Formation of depurinating N3adenine and N7guanine adductsafter reaction of 1,2-naphthoquinone or enzyme-activated
1,2-dihydroxynaphthalene with DNAImplications for the mechanism of tumor
initiation by naphthalene
Muhammad Saeed, Sheila Higginbotham, Eleanor Rogan, Ercole Cavalieri ∗Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center,
986805 Nebraska Medical Center, Omaha, NE 68198-6805, United States
Received 17 August 2006; received in revised form 4 December 2006; accepted 5 December 2006
Available online 16 December 2006
Abstract
Naphthalene is considered by the US Environmental Protection Agency to be a carcinogenic compound based on inhalationstudies in rats. The primary metabolite of naphthalene is naphthalene 1,2-arene oxide. This unstable intermediate can lead toformation of 1-naphthol and naphthalene-1,2-dihydrodiol. Secondary metabolites include 1,2-dihydroxynaphthalene (1,2-DHN),which can be further oxidized to 1,2-naphthoquinone (1,2-NQ). Based on the metabolism of naphthalene and its similarity to themetabolic activation of carcinogenic natural estrogens, synthetic estrogens and benzene, we hypothesize that naphthalene is activatedto initiate cancer by reaction of 1,2-NQ with DNA to form the depurinating adducts 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua.These adducts were synthesized by reaction of 1,2-NQ with Ade or dG in acetic acid/water/DMF (1:1:1). 1,2-NQ was reactedwith DNA, and the depurinating 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua adducts were analyzed by ultraperformance liquidchromatography/tandem mass spectrometry and HPLC with electrochemical detection. After the reaction of 1,2-NQ with DNA,the N3Ade and N7Gua adducts were found. Similarly, when 1,2-DHN was activated by tyrosinase in the presence of DNA, higheramounts of the N3Ade and N7Gua adducts were detected. These same adducts were also formed when 1,2-DHN was activatedby prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. These depurinatingadducts are analogous to those obtained from the ortho-quinones of natural estrogens, synthetic estrogens and benzene. These resultssuggest that reaction of ortho-quinones with DNA by 1,4-Michael addition is a general mechanism of weak carcinogenesis that
react with DNA preferably at the N3 position of ade-
176 M. Saeed et al. / Chemico-Biolo
1. Introduction
Naphthalene, a condensed two-benzene ring aromatichydrocarbon, is a component of coal tar, coal tar products(e.g., creosote) and moth repellents, and is extensivelyused as an intermediate in the production of plasticiz-ers, resins, insecticides and surface-active agents [1]. Itis a ubiquitous pollutant found mainly in ambient air andto a minor extent in effluent water. Although the majorcontributors of naphthalene in air are coal and oil gasi-fication sites, a significant amount is also released as apyrolytic product of both mainstream and side streamtobacco smoke. Human exposure to naphthalene wascorroborated when it was found in nearly 40% of humanfat samples tested [2] and in 75% of human breast milksamples [3]. These facts argue that the U.S. populationis exposed to this compound, and it has been included asone of 189 hazardous air pollutants under the Clean AirAct Amendments of 1990 (Title III) of the Environmen-tal Protection Agency [4]. Until recently, naphthalene inthe environment was thought to present no significantcancer risk to humans, in spite of the reported cancercases in employees working in a naphthalene purificationplant in the former East Germany [5,6].
Mice inhaling naphthalene chronically (10 or 30 ppm)exhibited diverse effects, including inflammation of thenose, metaplasia of the olfactory epithelium, and hyper-plasia of the respiratory epithelium [7–9]. No neoplasticeffects in male mice were noted. Female mice, however,showed a slight increase in alveolar/bronchiolar adeno-mas and carcinomas at the highest exposure level. More
recently, the U.S. National Toxicology Program releasedthe results of a 2-year bioassay study with rats exposedto doses of 10, 30 or 60 ppm naphthalene [10,11], show-
Fig. 1. Metabolic activation of naphthalene to
teractions 165 (2007) 175–188
ing a concentration-dependent increase in adenomas ofthe respiratory epithelium of the nose and of neurob-lastomas of the olfactory epithelium. These results areconsidered to be significant in rodent studies and haveraised concerns about naphthalene as a potential humancarcinogen [1].
As for many chemical carcinogens, the toxicity ofnaphthalene is dependent on its metabolic activation.Studies conducted in vitro and in vivo demonstrated thatthe first step in the metabolic conversion of naphthaleneis the cytochrome P450-dependent formation of the 1,2-epoxide (Fig. 1) [7,12–18]. This compound is unstableat physiological pH [14,15] and can either react withglutathione to form glutathione conjugates or convert tothe metabolites 1-naphthol by chemical isomerizationor naphthalene 1,2-dihydrodiol by epoxide hydrolase[16,17]. The conversion to 2-naphthol can occur after�-elimination of the sulphated/glucuronidated conju-gate of naphthalene 1,2-dihydrodiol (not shown inFig. 1). Naphthalene 1,2-dihydrodiol [19–21] or 1-naphthol [22] can be further metabolically oxidizedto 1,2-dihydroxynaphthalene (1,2-DHN) or its oxi-dized product, 1,2-naphthoquinone (1,2-NQ). 1,2-NQhas been reported to be the metabolite that binds cova-lently to proteins [22,23].
Being electrophilic in nature, 1,2-NQ can also bindcovalently to the cellular nucleophiles of DNA and RNA.Analogously to catechol estrogen quinones derived fromnatural estrogens [24–29], synthetic estrogens [30,31],and benzene [32], we hypothesize that 1,2-NQ can
the electrophilic 1,2-naphthoquinone.
nine and the N7 position of guanine, leading to theformation of the corresponding depurinating adducts(Fig. 2). Apurinic sites generated by depurination are
M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188 177
F hthalenes
mmdemfatadNicc
2
cg
2
aiaOCm
ig. 2. Proposed mechanism of cancer initiation by estradiol and naphown.
utagenic and may lead to initiation of cancer after accu-ulation of mutations in critical genes controlling cell
ivision [28,29,33]. In this article we provide the firstvidence concerning the ability of 1,2-NQ and enzy-atically activated 1,2-DHN to react with DNA and
orm depurinating and stable adducts. The depurinatingdducts formed were identified and quantified by usinghe standard synthesized adducts 1,2-DHN-4-N3Adend 1,2-DHN-4-N7Gua. Furthermore, the kinetics ofepurination were also investigated. Depurination of the3Ade adduct is rapid, whereas that of the N7Gua adduct
s slower. These findings may have important impli-ations for understanding the etiology of naphthalenearcinogenesis.
