reagent grade and, unless further specified, were used without further purification. 5 F. F. Kadlubar and T. J. Flammang, unpublished studies. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research. VOL. 41 DNA Modification by N-HO-1-NA of the absorbance at the specified temperature to the absorb ance at 50°.A line was fitted through the 7 lowest data points (a total interval of 1.2°) by quadratic least squares, and the HNN'OH derivative was calculated for the midpoint. The interval was then raised one data point (0.2°), the procedure was repeated, and so on, until the derivative had been calculated for the entire experimental range. RESULTS AND DISCUSSION Fluorescence Spectroscopic Analysis of NA-DNA. DNA modified at the exocyclic oxygen atom (O6) of guanine was prepared by reacting it with the carcinogen N-HO-1-NA; the individual NA-deoxyribonucleoside adducts were isolated by enzymatic hydrolysis and subsequent high-pressure liquid chromatography (cf. "Materials and Methods" and Ref. 17). Adduci I 2-(Deoxyguanosin-O 1-naphthylamine (30%) Adduci -yl)- U N-(Deoxyguanosin-Oyl) 1-naphthylamine (60%) Chart 1. Structures of the DNA adducts formed by reaction with the carcino gen N-HO-1-NA. dfl, deoxyribonucleoside. Space-filling models (CPK Precision Molecular Models) of DNA were obtained from the Baling Corp. (South Natwick, Mass.). Preparation of N-HO-1-NA-reacted DNA and Isolation of Adducts. The reaction of [3H]-N-HO-1-NA with DNA at pH 5, the purification of NA-DNA, and the isolation of the adducts were carried out as described previously (15, 17). The extent of DNA base modification was varied between 0.2 and 2.4% by incubation with varying concentrations of [3H}-N-HO-1 -NA (0.1 to 1.0 mw). Preparation and isolation of AAF-DNA (0.7 to The fluorescence spectra of the purified NA-DNA (0.33% base modification) and of NA-deoxyribonucleoside adducts are shown in Chart 2. 2-(Deoxyguanosin-O6-yl)-1-naphthylamine (Adduct I, the minor adduct), (V-(deoxyguanosin-O6-yl)-1-naphthylamine (Adduct II, the major adduct), and NA-DNA all show an absorbance above 300 nm, which is characteristic of the naphthyl moiety (12). The fluorescence emission maxima occur at 410 nm for Adduct I and at 450 nm for both Adduct II and NA-DNA (excitation at 325 nm). Thus, the naphthyl fluo rescence of the NA-DNA appears to be derived primarily from Adduct II, and this appears to be due to the lower fluorescence intensity of Adduct I as well as the greater abundance of Adduct II in the DNA. The fluorescence decay curve obtained by a single photoncounting technique (10) also indicates one dominant naphthyl fluorescent species in the modified DNA (Chart 3). Except for a small degree of nonexponentiality at early times which is probably due to light scattering within the apparatus (account- 3.3% base modification) were done under identical conditions using 0.13 to 1.0 mw [3H]-N-AcO-2-AAF. NA-DNA Analyses. Fluorescence and absorption spectroscopic measurements (25) and electric linear dichroism anal yses (9) were determined for NA-DNA (A26o = 1 to 10) in 5 mw sodium cacodylate buffer (pH 7.1) as described previously in detail for DNA that had been treated with benzo(a)pyrene 7,8diol-9,10-oxide. Fluorescence spectra were recorded on a Perkin-Elmer Hitachi MPF-2A spectrophotometer. Ag+ com plexes of NA-DNA were prepared by addition of appropriate amounts of AgNO3 to the DNA solutions. Thermal stability was studied photometrically at 260 nm with samples which had been dialyzed against 15 mM NaCI-1.5 m.M sodium citrate (pH 7), purged with helium, and placed in a cuvet under a layer of spectral-grade dodecane. A Gary Model 219 recording spectrophotometer with a heating rate of ap proximately 0.5°/min was used. All absorbance measurements were corrected for the thermal expansion of water. Tm was operationally defined as the temperature at the intersection of the absorbance curve with the line midway between extrapo lations of the linear portions of the curve at low and high temperature. Hyperchromicity was defined as the ratio of the extrapolated absorbances at Tm. For the derivative melting curves, hyperchromicity was more simply defined as the ratio JUNE 400 500 Chart 2. Fluorescence spectra of NA-DNA Adduct I (At. NA-DNA Adduct II (B), and NA-DNA (C; 0.33% base modification). Excitation wavelength, 325 nm. O6-dG, deoxyguanosine-O6-yl-1 -naphthylamine. 