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Products formed from the in vitro reaction of metabolites of 3-aminochrysene with calf thymus DNA
Chem.-Biol. Interactions, 86 (1993) 1-15
1
Elsevier ScientificPublishers IrelandLtd.
PRODUCTS FORMED FROM THE IN VITRO REACTION OF METABOLITES OF 3-AMINOCHRYSENE WITH CALF THYMUS DNA
DIOGENES HERRENO-SAENZ, FREDERICK E. EVANS, CHING-CHENGLAI, JOAQUIN ABIAN, PETER P. FU and K. BARRYDELCLOS National Center for Toxicological Research, Jefferson, AR 75079 (USA)
(Received April 13th, 1992) (Revision received October 20th, 1992) (Accepted October 20th, 1992)
SUMMARY
3-Aminochrysene, a mutagenic geometric isomer of the mutagenic and carcinogenic aromatic amine 6-aminochrysene, has been synthesized and its metabolic activation studied by characterization of the products formed from the reaction of metabolites with calf thymus DNA. DNA adducts produced by 3aminochrysene via N-oxidation were examined by preparing 3-nitrosochrysene and incubating the nitroso derivative with calf thymus DNA in the presence of ascorbic acid (to generate the N-hydroxy derivative) at pH 5. The major adduct, as determined by 1H-NMR and thermospray-mass spectrometry of the modified nucleoside obtained after enzymatic hydrolysis of the modified DNA, was N(deoxyguanosin-8-yl)-3-aminochrysene. Thus, the reaction of N-hydroxy-3aminochrysene with DNA differs from that of N-hydroxy-6-aminochrysene, which had previously been shown to generate N-(deoxyguanosin-8-yl)-6aminochrysene, 5~deoxyguanosin-N2-yl)-6-aminochrysene and N-(deoxyinosin-8yl)-6-aminochryseneas major adducts. 32p-Postlabeling analysis of DNA treated with 3-aminochrysene in the presence of liver microsomes from rats pretreated with phenobarbital indicated an adduct pattern identical to that seen with DNA that had been treated with 3-nitrosochrysene and ascorbic acid. However, DNA treated with 3-aminochrysene (3-AC) in the presence of liver microsomes from rats pretreated with 3-methylcholanthrene contained a major adduct that was chromatographically distinct from N-(deoxyguanosin-8-yl)-3-aminochrysene.
Correspondence to: K. BarryDelclos,Divisionof BiochemicalToxicology,HFT-110NationalCenter for ToxicologicalResearch,Jefferson,AR 72079, USA. Ab~eviations: 3-AC,3-aminochrysene;6-AC,6-aminochrysene;3-MC-microsomes,livermicrosomes from rats pretreatedwith 3-methylcholanthrene;6-NC, 6-nitrochrysene;PAH, polycyclicaromatic hydrocarbon;PB-microsomes,livermicrosomesfromrats pretreatedwith phenobarbital;TSP-MS, thermospray-massspectrometry.