. Materials and methods
Caution. Catechols and quinones are hazardoushemicals and should be handled according to NIHuidelines [34].
.1. Chemicals, reagents, and enzymes
1,2-NQ, citric acid, deuteriated DMSO, DMF anddenine (Ade) were purchased from Aldrich Chem-cal Co. (Milwaukee, WI). Deoxyguanosine (dG)
nd DNA were purchased from USB (Cleveland,H). Prostaglandin H synthase was purchased fromayman Chemicals (Ann Arbor, MI). Tyrosinase, 3-ethylcholanthrene (MC)-induced rat liver microsomes,
. The presumed major pathway of naphthalene to naphthoquinone is
methemoglobin, arachidonic acid, ammonium acetateand deoxyadenosine (dA) were purchased from Sigma(St. Louis, MO). 1,2-DHN was synthesized by reducing1,2-NQ with NaBH4 in ethanol [35]. All chemicals wereused as such without further purification.
2.2. Instrumentation
(1) UV. The UV spectra were obtained during HPLCby using a photodiode array (PDA) detector,Waters 996 (Milford, MA) for all synthesizedcompounds. HPLC separations were monitored at254 nm.
(2) NMR. NMR spectra were recorded on a VarianUnity-Inova 500 instrument operating at a resonancefrequency of 499.8 MHz for 1H and 125.6 MHz for13C spectra at 25 ◦C. Samples were dissolved in600 �L of DMSO-d6 and referenced to the sol-vent signals at 2.5 ppm for 1H and 39.7 ppm for13C. All 2D experiments were performed by usingthe standard Varian software (VNMR v6.1c). For2D experiments, relaxation delays of 1.2–2 s wereused; 128–512 t1 increments and 2048 complexdata points in t2 were recorded for a spectral widthof 8000 Hz in two dimensions. 1H–1H correlationswere recorded by using correlation spectroscopy
(COSY) techniques. In pulsed field gradient (PFG)1H–13C heteronuclear single quantum coherence(HSQC) and heteronuclear multiple bond correla-tion (HMBC) sequences, delays were optimized for
gical In
178 M. Saeed et al. / Chemico-Biolo
coupling constants around 140 and 8 Hz, respec-tively.
(3) Mass spectrometry (MS). Fast atom bombard-ment tandem mass spectrometry (FAB-MS/MS) wasconducted at the Nebraska Center for Mass Spec-trometry (University of Nebraska-Lincoln) using aMicroMass (Manchester, England) AutoSpec highresolution magnetic sector mass spectrometer. Theinstrument was equipped with an orthogonal accel-eration time-of-flight serving as the second massspectrometer. Xenon was admitted to the collisioncell at a level to attenuate the precursor ion sig-nal by 75%. Data acquisition and processing wereaccomplished by using OPUS software that was pro-vided by the manufacturer (Microcasm). Sampleswere dissolved in 5–10 �L of CH3OH; 1 �L aliquotswere placed on the sample probe tip along with 1 �Lof a 1:1 mixture of glycerol/thioglycerol.
(4) Ultra performance liquid chromatography–tandemmass spectrometry (UPLC–MS/MS). Samples wereanalyzed by a Waters Acquity UPLC equipped witha MicroMass QuattroMicro triple stage quadrupolesystem (Waters, Milford, MA). The 10 �L injec-tions were carried out on a Waters Acquity UPLCTM
BEHC18 column (1.7 �m, 1 mm × 100 mm). Theinstrument was operated in positive electrospray ion-ization mode. All aspects of system operation, dataacquisition and processing were controlled usingQuanLynx v4.0 software (Waters). The column waseluted starting with 5% CH3CN in H2O (0.1%formic acid) for 1 min at a flow rate of 150 �L/min;then raised to 55% CH3CN in 10 min. Ionizationwas achieved by using the following settings: cap-illary voltage 3 kV; cone voltage 15–40 V; sourceblock temperature 100 ◦C; desolvation temperature200 ◦C with a nitrogen flow of 400 L/h. Nitrogenwas used as both the desolvation and auxillary gas.MS/MS conditions were optimized prior to analy-sis using pure standards of 1,2-DHN-4-N3Ade and1,2-DHN-4-N7Gua. Argon was used as the collisiongas. Three-point calibration curves were run for eachstandard. Triplicate samples were analyzed for eachdata point.
(5) HPLC. Purification of adducts was conducted byusing preparative HPLC on a Waters DeltaPrep sys-tem equipped with a 2478 dual wavelength UVdetector. About 10 mL of reaction mixture wasloaded on a Luna-2 C-18 column (10 �m, 120 A,
21.2 mm × 250 mm, Phenomenex, Torrance, CA)and eluted with a mobile phase consisting of 10%CH3CN in H2O [0.4% trifluoroacetic acid (TFA)]which was increased linearly up to 40% CH3CN
teractions 165 (2007) 175–188
in 20 min, and to 100% in the next 5 min at aflow rate of 25 mL/min. Analyses of reaction mix-tures were performed on a Waters 2690 (Alliance)Separations Module equipped with a Waters 996PDA interfaced to a Digital Venturis Fx 5100 com-puter. A 50 �L aliquot of a reaction mixture waseluted on a Luna-2 C-18 column (5 �m, 120 A,250 mm × 4.6 mm, Phenomenex), using a linear gra-dient of 10% CH3CN/90% H2O (0.4% TFA), whichwas changed to 40% CH3CN in 20 min and then to100% CH3CN in 5 min at a flow rate of 1 mL/min.The eluants were monitored at 254 nm.