1981 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research. 2169 F. F. Kadlubar et al. 10,000-r function of the diffusional encounter rate and the probability of quenching upon an encounter between O2 and NA. It has been shown that K is about 1010 M~1 see"1 in aqueous solution where aromatic molecules and O2 can freely encounter one another (10, 18). Upon intercalation of a planar aromatic mol ecule between neighboring base pairs in native DNA, K de creases by a factor of 10 to 20 (10, 18). By determining T and TO(10, 25), K was calculated for NA-DNA and was found to be 1.0 x 1010 M~1 sec"1, while K for N-HO-1-NA in solution was 1.4 x 1010 M~1 sec"1. Because the former value for NA-DNA 1000- • is only slightly lower than the value obtained for N-HO-1 -NA in aqueous solution, we can conclude that the NA-O6-guanine Z 3 O u in u adduct is located in a region of the DNA helix that is freely accessible to molecular oxygen. This conclusion was further substantiated by determining the effect of Ag* ions on the relative fluorescent intensity of NA loo-: oc O covalently bound to DNA. Silver ions are known to bind pref erentially to guanine bases in DNA, particularly for r values (silver ions per base) below 0.2. We have shown previously (10, 25) that, when Ag+ was added to DNA containing inter 10- - calated polycyclic aromatic hydrocarbons, the fluorescence of the latter was quenched by 60 to 80% at r = 0.15. However, when Ag+ was added to DNA containing only covalently bound benzo(a)pyrene which is located in the open minor groove of the DNA helix, quenching of the pyrene moiety was not ob served. In Chart 4, the effect of [Ag+] on the fluorescence of 15.62ns H 1 1 1 1 1 1 1 1 (••—I1 1-2- TIME Chart 3. Fluorescence ns, nsec. decay curve for NA-DNA (0.33% base modification), ing for about 8% of the total emission), the fluorescence decay curve can be described reasonably well by a single exponential function, lF(t) oce~'/T, where r is the exponential lifetime, i is the time, and lF(t) is the instantaneous fluorescence intensity. These data indicate that, for times greater than 6 nsec, there is only one dominant fluorescent species originating from the aromatic naphthyl groups bound to the DMA. If there were 2 or more exponentially decaying fluorescent components super imposed on each other, a straight line as observed in Chart 3 would not be obtained. The fluorescence lifetime (T) calculated from these data is 18 nsec for NA-DNA; this compares to a lifetime of 13 nsec for N-HO-1-NA in solution. Thus, the decay time of N-HO-1-NA is shorter than that for the DNA-bound covalent adduci. This is in contrast to other aromatic hydrocarbons which, when noncovalently bound to DNA by an intercalation mechanism, display decreased fluorescent lifetimes (10). For example, in the case of the benzo(a)pyrene-DNA intercalation complex, the fluores cence is quenched by 90% or more, and the decay time is decreased significantly (10). These data indicate that the fluo rescence-emitting singlet of NA bound to DNA is not subject to strong interaction with or quenching by neighboring DNA bases and thus is not intercalated. The conclusion that the naphthyl moiety bound to DNA is freely accessible to the surrounding solution can be verified by considering the effects of a dynamic quencher, O2, on fluores cent lifetimes. According to the Stern-Volmer equation, the lifetimes in the absence of dissolved O2 (TO)and in the presence (T) of a certain concentration of O2, given by [O2], are related by the equation 1/T = 1/TO + K[O2]. The constant K is a 2170 NA-DNA is shown. At r > 0.05, there is only about a 20% reduction in the fluorescence intensity. This reduction is much less than that observed with intercalated complexes and is probably due to the binding of Ag+ to guanine moieties to