2
Key words: Aromatic amines; Metabolic activation; DNA adducts; 32p_ postlabeling
INTRODUCTION
A substantial body of evidence indicates that the primary mode of activation of carcinogenic aromatic amines involves N-oxidation as a critical metabolic transformation. The predominant lesions produced in DNA from activated Nhydroxyarylamines and the lesions that are believed to play major roles in the initiation of tumorigenesis by this class of compounds, have generally been found to be adducts formed by reaction of the ultimate carcinogen with the C8 position of deoxyguanosine [1]. There are, however, other possible activation pathways and other sites of modification on DNA that have been identified for several activated N-hydroxyarylamines [1]. 6-AC is an example of an aromatic amine that is activated in a manner that is atypical for this class of compounds. Grantham et al. [2] were unable to find N-oxidized metabolites of 6-AC in rat excreta and suggested that this could be due to either the dosing regimen used or the fact that this compound was metabolized in a manner analogous to PAHs. More recent work on the metabolic activation of 6-NC and 6-AC has indicated that 6-AC can be activated by N-oxidation or by ring oxidation and that both pathways can lead to toxicity as measured by the induction of mutations or tumors [3- 7]. In contrast to the majority of aromatic amines that have been studied, 6-AC appears to be preferentially N-oxidized by phenobarbital-inducible cytochromes P-450 IIB1 and IIB2 [8]. Furthermore, three major DNA adducts are produced on reaction of N-hydroxy-6-aminochrysene with calf t h y m u s DNA: 5(deoxyguanosin-N2-yl)-6-aminochrysene, N-(deoxyguanosin-8-yl)-6-aminochrysene and N-(deoxyinosin-8-yl)-6-aminochrysene [9]. The latter adduct has been hypothesized to result from the oxidative deamination of the corresponding deoxyadenosine adduct [9]. While the roles of each of these adducts have not been determined, recent analyses of point mutations induced in the hprt gene of CHO cells by 6-nitrosochrysene, under conditions where the adduct pattern produced was identical to that produced from the reaction of N-hydroxy-6-AC with DNA, have indicated that the mutations in 14 of 17 independently isolated mutants with single base pair substitutions had sequence changes that were consistent with mutagen-induced damage at deoxyadenosine residues [10]. Indications of the potential biological significance of the modification of deoxyadenosine residues by arylamine metabolites have also been reported for 2-nitropyrene [11], 4-aminobiphenyl [12], 4,4'-methylenebis(2-chloroaniline) [13] and aristolochic acid [14]. We are interested in determining the structural features that determine both the reactivity of N-hydroxyamino PAHs with DNA and the activation of these compounds by ring oxidative pathways as opposed to N-oxidation. These differences in metabolic activation can then be related to differences in biological activity. As a first step toward this goal, we report here the synthesis of 3-AC
and 3-nitrosochrysene, the chemical preparation of DNA adducts from Nhydroxy-3-AC by reaction of 3-nitrosochrysene with calf thymus DNA in the presence of ascorbic acid and in vitro formation of DNA adducts from 3-AC mediated by PB- and 3-MC-microsomes from rat liver. These results are compared with the results that have previously been reported for the geometric isomer, 6-aminochrysene. MATERIALS AND METHODS
Materials 3-Aminochrysene was synthesized as follows: Friedel-Crafts acetylation of chrysene with acetyl chloride in carbon disulfide [15] yielded 6-acetylchrysene as the major product (approx. 60% yield) and 3-acetylchrysene as a minor product (10% yield). 3-Acetylchrysene was reacted with hydroxylamine hydrochloride in pyridine and absolute ethanol at 80°C to give 3-acetylchrysene oxime in a 90% yield. Beckmann rearrangement of the oxime to 3-acetylaminochrysene followed by hydrolysis to 3-aminochrysene (38% yield) was achieved by heating at 120°C in acetic anhydride and acetic acid for 8 h. The 1H-NMR data for 3aminochrysene are given in Table I. For the synthesis of 3-nitrosochrysene, 3-AC (0.359 mmol) was dissolved in 25 ml of acetone and treated under argon with 2 M equivalents of m-chloroperoxybenzoic acid at 0°C for 2 h. Ice-cold ethyl acetate (100 ml) was added to the reaction mixture and the solution was washed three times with saturated sodium bicarbonate and three times with water. The solution was dried over anhydrous magnesium sulfate and solvent was removed under reduced pressure. Purification by open-column silica gel chromatography with benzene as the eluting solvent gave 3-nitrosochrysene in 50% yield. The identity of the product as 3-nitrosochrysene was confirmed by mass spectrometry [m/z (relative intensity): 257 (60%); 227 (100%)]. All enzymes used in the hydrolysis of DNA for adduct analysis by HPLC or by 32P-postlabeling were obtained from Sigma Chemical Co., St. Louis, MO. T4 polynucleotide kinase was from United States Biochemical Corporation, Cleveland, OH. [7-S2p]ATP (-> 3000 Ci]mmol) was synthesized from carrier-free [32P]phosphate (ICN Biomedicals, Costa Mesa, CA) as described by Gupta et al. [16].