Analyses of depurinating adducts were con-ducted on an HPLC system equipped with dualESA Model 580 solvent delivery modules, anESA Model 540 auto-sampler and a 12-channelCoulArray electrochemical detector (ESA, Chelms-ford, MA). The two mobile phases used were(A) CH3CN:CH3OH:buffer:H2O (15:5:10:70) and (B)CH3CN:CH3OH:buffer:H2O (50:20:10:20). The bufferwas composed of a mixture of 105 g citric acid, 78 gammonium acetate in triple-distilled H2O, and the pHwas adjusted to 3.6 with acetic acid. The 50 �L injec-tions were carried out on a Phenomenex Luna-2 C-18column (5 �m, 120 A, 4.6 mm × 250 mm) were initiallyeluted isocratically at 90% A/10% B for 15 min, fol-lowed by a linear gradient to 90% B in the next10 min at the rate of 1 mL/min. The serial array of12 coulometric electrodes was set at potentials of−50, −10, 10, 50, 100, 150, 200, 250, 300, 350,400 and 450 mV. The system was controlled and thedata were acquired and processed using the CoulAr-ray software package. Peaks were identified by bothretention time and peak height ratios between the dom-inant peak and the peaks in the two adjacent channels.The depurinating adducts were quantified by compar-ison of peak response ratios with known amounts ofstandards.
2.3. Synthesis of standard adducts
2.3.1. Reaction of 1,2-NQ with dG, Ade or dATo a stirred solution of Ade (405 mg, 3.0 mmol),
dG (450 mg, 1.5 mmol) or dA (807 mg, 3.0 mmol) inacetic acid/H2O (1:1, 15 mL) was added a solutionof 1,2-NQ (50 mg, 0.3 mmol) in DMF (7.5 mL) atroom temperature. The reaction mixture was stirred
at the same temperature for 12 h and filtered toremove any insoluble material. Analysis and purifica-tion of adducts were performed on HPLC, as describedabove.
2.3.2. Kinetics of the appearance anddisappearance of 1,2-DHN-4-N7dG adduct
To a stirred solution of dG (90 mg, 0.3 mmol) in aceticacid/H2O (1:1, 3 mL) was added a solution of 1,2-NQ(10 mg, 0.06 mmol) in DMF (1.5 mL). The pH of thissolution was ∼2. The reaction mixture was stirred eitherat room temperature (22 ◦C) or at 37 ◦C. For reactionsat pH 4.6, dG (90 mg) was stirred in 0.1 M phosphatebuffer (pH 4.6, 3 mL) with a solution of 1,2-NQ (10 mg)in DMF (1.5 mL) at either 22 ◦C or 37 ◦C. Aliquots ofthe reaction mixture were removed after 0, 0.5, 1, 2,5, 7, 10, and 15 h and analyzed quickly on HPLC andmass spectrometry. Identification of 1,2-DHN-4-N7dGadduct was confirmed by isolating the labile peak bypreparative HPLC, recording its molecular weight bymass spectrometry and observing its conversion into theN7Gua adduct by analytical HPLC. Quantification wascarried out by comparing the area under the peak ofthe N7dG adduct with that of the standard depurinat-ing 1,2-DHN-4-N7Gua adduct. The rationale for thiscomparison derives from the identical molar extinctioncoefficients of both adducts.
2.3.3. Covalent binding of 1,2-NQ to DNAA solution of 1,2-NQ (5 mg, 32 �mol) in 500 �L
DMSO was mixed with DNA (3 mM in 0.067 M sodiumpotassium phosphate buffer, pH 7.0) in 10 mL total reac-tion mixture and incubated at 37 ◦C for 10 h. Afterincubation, DNA was precipitated with two volumesof ethanol, and the supernatant, containing depurinat-ing adducts, was evaporated to dryness. The residue wasdissolved in 10 mL of a mixture of DMF/CH3OH, fil-tered and injected to preparative HPLC as describedbefore. Blind fractions were collected one minute beforeand after the pre-established retention times of the stan-dard depurinating adducts. The retention times of theadducts were established after injecting a mixture ofstandard 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua onthe HPLC column and recording the retention times forboth adducts. The collected fractions were evaporatedand the residue reconstituted in 200 �L of 50% CH OH
3in 0.1 M NH4OAc (pH 4.4). This solution (100 �L) wasanalyzed by HPLC connected to the electrochemicaldetector and 10 �L was analyzed on UPLC–MS/MS.The levels of depurinating adducts were determined by
gical In
180 M. Saeed et al. / Chemico-Biolo
comparing peak heights with known adduct standards.Recovery of the depurinating adducts in the reactionmixtures was assessed by spiking a known amount ofstandard 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua ina reaction mixture containing only DNA. The DNAwas precipitated immediately and the supernatant wasprocessed for depurinating adducts as described above.Under these conditions, the recoveries of the adductswere from 80 to 95%.
Precipitated DNA from each experiment was pro-cessed for 32P-post-labeling analysis of stable adducts[36] after purification of the DNA. The purified DNA washydrolyzed with micrococcal nuclease, spleen phospho-diestrase and nuclease P1. The labeled nucleotides wereseparated by using polyacrylamide gel electrophoresis[37]. Four bands were observed; they were cut out of thegel and the 32P counted.
2.3.4. Kinetics of DNA depurination1,2-NQ (2.5 mg, 16 �mol) in 500 �L DMSO was
mixed with DNA (3 mM in 0.067 M sodium potassiumphosphate buffer, pH 7.0) in 10 mL of total reaction mix-ture and incubated at 37 ◦C for 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10,15, 20, or 24 h. DNA at each time point was precipitatedby ethanol and the supernatant processed for measure-ment of the level of depurination by UPLC–MS/MS andHPLC with electrochemical detection.
2.3.5. Covalent binding of 1,2-DHN to DNA in thepresence of air or an enzyme
1,2-DHN (2.5 mg in 500 �L DMSO) was reacted with3 mM calf thymus DNA (pH 7.0) in 10 mL reaction mix-tures in the presence of air. Alternatively, 1,2-DHN wasbound to DNA in 10 mL reaction mixtures catalyzed bytyrosinase, prostaglandin H synthase, or MC-induced ratliver microsomes during 10 h of incubation at 37 ◦C.In the tyrosinase experiments, the mixture containing3 mM calf thymus DNA in 0.067 M sodium–potassiumphosphate (pH 7.0), 2.5 mg of 1,2-DHN (in 500 �L ofDMSO) and 1 mg of enzyme (2577 units) was incubatedat 37 ◦C for 10 h. For prostaglandin H synthase-catalyzedreactions, the mixture containing 3 mM DNA, 2.5 mgof 1,2-DHN (in 500 �L of DMSO), 1 mL of methe-moglobin (1.47 mg/mL in 75 mM KH2PO4, pH 7.5),152 �L of arachidonic acid (commercial 100 mg/mLsolution in ethanol) and 800 �L of prostaglandin H syn-thase (400 units) was incubated at 37 ◦C for 10 h. For theMC-induced microsomes, the reaction mixture contain-
ing 3 mM DNA in 150 mM Tris-HCl (pH 7.5), 150 mMKCl, 5 mM MgCl2, 2.5 mg of 1,2-DHN (in 500 �L ofDMSO), 10 mg of microsomal protein, and NADPH(0.6 mM) was incubated at 37 ◦C for 10 h. After the
teractions 165 (2007) 175–188
incubation period, DNA was precipitated with 2 vol-umes of ethanol, and processed for 32P-post-labelinganalysis of stable adducts [36,37]. The supernatantwas used for the analysis of depurinating adducts, asdescribed above. Control reactions were carried outunder identical conditions either with no enzyme or nocofactor.