which NA is covalently bound. This result further indicates that the NA adduct exists in a relatively open environment in the DNA molecule. Electric Linear Dichroism Studies. By analysis of electric field-induced linear dichroism data, the relative orientations of the NA moiety and the DNA bases were determined. The methods of analysis, which have been described in detail (9), involve a measurement of the orientations of the transition moments of the DNA bases (at 260 nm) and of the NA moiety (at 335 nm). The directions of these transition moments for NADNA were obtained by orienting the DNA in an electric field , = Au' BAS! Chart 4. Effect of Ag* ions on the relative fluorescence of NA covalently bound to DNA. F(Ag*)/F0, ratio of the fluorescence intensity with and without added Ag*; r, mol Ag* divided by mol DNA base. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research. VOL. 41 DNA Modification and utilizing polarized light to measure the absorbance at various wavelengths with the electric vectors of the polarizers oriented either parallel (A() or perpendicular (A J to the applied electric field (£). The various orientations and directions of transition moments are summarized in Chart 5 for the DNA bases (Vector a) and for the naphthyl moiety (Vector b). For the DNA bases, the vector is parallel to the plane of the bases and perpendicular to the helical axis. For the NA moiety, the transition moment lies along the short axis of the naphthyl ring across carbon atoms 9 and 10. The linear dichroism, AA, which is defined by AA = AB- A±, measures the relative orientations of the transition moments a and b and can be either positive or negative. The linear dichro ism spectrum (AA versus wavelength) for NA-DNA is shown in Chart 6. Note that AA is negative both for the DNA bases (AA by N-HO-1-NA values at <320 nm) and for the NA group (AA values at >320 nm). Thus, the 2 transition moments tend to be oriented at an angle >55° with respect to the electric field vector £.A detailed analysis (9) shows that the angle between these 2 directions (Chart 5, Transition Moments a and b) is between 0 and 20°. Thus, these data indicate that the short axis of the naphthyl ring tends to be perpendicular to the helical axis. It should be noted that the relative orientation of the naphthyl ring repre sents an average of the conformational angles of both Adducts I and II. At the wavelengths used in the analysis, Adducts I and II would contribute 35 and 65%, respectively, to the total absorbance (17). Molecular Model Studies of NA-ONA. Construction of CPK space-filling models of DNA containing NA bound at the O6 atom of a guanine base result in a configuration that is in excellent agreement with the physicochemical analyses. As shown (Fig. 1) for the major adduct (II), the naphthyl group is situated in the major groove of the DNA, and its construction does not require distortion of normal bond angles or alterations in the native B-conformation of DNA. In accord with the fluo rescence studies, the position of the naphthyl moiety indicates that it would be freely accessible to O2 and relatively unaffected by Ag+-base quenching. Furthermore, as evidenced by the linear dichroism studies, the short axis of the naphthyl ring is (a) Chart 5. Relative orientations of the transition moments of the DNA bases (a) and of the bound NA residue (b) obtained at 260 and 335 nm, respectively. Although the electric field (E) induces the DNA to align with £as shown, only about 1% is oriented in an actual experiment; thus, only the relative orientations of a and b are compared. 300 350 WAVELENGTH (nm) Chart 6. Electric field-induced JUNE 400 linear dichroism spectra of NA-DNA. Fig. 1. CPK space-filling molecular model of the DNA helix containing N(deoxyguanosin-O6-yl)-1-naphthylamine (dG). The position of the NA moiety (1NA) at the O6 atom of guanine is indicated. 1981 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research. 