Mutagenicity assays Reversion to prototrophy was measured using Salmonella typhimurium histidine auxotrophic strain TA98 by the method outlined by Maron and Ames [17]. Assays were carried out in quadruplicate using 1 - 20 nmol/plate. The postmitochondrial supernatant fraction ($9) was prepared from a liver homogenate of Aroclor 1254-pretreated male Sprague- Dawley rats and used at a concentration of 50 ~l/plate. The number of revertants per plate observed in solvent controls ranged from 25 to 40. The number of revertants per nmol was calculated from linear regression analysis of the increasing portion of the dose-response curve.
4 TABLE I 1H-NMR C H E M I C A L S H I F T S O F 3 - A M I N O C H R Y S E N E A N D N-(DEOXYGUANOSIN-8-YL)-3A M I N O C H R Y S E N E IN M E T H A N O L - d 4 A N D DMSO-d6 a Assignment
3-AC 1 2 3 (NHfNH2) 4 5 6 7 8 9 10 11 12
3-Aminochrysene
N-(deoxyguanosin-8-yl)-3-AC
Methanol-d 4
DMSO-d 6
Methanol-d 4
7.76 7.12 -8.01 8.62 7.93 7.96 7.59 7.66 8.78 8.46 7.85
7.75 7.05 5.56 7.85 8.53 7.99 8.02 7.63 7.69 8.83 8.44 7.86
7.90 7.90 -9.23 8.80 8.01 7.99 7.61 7.68 8.81 8.62 7.94
7.99 8.09 9.05 9.25 8.63 8.11 8.08 7.68 7.74 8.91 8.69 8.02
----------
-----------
6.53 2.78 2.23 4.64 4.06 4.03 3.96 -----
6.40 2.62 2.08 4.49 3.98 3.86 3.86 10.61 6.38 5.35 6.13
DMSO-d 6
dG 1' 2' 2" 3' 4' 5' 5" 1-NH 2-NH2 3'-OH 5'-OH
a Chemical shifts a r e given in ppm. A s s i g n m e n t s for 5 ' a n d 5 " p r o t o n s of t h e 2 '-deoxyribose moiety of t h e adduct m a y be reversed. Coupling c o n s t a n t s (J, Hz) for 3-aminochrysene in methanol-d 4 are as follows: J1-2, 8.5; J2-4, 2.2; J5-6, 9.0; J7-8, 8.0; J7-9, 1.4; J8-9, 6.9; Jg-lo, 8.4; Jll-12, 9.0. Coupling c o n s t a n t s for N-(deoxyguanosin-8-yl)-3-AC in methanol-d 4 are as follows: J1-2, not detected due to m a g n e t i c equivalence; J5-6, 9.2; J7-8, 8.0; J8-9, 6.9; J9-1o, 8.4; Jl1-12, 9.0; J l ' - 2 ' , 9.3; J 1 ' - 2 " , 6.0; J 2 ' - 2 " , - 1 3 . 3 ; J 2 ' - 3 ' , 6.5; J 2 " - 3 ' , 2.0; J 3 ' - 4 ' , 2.6; J 4 ' - 5 ' , 2.4; J 4 ' - 5 " , 1.8; J 5 ' - 5 " , - 11.7. First- order m e a s u r e m e n t s indicate t h a t t h e coupling c o n s t a n t s are the s a m e within experim e n t a l error for both solvents, m e t h a n o l - d 4 and DMSO-d 6.