3. Results and discussion
3.1. Synthesis and structure elucidation of DNAadducts
Previous studies from our laboratory have demon-strated that ortho-quinones, derived from naturalestrogens [24–29], synthetic estrogens [30,31] and ben-zene [32], react with dG predominantly at the N-7position and with Ade at the N-3 position, leading to theformation of depurinating adducts. Analogously, 1,2-NQwas reacted with 5 equiv. of dG at room temperature for12 h (Fig. 3). Analysis by reverse phase HPLC duringthis time indicated the formation of three compoundsdetected in the adduct area of the chromatogram. How-ever, analysis after 12 h indicated the disappearance ofthe middle peak corresponding to the labile 1,2-DHN-4-N7dG adduct (see below for details), leaving a majoradduct, 1,2-DHN-4-N7Gua (10%), and a minor adduct,1,2-NQ-4-N7Gua (0.7%). The structures of the synthe-sized adducts were elucidated based on detailed NMRspectroscopy and mass spectrometry (MS) as describedbelow.
Purification of adducts was attempted by using a solidphase extraction cartridge, as described [38]. However,HPLC analysis of the sample after elution from thecartridge showed only one compound, with completeelimination of the major adduct, 1,2-DHN-4-N7Gua,and exclusive formation of the oxidized adduct, 1,2-NQ-4-N7Gua. This indicated that the use of solid phaseextraction facilitates the autoxidation of the catecholadduct to the corresponding quinone adduct. Therefore,we decided to skip the extraction step and employedpreparative reverse phase chromatography directly afterfiltration of the reaction mixture. The HPLC conditionsfor both analytical and preparative scales are describedin Section 2. Along with recovered starting material, twoproducts could be isolated, eluting at retention times of7 and 9 min and identified as 1,2-NQ-4-N7Gua (0.7%)and 1,2-DHN-4-N7Gua (10%), respectively. The recov-
ered starting material (52%) was recycled to increase theoverall yield of the reaction.
Preliminary NMR and MS analyses of 1,2-NQ-4-N7Gua indicated that it is the same compound
M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188 181
by reac
ctwattoatw1aId
mwNpwsoN7
Fig. 3. Synthesis of standard adducts
haracterized by McCoull et al., as a sole product ofhe reaction [38]. In addition to the quinone adduct,e were able to isolate and characterize the catechol
dduct. The partial oxidation of the catechol adduct tohe quinone adduct, in the reaction mixture, was due tohe 1,2-NQ, which was reduced to 1,2-DHN, as observedn HPLC. This was previously observed for the N6Adedduct of hexestrol-3′,4′-quinone [39]. In that case, onlyhe quinone adduct was observed. Moreover, treatmentith NaBH4 completely reduced 1,2-NQ-4-N7Gua to,2-DHN-4-N7Gua. The 1,2-NQ-4-N7Gua is formed byutoxidation of 1,2-DHN-4-N7Gua during purification.n fact, 1,2-DHN is not very stable and tends to autoxi-ize in the presence of air.
FAB-MS of 1,2-DHN-4-N7Gua showed a protonatedolecular ion [M + H]+ at m/z 310, which is consistentith the addition of 1,2-DHN to the Gua moiety. The 1HMR spectrum of the adduct showed two exchangeablehenolic protons resonating at 9.79 and 9.49 ppm, alongith the characteristic chemical shifts of the naphthalene
tructure. The spectrum also demonstrated the absencef the deoxyribose moiety of dG and the presence of H-8,H-1 and exocyclic NH2, resonating at 8.64, 11.72 and.32 ppm, respectively, thus confirming the identifica-
tion of 1,2-naphthoquinone with dG.
tion of the adduct as N7Gua. The attachment of the Guamoiety at C-4 of the naphthalene structure was estab-lished as follows: (1) two doublets in the first ring of thenaphthalene moiety were replaced with one singlet res-onating at 7.32 ppm, indicating the bond is at either C-3or C-4. (2) The doublet at 7.21 ppm was significantlyshifted upfield, relative to that in the parent 1,2-DHN(i.e., 7.70 ppm) due to its location in the shielding zoneof the Gua C8-N9 double bond. (3) Finally, in the longrange HMBC experiment, H-8 of Gua showed a corre-lation with C-4 at 122.4 and not at 118.0 ppm, assignedto C-3, thus ruling out the C-3 position as the site ofattachment.
The reaction of 1,2-NQ with Ade (Fig. 4) affordedthree major adducts, as well as some minor humps onHPLC (not shown). Based on their on-line UV spectra,we assumed these minor compounds (∼10% of the totaladduct yield) were the oxidized adducts of the majorcatechol adducts. This assumption was based on our pre-vious observation of such oxidized adducts formed in
the reaction between dG and 1,2-NQ. A brief treatmentwith NaBH4 removed these oxidized adducts, renderingthe original major adducts as the only products of thereaction. Purification by preparative HPLC and structure
182 M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188
reaction
Fig. 4. Synthesis of standard adducts by
elucidation by NMR and MS yielded the major adduct(20%) as 1,2-DHN-4-N1Ade and two other adducts as1,2-DHN-4-N3Ade (8%) and 1,2-DHN-4-N6Ade (9%).FAB-MS analysis of all three compounds produced thesame [M + H]+ at m/z 294.0946, corresponding to theproposed elemental composition.