2171 F. F. Kadlubar et al. indeed perpendicular to the helical axis. In the configuration shown in Fig. 1, the naphthyl plane is nearly parallel to the DNA bases. However, the naphthyl group can freely rotate about the short axis but maintain its perpendicularity to the DNA helix. Although not shown, construction of the minor adduct (Adduct I) in the DNA gave similar results. In its least sterically hindered position, the short axis of the naphthyl ring in Adduct I is also perpendicular to the helical axis. Thermal Stability of NA-DNA. Thus, it was anticipated that DNA modified by N-HO-1 -NA would show only slight decreases in its thermal stability due to the loss of hydrogen bonding between the O6-substituted guanine and the cytosine in the complementary strand (17). However, N-HO-1-NA strongly de creased both the thermal stability of DNA (Chart 7) and the hyperchromicity associated with the helix-coil transition. Insta bility and partial denaturation of the helix, even at low temper ature, increased approximately linearly with increasing substi tution. Surprisingly, the changes in thermal stability of AAFDNA were of about the same magnitude, suggesting similar effects of the 2 compounds on DNA structure. However, derivative melting curves (the derivative of hyper chromicity with respect to temperature; for a review of this technique, see Ref. 2) provided additional information. These studies indicated that the lowered thermal stability and hyper chromicity caused by N-HO-1-NA modification were not spe cifically associated with denaturation of DNA regions contain ing high-melting G:C base pairs (Chart 8). In contrast, N-AcO2-AAF, markedly destabilized the high-melting regions of the helix (Chart 8). The difference is so striking that it suggests that DNA adducts formed by the 2 compounds have quite different structural consequences. Chart 8 clearly demon strates the highly empirical nature of 7"m's.Whether defined as the temperature at the maximum of the derivative curve or, as here, the temperature dividing the derivative curve into 2 equal areas, the Tmstrongly reflects both the source of the DNA and the nature of the damage. Conclusions about structural l 2 Base 3 Modification Chart 7. T„ as a function of percentage of base modification. DNA treated with N-HO-1-NA (•)or N-AcO-2-AAF (•)was melted in 15 mM NaCI-1.5 mu sodium citrate (pH 7). The decrease in Tmas a function of the percentage of base modification was —1.2°and -1.4° per percent/base modification for NA-DNA and AAF-DNA, respectively. The percentage of hyperchromicity was 34.5% for unmodified DNA. and the decrease in percentage of hyperchromicity as a function of the percentage of base modification was —2.3and -1.8% per percent/base modification for NA-DNA and AAF-DNA, respectively. 2172 0.05 0.04- - 0.03- • X •o 0.02- - 0.01-- 50 Chart 8. Derivative melting curves of calf thymus DNA modified by N-HO-1 NA or N-AcO-2-AAF. dH/dT, derivative of fractional hyperchromicity divided by derivative of temperature; a, unmodified DNA; b, 1.6% NA-modified DNA; c. 2.4% NA-modified DNA; d. 3.3% 2-acetylaminofluorene-modlfied DNA. changes should be treated cautiously when they are based solely on Tmdata. Besides the difference between N-HO-1-NA and N-AcO-2AAF, the derivative plots show a second interesting phenome non. The prominent peaks on the high-temperature side of the derivative melting profile of untreated calf thymus DNA (Chart 8) are caused by some of the calf satellite DNA's, which have G:C contents higher than the bulk of the DNA (6). N-HO-1-NA specifically destabilizes these satellites, causing their peaks to disappear from the profile, while having a much smaller effect on other DNA with the same initial stability. N-AcO-2-AAF similarly destabilizes the G:C-rich satellites but also has major effects on the bulk of the DNA. Available mutagenicity data for NA and AcO-2-AAF appear to be consistent with these results (28, 29). The binding of NA to O6 of guanine and local destabilization of the DNA helix may account respectively for the base pair substitution and chain termination (with subsequent misrepair) that is believed to result in mutations in the Salmo nella typhimurium test system (28). The more generally desta bilized