In vitro DNA binding and analysis of modified DNA Reaction of 3-nitrosochrysene with DNA in the presence of ascorbic acid. Calf thymus DNA (1 mg/ml) was dissolved in argon-purged 10 mM sodium citrate buffer (pH 5) and treated overnight at 37°C with 3-nitrosochrysene (0.1 mg/ml) and ascorbic acid (5-fold molar excess over 3-nitrosochrysene). The incubation mixture was extracted with equal volumes of solvents as follows: 3 times with ethyl acetate, 3 times with phenol, 2 times with phenol/chloroform/isoamyl alcohol
(25:24:1 by vol.) and 1 time with chloroform/isoamyl alcohol (24:1 v/v). The phenol-containing solutions were saturated with 50 mM Tris-HCI (pH 8) prior to use. DNA was precipitated with 2 vols. of cold ethanol after addition of NaC1 to a final concentration of 0.5 M. The modified DNA was dissolved in 5 mM BisTris, 0.1 mM EDTA (pH 7.1), and its concentration was determined by scanning with a Beckman Model DU-65 spectrophotometer (1 mg/ml DNA = 20 absorbance units at 260 nm). For HPLC analysis and collection of 3-aminochrysenenucleoside adducts, modified DNA was treated with DNase I (Type VII, 0.1 mg/mg DNA) in the presence of magnesium chloride (10 ~l of a 1-M solution/mg DNA) for 3 h at 37°C. Snake venom phosphodiesterase (Type VII, 0.01 U/mg DNA) and bacterial alkaline phosphatase (Type III-S, 0.6 U/mg DNA) were added and the incubation was continued overnight. All DNA hydrolysis incubations were carried out under an argon atmosphere. The hydrolysis mixture was extracted three times with equal volumes of water-saturated n-butanol, the pooled n-butanol extracts were washed once with n-butanol-saturated water and the solvent was evaporated under reduced pressure. The residue was dissolved in methanol and separations were carried out on a 5-~m Vydac C18 analytical column (Separation Group, Hesperia, CA) using a 40-min linear gradient from 40% methanol in water to 100% methanol at a flow rate of 1.5 ml/min. To prepare samples for NMR spectroscopy, additional purification of the peaks was carried out isocratically (55% or 70% methanol in water) using the same analytical column described above and a flow rate of 1.5 ml/min. HPLC analyses were carried out with instrumentation from Waters Associates, Inc., Milford, MA (two model 510 pumps, a model 660 solvent programmer, a model U6K injector and a model 440 absorbance monitor). In some cases, ultraviolet spectra were taken during elution with a Hewlett-Packard 1040B photodiode array detector. For analysis by s2p-postlabeling, the modified DNA was hydrolyzed by incubating with micrococcal nuclease (0.012 U/#g) and spleen phosphodiesterase (0.001 U/~g) in 20 mM sodium succinate, 10 mM CaC12 (pH 6.0) at 37°C for 3.5 h as described by Gupta et al. [16]. The DNA digest was then treated with nuclease P1 (0.54 U/~g DNA) for 1 h at 37°C [18l or extracted with n-butanol [19], [5'-S2p]phosphorylated at 37°C for 40 min with T4 polynucleotide kinase (1 U/~g DNA) and 200 ~Ci [7-S2P]ATP and treated for 30 rain with 30 mU apyrase [16]. 32p-Labeled mutagen-nucleotide adducts were separated from unmodified nucleotides (D1) using Machery-Nagel PEI cellulose plates from Alltech Associates, Inc., Deerfield, IL with 0.65 M sodium phosphate buffer (pH 6.0) as the developing solvent. The adducts, which remained as a compact spot at the origin of the D1 plate, were excised and placed in contact [20] with the origin (1.5 cm from bottom, 1.5 cm from left border) of a 10 × 10 cm E. Merck PEI cellulose plates (Alltech Associates, Inc.) for transfer and development in the D3 (3.3 M lithium formate, 7.7 M urea, pH 8.2) and D4 (0.72 M sodium phosphate, 0.45 M Tris, 7.6 M urea, pH 8.2) directions. Following development in the D4 direction, a wick (Whatman 1 filter paper, 1.2 cm in width) was attached to the top of the plate (top with respect to D4 development direction) and plates were washed with 0.9 M sodium phosphate buffer (pH 6.8) until the solvent reached the top of the wick. The wicks were removed and the corners of the plate marked