The 1H NMR spectrum of the major compound, 1,2-DHN-4-N1Ade, showed four proton signals resonatingat 10.00, 9.69, 9.54 and 9.29 ppm, which disappearedafter D2O exchange. Thus, two of them were assigned tothe phenolic OH’s and the remaining two were assignedas NH protons of Ade. This compound was initiallythought to be 1,2-DHN-4-N6Ade, in which the exocyclicNH2 group was attached to the naphthalene moiety atC-4. However, further detailed 2D NMR experiments,including HSQC and HMBC, ruled out this compoundbeing 1,2-DHN-4-N6Ade. In the HMBC experiment, acorrelation between H-2 of Ade at 8.89 ppm with C-4of naphthalene at 123.2 ppm suggested the compound tobe either an N3Ade or N1Ade adduct. Previous resultsfrom our laboratory have shown the splitting of theNH2 signal into two separate singlets when the bondis at the N-1 position of Ade [40,41]. On this basis, thestructure was assigned as 1,2-DHN-4-N1Ade. The pres-ence of two characteristic doublets and two triplets at8.19, 7.22, 7.48 and 7.28 ppm, respectively, indicatedthat the protons of ring B of the naphthalene moietyremained unattached after nucleophilic reaction of Adewith 1,2-NQ. However, the appearance of one singletat 7.52 ppm indicated the attachment of Ade at ring Aof the naphthalene moiety. Assignment of this proton
as H-3 was proposed based on the 1H/13C correlationbetween this proton and two carbons at C-10 (124.5 ppm)and C-1 (140.8 ppm) in an HMBC experiment with theadduct.
of 1,2-naphthoquinone with Ade or dA.
The 1H NMR spectrum of 1,2-DHN-4-N6Adeshowed two sharp singlets (10.00, 9.73 ppm) and twobroad singlets at 9.78, and 8.37 ppm, which disappearedafter D2O exchange. The two sharp singlets at 10.00 and9.73 ppm were assigned to the phenolic OH’s and theremaining two at 9.78 and 8.37 ppm were assigned asthe N-9 and exocyclic N6H protons of Ade. All otherchemical shifts were assigned with the help of detailed1D and 2D NMR data.
The 1H NMR spectrum of 1,2-DHN-4-N3Adeshowed a two-proton broad singlet resonating at7.85 ppm, assigned as the exocyclic NH2 group of Ade,based on its disappearance after D2O exchange. All otherchemical shifts were assigned with the help of detailed1D and 2D NMR data.
In the reaction between 1,2-NQ and dA (Fig. 4), thereaction mixture was treated with NaBH4 to remove theminor oxidized adducts and then subjected to prepara-tive HPLC. The major adduct formed was elucidated byNMR and MS and found to be 1,2-DHN-4-N6dA.
FAB-MS analysis of the adduct showed an [M + H]+
at m/z 410, which is consistent with the addition of 1,2-DHN to the dA moiety. Moreover, the 1H NMR showedsignals for the deoxyribose unit, indicating the formationof an adduct containing a stable glycosyl bond. Assign-ments of all chemical shifts were carried out as describedabove for reaction between 1,2-NQ and Ade.
3.2. Kinetics of the loss of the deoxyribose moiety
After 2 h, the reaction of 1,2-NQ with dG in a mixture
of DMF/water/acetic acid (1:1:1) afforded three adducts,namely, 1,2-DHN-4-N7dG, 1,2-DHN-4-N7Gua and 1,2-NQ-4-N7Gua as shown in Fig. 5A. When the reactionmixture was analyzed by HPLC after 12 h, the mid-
M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188 183
F e of 1,2( -DHN-4
d(ttsstunlwciDMstwww1iNa
dTate
ig. 5. Reaction of 1,2-NQ with dG. (A) HPLC showing the presencB) presence of 1,2-DHN-4-N7Gua after 12 h, and (C) MS/MS of 1,2
le peak corresponding to 1,2-DHN-4-N7dG was absentFig. 5B), and only two peaks could be seen. To iden-ify the initially formed labile adduct, an aliquot ofhe reaction mixture was directly infused into the masspectrometer after 2 h and parent molecular ions werecanned. A molecular ion peak at m/z 426 indicatedhe presence of an adduct in which the deoxyribosenit is still bonded to the Gua moiety. We tentativelyamed this adduct 1,2-DHN-4-N7dG, based on the fol-owing observations: (a) the reaction mixture after 2 has subjected to preparative HPLC, the labile peak was
ollected and analyzed quickly on MS by direct infusion;t produced an ion at m/z 426, corresponding to [1,2-HN-4-N7dG]+, which upon further fragmentation byS/MS gave a daughter ion at m/z 310 (Fig. 5C), corre-
ponding to 1,2-DHN-4-N7Gua, i.e., the adduct withouthe deoxyribose moiety; (b) when the isolated peakas re-injected in analytical HPLC, the labile adductas completely converted to 1,2-DHN-4-N7Gua; (c)e were unable to record the NMR spectrum of the,2-DHN-4-N7dG adduct because it decomposed dur-ng evaporation/lyophilization of the solvent. Analogous7dG adducts have been described in similar studies of
dducts formed by natural and synthetic estrogens [42].Next, we undertook the kinetics of formation and
ecomposition of the labile 1,2-DHN-4-N7dG adduct.
he reaction was conducted at different pHs (1.9 and 4.6)nd different temperatures (22 and 37 ◦C). Aliquots ofhe reaction mixtures were analyzed by HPLC at differ-nt time points, as shown in Fig. 6. The greatest amount
-NQ-4-N7Gua, 1,2-DHN-4-N7dG and 1,2-DHN-4-N7Gua after 2 h,-N7dG.
of 1,2-DHN-4-N7dG adduct was formed at the lowertemperature (22 ◦C) and lower pH (1.9) (Fig. 6A). Thisphenomenon may be explained by assuming a relativestabilization of the glycosyl bond at lower tempera-ture so that more of the N7dG adduct accumulated overtime. Alternatively, more of the N7dG adduct may haveformed at lower pH in the acid-catalyzed Michael addi-tion. When the temperature was raised to 37 ◦C keepingthe pH fixed at 1.9, a high rate of loss of deoxyribosewas observed. Even though the level of N7dG adduct at37 ◦C was not as high as that at 22 ◦C (Fig. 6A), a rel-atively high level of the 1,2-DHN-4-N7Gua adduct wasobserved (Fig. 6B). This indicates a faster 1,4-Michaeladdition (first step), as well as faster loss of deoxyribosefrom the labile 1,2-DHN-4-N7dG adduct. Increasing thepH to 4.6 afforded a much lower yield of 1,2-DHN-4-N7Gua at both the lower (22 ◦C) and higher (37 ◦C)temperature (Fig. 6B). Again, this can be explained byassuming less efficient 1,4-Michael addition at the higherpH.
