Download Structural Consequences of Modification of the Oxygen Atom of

[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. N-HO-1-NA may also have a specific effect on
satellite and repetitive sequences, with resultant biological
consequences. Additional studies on the formation and per
sistence of these N-HO-1-NA-DNA adducts in vivo are in prog
ress, as are studies of sequence-specific
binding of carcino
gens.
22.
23.
24.
25.
REFERENCES
26.
1. Abbott, P. J., and Saffhill, R. DNA synthesis with methylated poly (dC-dG)
templates. Evidence for a competitive nature to miscoding by O6-methyl-
JUNE
guanylic acid and its copolymers with deoxycytidylic acid. Biochim. Biophys.
Acta, 527. 770-778, 1978.
Metzger, G., Wilhelm. F. X., and Wilhelm, M. L. Non-random binding of a
chemical carcinogen to the DNA in chromatin. Biochem. Biophys. Res.
Commun., 75. 703-710, 1977.
Moore, P. D., Rabkin, S. D., and Strauss, B. S. Termination of in vitro DNA
synthesis of AAF adducts in the DNA. Nucleic Acids Res., 8. 4473-4484,
1980.
Perez. G., and Radomski, J. L. The mutagenicity of the fV-hydroxynaphthylamines in relation to their carcinogenicity.
Ind. Med. Surg., 34: 714716, 1965.
Prusik, T.. Geacintov, N. E., Tob;a<", C., Ivanovic, V., and Weinstein, I. B.
Fluorescence study of the physico-chemical properties of a benzo(a)pyrene
7,8-dihydrodiol 9,10-oxide derivative bound covalently to DNA. Photochem.
Photobiol.. 29. 223-232, 1979.
Pulkrabek, P., Leffler, S., Weinstein, I. B., and Grunberger, D. Conformation
of DNA modified with a dihydrodiol epoxide derivative of benzo(a)pyrene.
Biochemistry, 76. 3127-3132,
1977.
1981
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research.
2173
F. F. Kadlubar et al.
27. Radomski. J. L, Brill, E.. Deichmann, W. B., and Glass, E. M. Carcinogenicity
testing of N-hydroxy and other oxidation and decomposition products of 1and 2-naphthylamine. Cancer Res., 3). 1461-1467,
1971.
28. Scribner, J. D., Fisk, S. R.. and Scribner, N. K. Mechanisms of action of
carcinogenic aromatic amines: an investigation using mutagenesis in bac
teria. Chem. Biol. Interact., 26. 11-25, 1979.
29. Simmon, V. F. In vitro mutagenicity assays of chemical carcinogens and
related compounds with Salmonella typhimurium. J. Nati. Cancer Inst., 62.
2174
893-899, 1979.
30. Skinner, D. M. Satellite DNA's. BioScience,
27. 790-796,
1977.
31. Waring, M. J., and Wakelin, L. P. G. Echinomycin: a bifunctional intercalating
antibiotic. Nature (Lond.), 252 653-657, 1974.
32. Wells, R. D., and Larson, J. E. Studies on the binding of actinomycin D to
DNA and DNA model polymers. J. Mol. Biol., 49: 319-342, 1970.
33. Willstatter, R., and Kubli, H. Ãœberdie Reduktion von Nitroverbindungen nach
der Methods von Zinin. Chem. Ber.. 41: 1936-1940.
1908.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1981 American Association for Cancer Research.
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.