with radioactive ink spots. Adduct spots were visualized by placing the plates in contact with Cronex X-ray film (DuPont, Wilmington, DE) for a length of time determined by scanning the plate with a survey meter equipped with a GeigerMueller detector. In some cases, the adduct spots were cut from the plates, placed in 20-ml scintillation vials with 10 ml Ultima Gold (Packard, Downers Grove, IL) and counts were determined in a scintillation counter. Binding of 3-AC to DNA mediated by PB- or 3-MC-microsomes from rat liver. DNA modified by microsomal metabolites of 3-aminochrysene was prepared by incubating for 1 h at 37°C a mixture containing, 50 nmol/ml 3-AC, 50 #mol/ml Tris-HC1 buffer (pH 7.5), 3 #mol/ml magnesium chloride, 1 U/ml glucosephosphate-dehydrogenase (Type XII, Sigma Chemical Co., St. Louis, MO), 2 ~mol glucose 6-phosphate, 125 nmol NADP +, 1 mg rat liver microsomal protein and 2 mg calf thymus DNA. In some cases, 50 nmol of 6-AC was used in place of 3-AC. The rat liver microsomes were prepared as described previously from rats that had been pretreated for 3 consecutive days prior to sacrifice with intraperitoneal injections of 3-methylcholanthrene (25 mg/kg) or phenobarbital (80 mgfkg) [21]. DNA was purified from the microsomal incubations by solvent extractions and precipitation (approx. 70% recovery of DNA added to reaction), hydrolyzed and analyzed by 32p-postlabeling as described above. To ensure that any adduct spots observed were due to chemical treatment, DNA was incubated under identical conditions in the absence of test compound, prior to isolation, hydrolysis and analysis. Characterization of the major DNA adduct derived from N-hydroxy-3-A C by mass and nuclear magnetic resonance spectroscopy. For analysis of the products of DNA hydrolysis by thermospray (TSP) HPLC-mass spectrometry, a Finnigan (San Jose, CA) TSQ 70 triple quadrupole mass spectrometer equipped with a Finnigan TSP source and interface was used in conjunction with a Spectra-Physics SP8700XR programmer and pumping system with a 5-m Spherisorb ODS-2 reverse phase column (0.46 x 15 cm, Phase Separations Inc., Norwalk, CT). Samples were dissolved in methanol for injection and were separated with a linear gradient from 70% solvent B: 30% solvent A to 100% solvent B in 5 min at a flow rate of 1 ml/min. (Solvent A: 0.05 M ammonium acetate buffer (1% formic acid); solvent B: 70% methanol/30% 0.05 M ammonium acetate buffer (1% formic acid)). It was determined from the HPLC-UV data, using the molar absortivity of 3-aminochrysene at 254 nm as the reference, that the quantities of adduct injected were in the range of 100- 500 ng. Interface and jet temperatures were set at 80°C and 230°C, respectively. Mass spectrometric analyses were carried out by scanning the third quadrupole of the instrument (Q3) between 130 and 600 u at a rate of 0.5 scans/s. To enhance the signal, spectra were obtained in the filament on mode. 1H-NMR measurements were carried out on a Bruker AM500 spectrometer. Samples of about 400 ug of adduct were dissolved in 0.6 ml of methanol-dt or DMSO-d6 and NMR spectra were recorded at 29°C. The chemical shifts are reported in ppm by assigning the methanol resonance to 3.30 ppm or the DMSO resonance to 2.49 ppm. Resonance assignments are based on chemical shift and coupling constant measurements, integrations, selective decoupling experi-
ments, long-range coupling constants, nuclear Overhauser effect (NOE) measurements and comparison to model compounds. RESULTS
Mutagenicity of S-aminochrysene The mutagenic activity of 3-AC was compared to that of 6-AC in Salmonella typhimurium strain TA98 in the absence and presence of liver $9 from Aroclorpretreated rats. In the absence of $9, 3-AC treatment resulted in 183 revertants/nmol while 6-AC treatment gave 115 revertants/nmol. The direct-acting mutagenicity of 6-AC has been previously reported [5]. In the presence of $9, 3-AC treatment gave 170 revertants/nmol in comparison to 710 revertants/nmol for 6-AC treatment.