3.3. Covalent binding of 1,2-NQ orenzyme-activated 1,2-DHN to DNA
Formation of depurinating adducts was observed byreacting 1,2-NQ or enzyme-activated 1,2-DHN with
calf thymus DNA at 37 ◦C for 10 h. The supernatantcontaining the depurinating DNA adducts was ana-lyzed by HPLC with electrochemical detection and byUPLC–MS/MS. Quantitation of the adducts was car-
184 M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188
(B) fo
Fig. 6. (A) Formation and disappearance of 1,2-DHN-4-N7dG andtemperatures and pHs.
ried out by monitoring one of the major fragments of1,2-DHN-4-N7Gua (m/z 310 > 185.9) for the N7Guaadduct and of 1,2-DHN-4-N3Ade (m/z 294 > 231) for theN3Ade adduct, as shown in Figs. 7 and 8, respectively.
Reaction of 1,2-NQ with DNA produced thedepurinating adducts 1,2-DHN-4-N7Gua and 1,2-DHN-4-N3Ade at similar levels (3.1 ± 0.7 and2.5 ± 0.9 �mol/mol DNA-P, respectively, Table 1). Thepotential of 1,2-DHN for autoxidation was assessedby reacting 1,2-DHN with DNA in the presence ofair. We observed formation of depurinating adductsat low levels, i.e., 0.5 ± 0.1 �mol N3Ade/mol DNA-Pand 0.4 ± 0.1 �mol N7Gua/mol DNA-P. Oxidation of1,2-DHN in the presence of tyrosinase (with oxygen
of the air as cofactor) was found to be highly efficient,producing 25.6 ± 2.1 �mol N3Ade/mol DNA-P and17.5 ± 1.0 �mol N7Gua/mol DNA-P. The level ofdepurinating adducts was intermediate after activation
rmation of 1,2-DHN-4-N7Gua from 1,2-DHN-4-N7dG at various
of 1,2-DHN with prostaglandin H synthase (and arachi-donic acid as cofactor) and lower with MC-inducedrat liver microsomes (and NADPH). The lower levelof formation of depurinating adducts with the latterenzymes may be due to the in situ reaction of 1,2-NQwith proteins.
The stable adducts were quantified by the 32P-post-labeling method [36,37]. In each case, four adductbands were detected upon separation by polyacry-lamide gel electrophoresis. The total amounts of stableDNA adducts were similar to those of the depuri-nating adducts, except when 1,2-DHN was activatedwith tyrosinase, which formed much higher levels ofdepurinating adducts (Table 1). Although exact quan-
tification was not achieved due to differences in adductrecovery, the pattern of adduct formation suggests thatdepurinating adducts are an important fraction of totaladducts.
M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188 185
Fig. 7. Multiple reaction monitoring (MRM) profile of the standard 1,2-DHN-4-N7Gua and 1,2-DHN-4-N7Gua formed in tyrosinase-catalyzedreaction of 1,2-DHN with calf thymus DNA. The upper spectrum is the MS/MS of the biologically formed adduct.
Fig. 8. Multiple reaction monitoring (MRM) profile of the standard 1,2-DHN-4-N3Ade and 1,2-DHN-4-N3Ade formed in tyrosinase-catalyzedreaction of 1,2-DHN with calf thymus DNA. The upper spectrum is the MS/MS of the biologically formed adduct.
186 M. Saeed et al. / Chemico-Biological Interactions 165 (2007) 175–188
Table 1Formation of adducts after reaction of 1,2-NQ with DNAa
a These results are the average of three reactions.b Not determined.
Fig. 9. Rate of depurination of 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua from DNA after reaction of 1,2-NQ with DNA.
3.4. Kinetics of DNA depurination
The rate of depurination of DNA adducts was deter-mined by reacting 1,2-NQ with DNA at 37 ◦C and pH7.0 and analyzing the depurinating adducts at differ-ent time points, i.e., 0.5, 1, 2, 3, 4, 5, 7, 9 and 10 h(Fig. 9). At each time point, the DNA was precipitatedwith 2 vol. of ethanol and the supernatant was analyzedby UPLC–MS/MS. Depurination of the N3Ade adductwas almost complete after 1 h; after that it remainedalmost constant. The depurination of the N7Gua adduct,however, was found to be slower and was complete in 3 h.The slower depurination of the N7Gua adduct is commonfor natural and synthetic estrogens [26,42], and benzene[43].
4. Conclusions
1,2-NQ and enzymatically activated 1,2-DHN, bothsecondary metabolites of naphthalene [19,22,23], reactwith DNA by 1,4-Michael addition to form specifi-cally the depurinating N7Gua and N3Ade adducts, aswell as unidentified stable adducts. Furthermore, the
N3Ade adduct depurinates instantaneously, whereas theN7Gua adduct depurinates with a half-life of ca. 1.5 h.These chemical and biochemical properties are simi-lar to those of the catechol-3,4-quinones of the natural
1.0 ± 0.4 3.1 ± 0.8
estrogens [24–27,42], the catechol quinones of the syn-thetic estrogens diethylstilbestrol and its hydrogenatedderivative hexestrol [30,31,42] and the catechol quinoneof the leukemogen benzene [32,43]. The apurinic sitesformed by the depurinating DNA adducts may be con-verted by error-prone base excision repair [28,29,44]into mutations that can initiate cancer. This unifyingmechanism renders plausible the hypothesis that naph-thalene initiates cancer via the same mechanism ofactivation, in which 1,2-NQ is its ultimate carcinogenicmetabolite.
Acknowledgements
This research was supported by U.S. Public HealthService Grant P01 CA49210. Core support at the EppleyInstitute was provided by Grant P30 CA36727 from theNational Cancer Institute.
References
[1] IARC, IARC Monographs on the Evaluation of CarcinogenicRisks to Human: Some Traditional Herbal Medicines, SomeMycotoxins, Naphthalene and Styrene, vol. 82, Lyon, France,2002, pp. 367–435.