AcO-2-AAF-modified DNA, while being quite sensitive to termination of DNA replication and misrepair (23, 28), also results in extensive frame-shift mutagenicity (28, 29). The latter is probably due to the major C-8 guanine-substituted 2-acetylaminofluorene adduct that is believed to cause intercalationunwinding (5) or insertion-denaturation [base displacement (7, 8,11)]. It remained to be determined if the thermal instability of NADNA was due to preferential reaction with satellite sequences instead of greater destabilization of satellite DNA which had reacted to the same extent (corrected for base composition) as bulk DNA. However, the preferential reaction hypothesis is consistent with known sequence-specific interactions, e.g., those of silver and mercury ions (30) and some antibiotics, such as actinomycin D (32) and echinomycin (31). There is also a recent report that the covalent binding of the diolepoxide of benzo(a)pyrene to DNA may be influenced by dif- CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research. VOL. 41 DNA Modification Table 1 with total calf thymus DNA and with calf thymus satellite DNA Calf thymus DNA (guanine + cytosine = 44.5%) or satellite DNA, 1.705 g/cu cm (guanine + cytosine = 49%), was prepared and reacted with N-HO-1-NA as described in "Materials and Methods." The concentrations of DNA and N-HO-1Reaction of N-HO-1-NA by N-HO-1-NA guanine. Biochim. Biophys. Acta, 562. 51-61, 1979. 2. Ansevin, A. T., Vizard, D. L., Brown, B. W., and McConathy. J. Highresolution thermal denaturation of DNA. I. Theoretical and practical consid erations for the resolution of thermal subtransitions. Biopolymers, 75. 153- 174, 1976. 3. Belman, S., Troll, W., Teebor, G.. and Mukai, F. The carcinogenic and mutagenic properties of W-hydroxy-aminonaphthalenes. Cancer Res., 28. NA in the reaction mixture for Experiment 1 were 30 fig/ml and 0.01 mM, 535-542, 1968. respectively; for Experiment 2, these values were 30 ,«g/ml and 0.10 mM, 4. Chang, C. T., Miller, S. J., and Wetmur, J. G. Physical studies of N-acetoxyrespectively. W-2-acetylaminofluorene-modified deoxyribonucleic acid. Biochemistry, 73. 2142-2148, 1974. bound/103 nubound/ 103 gua5. Drinkwater, N. R., Miller, J. A., Miller, E. C., and Yang, N.-C. Covalent intercalate binding to DNA in relation to the mutagenicity of hydrocarbon cleotides3.52 nines0.781.208.0 Experiment12DNA sampleTotal epoxides and /V-acetoxy-2-acetylaminofluorene. Cancer Res., 38. 3247DNA 3255, 1978. 4.8835.8 DNATotal Satellite 6. Filipski, J., Thiery, J.-P., and Bernardi, G. An analysis of the bovine genome by Cs2SO«-Ag* density gradient centrifugation. J. Mol. Biol., 80. 177-197, DNA 1973. Satellite DNAResidues 49.0Residues 12.0 7. Fuchs. R. P. P., and Duane, M. P. Dynamic structure of DNA modified with the carcinogen N-acetoxy-N-2-acetylaminofluorene. Biochemistry, 13: 4435-4440, 1974. 8. Fuchs, R. P. P., Lefevre, J. F., Peuyet, J., and Duane, M. P. Comparative ferent base sequences (13). To test this hypothesis, we com orientation of the fluorene residue in native DNA modified by N-acetoxy-2pared the reaction of N-HO-1-NA with total calf thymus DNA acetylaminofluorene and two 7-halogeno derivatives. Biochemistry, 75. and with the 1.705-g/cu cm satellite (Table 1). Over a 10-fold 3347-3351. 1976. 9. Geacintov, N. E., Gagliano, A., Ivanovic, V., and Weinstein, I. B. Electric range of DNA modification, reaction occurred to a greater linear dichroism study on the orientation of benzo(a)pyrene-7,8-dihydrodiolextent (37 to 39% greater) with the satellite DNA than would 9,10-oxide covalently bound to DNA. Biochemistry, 77. 5256-5262, 1978. 10. Geacintov, N. E., Prusik, T., and Khosrofian, J. M. Properties of benzobe expected on the basis of its increased G:C content (10% pyrene-DNA complexes investigated by fluorescence and triplet flash pho greater). Thus, these data further suggest that N-HO-1 -NA not tolysis techniques. J. Am. Chem. Soc., 98. 