Characterization of the major DNA adduct formed from the ascorbic acidenhanced binding of 3-nitrosochrysene to calf thymus DNA Nitrosoarenes have generally been found to react poorly with DNA relative to N-hydroxyarylamines [22,23]. However, ascorbic acid has been shown to be an effective reducing agent for generating N-hydroxyarylamines from nitroso derivatives of nitro PAHs [9,24]. In order to determine the profile of DNA adducts formed from N-hydroxy-3-AC, 3-nitrosochrysene was incubated with calf thymus DNA at pH 5 in the presence of ascorbic acid (see Materials and Methods). HPLC analyses of enzymatic hydrolysates of DNA recovered from this reaction revealed two potential modified nucleosides with retention times of
%_ MINUTES Fig, 1. Reversed-phase HPLC profile of an n-butanol extract of an enzymatic hydrolysate of calf thymus DNA modified by reaction with 3-nitrosochrysene in the presence of ascorbic acid. The conditions for the reaction, enzymatic digestion and HPLC separation are described in Materials and Methods.
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Fig. 2. 500 MHz 1H-NMR spectra with resonance assignments of N-(deoxyguanosin-8-yl)-3aminochrysene. (A) Spectrum recorded in methanol-d 4. (B) Spectrum recorded in DMSO-d6. Insets show an expanded view of the region of the spectra containing the resonances of chrysene protons H1, H2, H6, H7 and H12.
17.6 and 27.9 min (Fig. 1). Attempts to definitively identify the material eluting at 17.6 min were unsuccessful due to the small quantity of material available, the impurity of the material collected, which was indicated by the electon impact mass spectrum and unusual broadening of resonances in the ;H-NMR spectrum (data not shown). 1H-NMR spectra of the adduct eluting at 27.9 min were recorded at 500 MHz in both methanol-d4 (Table I and Fig. 2A) and DMSO-d6 (Table I and Fig. 2B) under various experimental conditions to identify the adduct. Decoupling experiments and coupling constant measurements, as well as an NOE observed for H5 as a result of saturation of H4, confirmed the presence of all expected chrysene protons and established the C3 position of the chrysene ring as the site of substitution. Fine structure observed in resolution enhanced spectra due to small long-range coupling (JH-H = 0.5 - 1.5 Hz) from H4 to H1, H2 and H12 also establish that the apparent singlet at 7.90 arises from the chrysene ring and not from a nucleic acid base [25]. Both chemical shift and coupling constant data (Table I) establish the presence of a 2'-deoxyribose ring. Moreover, the magnitude of the coupling constants of the sugar ring are indicative of an unusually high population of C2 '-endo sugar ring conformation and a predominant gauche+ (~ = 60 °) conformation about the C4' - C5' bond. This is a characteristic of CS-amino-substituted purine nucleosides and nucleotides [26] and the results are nearly identical to those reported for the closely related adduct N-(deoxyguanosin-8-yl)-2-aminofluorene [27]. Thus, the coupling constant data alone strongly suggest a substitution via nitrogen at the C8 position of a
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Fig. 3. Mass spectrum of N-(deoxyguanosin-8-yl)-3-aminochyrsene. DNA was treated with 3nitrosochrysene and ascorbic acid, enzymatically hydrolyzed to nucleosides and analyzed by thermospray HPLC-mass spectrometry as described in Materials and Methods.