[2] J.S. Stanley, Broad Scan Analysis of the FY82 National HumanAdipose Tissue Survey Specimens, Executive Summary (EPA-560/5-86-035), Environmental Protection Agency, vol. 1, Officeof Toxic Substances, Washington, DC, 1986.
[4] Environmental Protection Agency. Clean Air Act Amendmentsof 1990 Conference Report to Accompany S. 1630 (Report No.101-952), Washington, DC, Office of Pollution Prevention andToxics, 1990.
[5] O. Wolf, Cancers in Chemical Workers in a Former Naphtha-lene Purification Plant, vol. 31, Dt. Gesundh, Wesen, 1976, pp.996–999.
[6] O. Wolf, Carcinoma of the larynx in naphthalene purifiers, Z. Get.Hyg. 24 (1978) 737–739.
gical In
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
M. Saeed et al. / Chemico-Biolo
[7] A. Buckpitt, B. Boland, M. Isbell, D. Morin, M. Shultz, R. Bald-win, K. Chan, A. Karlsson, C. Lin, A. Taff, J. West, M. Fanucchi,L. Van Winkle, C. Plopper, Naphthalene-induced respiratory tracttoxicity: metabolic mechanisms of toxicity, Drug Metab. Rev. 34(2002) 791–820.
[8] National Toxicology Program (NTP), Toxicology and Carcino-genesis Studies of Naphthalene (CAS No. 91-20-3) in B6C3F1Mice (inhalation studies), Technical Report Series No. 410, NIHPublication No. 92-3141; U.S. Department of Health and HumanServices, Public Health Service, National Institutes of Health,Research Triangle Park, NC, 1992.
[9] K.M. Abdo, S.L. Eustis, M. McDonald, M.P. Joiken, B. AdkinsJr., J.K. Haseman, Naphthalene: a respiratory tract toxicant andcarcinogen for mice, Inhal. Toxicol. 4 (1992) 393–409.
10] K.M. Abdo, S. Grumbein, B.J. Chou, R. Herbert, Toxicity andcarcinogenicity study in F344 rats following 2 years of whole-body exposure to naphthalene vapors, Inhal. Toxicol. 13 (2001)931–950.
11] National Toxicology Program (NTP), Toxicology and Carcino-genesis Studies of Naphthalene (CAS No. 91-20-3) in F344/NRats (Inhalation Studies), CNTP Technical Report Series No. 500;NIH Publication No. 01-4434, Research Triangle Park, NC, 2000.
12] A.R. Buckpitt, N. Castagnoli Jr., S.D. Nelson, A.D. Jones,L.S. Bahnson, Stereoselectivity of naphthalene epoxidation bymouse, rat and hamster pulmonary, hepatic and renal microsomalenzymes, Drug Metab. Disp. 15 (1987) 491–498.
13] D.M. Jerina, J.W. Daly, B. Witkop, Z. Zaltzman-Nirenberg, S.Udenfriend, 1,2-naphthalene oxide as an intermediate in themicrosomal hydroxylation of naphthalene, Biochemistry 9 (1970)147–156.
14] P.J. Van Bladeren, K.P. Vyas, J.M. Sayer, D.E. Ryan, P.E. Thomas,W. Levin, D.M. Jerina, Stereoselectivity of cytochrome P-450c inthe formation of naphthalene and anthracene 1,2-oxides, J. Biol.Chem. 259 (1984) 8966–8973.
15] S. Kanekal, C. Plopper, D. Morin, A. Buckpitt, Metabolism andcytotoxicity of naphthalene oxide in the isolated perfused mouselung, J. Pharmacol. Exp. Ther. 256 (1991) 391–401.
17] M.D. Tingle, M. Pirmohamed, E. Templeton, A.S. Wilson, S.Madden, N.R. Kitteringham, B.K. Park, An investigation ofthe formation of cytotoxic, genotoxic, protein-reactive and sta-ble metabolites from naphthalene by human liver microsomes,Biochem. Pharmacol. 46 (1993) 1529–1538.
18] E. Boyland, P. Sims, Metabolism of polycyclic compounds,Biochem. J. 68 (1958) 440–447.
19] T.E. Smithgall, R.G. Harvey, T.M. Penning, Spectroscopic identi-fication of ortho-quinones as the products of polycyclic aromatictrans-dihydrodiol oxidation catalyzed by dihydrodiol dehydroge-nase, J. Biol. Chem. 263 (1988) 1814–1820.
20] R.M. Turkall, G.A. Skowronski, A.M. Kadry, M.S. Abdel-Rahman, A comparative study of the kinetics and bioavailabilityof pure and soil-absorbed naphthalene in dermally exposed malerats, Arch. Environ. Toxicol. 26 (1994) 504–509.
21] J. Bakke, C. Struble, J.-A. Gustafsson, B. Gustafsson, Catabolismof premercapturic acid pathway metabolites of naphthalene tonaphthols and methylthio-containing metabolites in rats, Bio-
chemistry 82 (1985) 668–671.
22] M. d’Arcy Doherty, R. Makowski, G.G. Gibson, G.M. Cohen,Cytochrome P-450 dependent metabolic activation of 1-naphtholto naphthoquinones and covalent binding species, Biochem. Phar-macol. 34 (1985) 2261–2267.
[
teractions 165 (2007) 175–188 187
23] M. Flueraru, A. Chichirau, L.L. Chepelev, W.G. Willmore, T.Durst, M. Charron, L.R. Barclay, J.S. Wright, Cytotoxicity andcytoprotective activity in naphthalenediols depends on their ten-dency to form naphthoquinones, Free Radic. Biol. Med. 39 (2005)1368–1377.
24] E.L. Cavalieri, D.E. Stack, P.D. Devanesan, R. Todorovic, I.Dwivedy, S. Higginbotham, S.L. Johansson, K.D. Patil, M.L.Gross, J.K. Gooden, R. Ramanathan, R.L. Cerny, E.G. Rogan,Molecular origin of cancer: catechol estrogen-3,4-quinones asendogenous tumor initiators, Proc. Natl. Acad. Sci. USA 94(1997) 10937–10942.
25] K.-M. Li, R. Todorovic, P. Devanesan, S. Higginbotham, H.Kofeler, R. Ramanathan, M.L. Gross, E.G. Rogan, E.L. Cavalieri,Metabolism and DNA binding studies of 4-hydroxyestradiol andestradiol-3,4-quinone in vitro and in female ACI rat mammarygland in vivo, Carcinogenesis 25 (2004) 289–297.