6444-6452, 1976. only reacts selectively with the O6 of guanine in DNA but also 11. Grunberger, D., and Weinstein, I. B. The base displacement model: an explanation for the conformational and functional changes in nucleic acids binds preferentially to a satellite sequence. modified by chemical carcinogenesis. In: J. M. Yuhas, R. Tennant, and J. D. The implications of these findings for studies of genotoxic Regan (eds.). Biology of Radiation Carcinogenesis, pp. 175-187. New York: compounds may be major, regardless of whether the com Raven Press. 1976. 12. Hirshberg, Y., and Jones, R. N. The ultraviolet absorption spectra of some pound reacts preferentially with certain sequences or prefer carboxy derivatives of naphthalene. Can. J. Res. Sect. B. Chemical Sci. 27. entially destabilizes them. Studies of the characteristics of 437-461, 1949. modified DNA, such as its hydrodynamic properties or its 13. Iyer, R.. Triplett, L. L., Slaga, T. J., and Papaconstantinou, J. Interaction of (±)benzo[a]pyrene-7/î.8a-diol 9a,10o-epoxide with fractionated eukaryotic thermal stability, may easily be misinterpreted due to such DNA. In: P. W. Jones and P. Leber (eds.). Polynuclear Aromatic Hydrocar nonrandom effects. Additionally, sequence-specific reactions bons, pp. 805-818. Ann Arbor, Mich.: Ann Arbor Science Publishers, 1979. 14. John, B., and Miklos, G. L. G. Functional aspects of satellite DNA and may have unsuspected biological consequences affecting heterochromatin. Int. Rev. Cytol., 58. 1-114, 1979. some of the many suggested functions of satellite DNA, such 15. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Microsomal N-oxidation of the as chromosome assortment, recombination, and the synthesis hepatocarcinogen /V-methyl-4-aminoazobenzene and the reactivity of Whydroxy-N-methyl-4-aminoazobenzene. Cancer Res., 36. 1196-1206, of macromolecules like histones and rRNA (14, 30). The large 1976. interspecies differences in the amount and nature of satellite 16. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Hepatic microsomal Nglucuronidation and nucleic acid binding of N-hydroxy arylamines in relation DNA may also be one source of species differences in response to urinary bladder carcinogenesis. Cancer Res., 37. 805-814, 1977. to carcinogens. Finally, sequence-specific binding may contrib 17. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Guanyl O6-arylamination and ute to the nonrandom binding of compounds, such as aminoO6-arylation of DNA by the carcinogen AMiydroxy-1-naphthylamine. Cancer Res., 38. 3628-3638, 1978. fluorene and its derivatives, to chromatin DNA (22). 18. Lakowicz, J. R.. and Weber, G. Quenching of fluorescence by oxygen. A In conclusion, heat denaturation, fluorescence spectrosprobe for structural fluctuations in macromolecules. Biochemistry, 12: copy, linear dichroism, and molecular model studies have 4161-4170, 1973. 19. Lawley, P. D. Carcinogenesis by alkylating agents. In. C. E. Searle (ed.). permitted an assessment of the conformation of a carcinogen Chemical Carcinogens, American Chemical Society Monograph 173, pp. that is covalently bound to the O6 atom of guanine in DNA. 83-244. Washington, D. C.: American Chemical Society. 1976. From these data, it is anticipated that the A/-(deoyguanosin-O620. McCann, J., Choi. E., Yamasaki, E., and Ames, B. N. Detection of carcino gens and mutagens in the Sa/moneHa/microsome test: assay of 300 chem yl)-1-naphthylamine residue will not cause a significant pertur icals. Proc. Nati. Acad. Sei. U. S. A., 72: 5135-5139, 1975. bation in the bulk of the DNA in vivo during N-HO-1-NA carci21. Mehta. J. R., and Ludlum, D. B. Synthesis and properties of O6-methyldeoxy- nogenesis. Consequently, such an adduct may not be recog nized by repair enzymes and may thus induce a heritable change in the DNA during replication that could give rise to neoplasia. 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VOL. 41 Structural Consequences of Modification of the Oxygen Atom of Guanine in DNA by the Carcinogen N-Hydroxy-1-naphthylamine F. F. Kadlubar, W. B. Melchior, Jr., T. J. Flammang, et al. Cancer Res 1981;41:2168-2174. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/6/2168 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research.