26] M. Zahid, E. Kohli, M. Saeed, E. Rogan, E. Cavalieri, The greaterreactivity of estradiol-3,4-quinone vs estradiol-2,3-quinone withDNA in the formation of depurinating adducts: implications fortumor-initiating activity, Chem. Res. Toxicol. 19 (2006) 164–172.
27] E. Cavalieri, E. Rogan, D. Chakaravarti, The role of endogenouscatechol quinones in the initiation of cancer and neurodegen-erative diseases, in: H. Sies, L. Packer (Eds.), Methods inEnzymology, Quinones and Quinone Enzymes, Part B, vol. 382,Elsevier, Duesseldorf, Germany, 2004, pp. 293–319.
28] D. Chakravarti, P. Mailander, K.-M. Li, S. Higginbotham, H.Zhang, M.L. Gross, E. Cavalieri, E. Rogan, Evidence that a burstof DNA depurination in SENCAR mouse skin induces error-pronerepair and forms mutations in the H-ras gene, Oncogene 20 (2001)7945–7953.
29] P.C. Mailander, J.L. Meza, S. Higginbotham, D. Chakravarti,Induction of A.T to G.C mutations by erroneous repair of depuri-nated DNA following estrogen treatment of the mammary glandof ACI rats, J. Steroid Biochem. Mol. Biol. 101 (2006) 204–215.
30] M. Saeed, S.J. Gunselman, S. Higginbotham, E. Rogan, E. Cav-alieri, Formation of the depurinating N3Adenine and N7Guanineadducts by reaction of DNA with hexestrol-3′,4′-quinone orenzyme activated 3′-hydroxyhexestrol. Implications for a uni-fying mechanism of tumor initiation by natural and syntheticestrogens, Steroids 70 (2005) 37–45.
31] M. Saeed, E. Rogan, E. Cavalieri, Mechanism of tumor initiationby the human carcinogen diethylstilbestrol: the defining link tonatural estrogens, Proc. Am. Assoc. Cancer Res. 46 (2005) 2129.
32] E.L. Cavalieri, K.-M. Li, N. Balu, M. Saeed, P. Devanesan, S.Higginbotham, J. Zhao, M.L. Gross, E.G. Rogan, Catechol ortho-quinones: the electrophilic compounds that form depurinatingDNA adducts and could initiate cancer and other diseases, Car-cinogenesis 23 (2002) 1071–1077.
33] Z. Zhao, W. Kosinska, M. Khmelnitsky, E.L. Cavalieri, E.G.Rogan, D. Chakravarti, P. Sacks, J.B. Guttenplan, Mutagenicactivity of 4-hydroxyestradiol, but not 2-hydroxyestradiol, inBB2 rat embryonic cells, and the mutational spectrum of 4-hydoxyestradiol, Chem. Res. Toxicol. 19 (2006) 475–479.
34] NIH Guidelines for the Laboratory Use of Chemical Carcinogens,NIH Publication No. 81-2385, US Government printing Office,Washington, DC, 1981.
35] K.L. Platt, F. Oesch, Efficient synthesis of non-K-region
trans-dihydro diols of polycyclic aromatic hydrocarbons fromo-quinones and catechols, J. Org. Chem. 48 (1983) 256–268.
36] L. Chen, P.D. Devanesan, S. Higginbotham, F. Ariese, R.Jankowiak, G.J. Small, E.G. Rogan, E.L. Cavalieri, Expandedanalysis of benzo[a]pyrene-DNA adducts formed in vitro and in
gical In
[
[
[
[
[
[
[
[dence that error-prone DNA repair converts dibenzo[a,l]pyrene-
188 M. Saeed et al. / Chemico-Biolo
mouse skin: their significance in tumor initiation, Chem. Res.Toxicol. 9 (1996) 897–903.
37] I. Terashima, N. Suzuki, S. Shibutani, 32P-postlabeling/polyacrylamide gel electrophoresis analysis: application to thedetection of DNA adducts, Chem. Res. Toxicol. 15 (2002)305–311.
38] K.D. McCoull, D. Rindgen, I.A. Blair, T.M. Penning, Syn-thesis and characterization of polycyclic aromatic hydrocarbono-quinone depurinating N7-guanine adducts, Chem. Res. Toxicol.12 (1999) 237–246.
39] S.T. Jan, P.D. Devanesan, D.E. Stack, R. Ramanathan, J. Byun,M.L. Gross, E.G. Rogan, E.L. Cavalieri, Metabolic activation andformation of DNA adducts of hexestrol, a synthetic nonsteroidalcarcinogenic estrogen, Chem. Res. Toxicol. 11 (1998) 412–419.
40] P.P.J. Mulder, L. Chen, B.C. Sekhar, M. George, M.L. Gross,
E.G. Rogan, E.L. Cavalieri, Synthesis and structure determi-nation of the adducts formed by electrochemical oxidation of1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in the pres-ence of deoxyribo-nucleosides or adenine, Chem. Res. Toxicol.9 (1996) 1264–1277.
teractions 165 (2007) 175–188
41] K.-M. Li, M. Byun, M.L. Gross, D. Zamzow, R. Jankowiak,E.G. Rogan, E.L. Cavalieri, Synthesis and structure determi-nation of the adducts formed by electrochemical oxidation ofdibenzo[a,l]pyrene in the presence of adenine, Chem. Res. Toxi-col. 12 (1999) 749–757.
42] M. Saeed, M. Zahid, S.J. Gunselman, E. Rogan, E. Cavalieri,Slow loss of deoxyribose from the N7deoxyguanosine adducts ofestradiol-3,4-quinone and hexestrol-3′,4′-quinone. Implicationsfor mutagenic activity, Steroids 70 (2005) 29–35.
43] A. Raghavanpillai, K. Olson, E. Rogan, E. Cavalieri, Slow lossof deoxyribose from N7deoxyguanosine adducts of catecholquinone: possible relevance for mutagenic activity, Proc. Am.Assoc. Cancer Res. 45 (2004) 1553.
44] D. Chakravarti, P.C. Mailander, E.L. Cavalieri, E.G. Rogan, Evi-
induced depurinating lesions into mutations: formation, clonalproliferation and regression of initiated cells carrying H-ras onco-gene mutations in early preneoplasia, Mutat. Res. 456 (2000)17–32.