10
purine ring. The absence of any singlet that can be attributed to a nucleic acid base is evidence that the substitution is to a guanine base. This was confirmed by recording spectra in DMSO-d6 (Fig. 2B) where the expected exchangeable protons at N1 (10.62 ppm) and NH2 (6.38 ppm) were found as well as that of the amine nitrogen at C8 of the substituted guanine ring (9.05 ppm). The exchangeable protons for the 3' (5.35 ppm) and 5' (6.13 ppm) hydroxyl groups were also identified in the spectrum. It is therefore concluded that the major adduct produced from the reaction of N-hydroxy-3-aminochrysenewith calf thymus DNA is N-(deoxyguanosin-8-yl)-3-aminochrysene. Further supporting evidence for the structure of the adduct and confirmation of its molecular weight were obtained from HPLC-MS analysis. Under the chromatographic conditions used for the analysis (see Materials and Methods), a single peak with a retention time of 12.5 min was obtained. The TSP spectrum of the adduct is shown in Fig. 3. The ion observed at m/z 509 is that expected for the [M + H] ÷ ion of N-(deoxyguanosin-8-yl)-3-aminochrysene(tool. wt. 508). The signal at m/z 393 can be assigned to the loss of deoxyribose (116 amu) from the molecular ion. The signals at m/z 134 and m/z 166 also support the presence of deoxyribose (S) in the adduct ([S + NHJ ÷ and [S + NH4 + CH3OH] ÷, respec-
Fig. 4. Autoradiogram of 32p-postlabeled n-butanol extract of an enzymatic digest of calf thymus DNA treated with 3-nitrosochrysene and ascorbic acid. DNA was hydrolyzed, extracted and labeled as described in Materials and Methods. 'O' marks the origin of the chromatogram. D3 was run from bottom to top and D4 and D5 were run from left to right.
11
tively). Other minor signals at m/z 116, 117 and 419 are also due to the deoxyribose moiety in the structure ([S + NH4-H20] +, [S + H] ÷ and [MH - 90] +, respectively). The ion at m/z 152 is indicative of the guanine moiety ([BH21 +). Except for a very low intensity signal at m/z 244, which may correspond to the [chrysene-NHs] ÷ ion, no other signals arising from the breakdown of the guanine-chrysene bonds are observed.
Fig. 5. Autoradiograms of 32p-postlabeled n-butanol extracts (Panel A) or nuclease Fl-treatec (Pane|s B - D) enzymatic digests of DNA from microsomal incubations conducted in the presence oJ absence of 3-AC. (A and B) Calf thymus DNA treated with 3-AC in the presence of PB microsomes (C) Calf thymus DNA from microsomal incubations with 3-AC in the presence of 3-MC microsomes (D) Calf thymus DNA from microsomal incubations with DMSO in the presence of 3-MC microsomes
12
s2p-Postlabeling analysis of calf thymus DNA modified by 3-nitrosochrysene in the presence of ascorbic acid or by microsomal metabolites of 3-aminochrysene 32P-Postlabeling analysis of calf thymus DNA modified by 3-nitrosochrysene in the presence of ascorbic acid, confirmed the presence of a single major adduct spot (Fig. 4, Spot 1; RfD3 = 0.45, RfD4 = 0.35) that was not present in control DNA. Only 30% of the N-(deoxyguanosin-8-yl)-3-AC-3 ',5 '-bisphosphate detected by the n-butanol method was recovered in the nuclease Pl-treated samples when equivalent amounts of N-hydroxy-3-AC-modified DNA were analyzed by these two enhancement techniques. 82P-Postlabeling analysis of calf thymus DNA that had been modified by incubation with 3-AC in the presence of PB-microsomes (Fig. 5A, Spot 1) indicated a single adduct spot that was chromatographically identical to that described above for DNA modified by N-hydroxy-3-AC and exhibited low recovery when the nuclease P1 enhancement procedure was used (Fig. 5B). This adduct was also observed in DNA recovered from an incubation of 3-AC with 3-MC-microsomes when the n-butanol enhancement method [20] was used prior to 32p_ postlabeling (not shown). However, when the nuclease P1 enhancement procedure was used, a second, chromatographically distinct, adduct spot (D3 Rf = 0.64, D4 Rf = 0.53) was the major product observed (Fig. 5C, Spot 2). This adduct spot was not present in DNA that was incubated with 3-MC microsomes with solvent alone (Fig. 5D). DISCUSSION
Like its geometric isomer, 6-AC, 3-AC is mutagenic in Salmonella strain TA98 both in the absence and in the presence of an exogenous metabolic activation system and we have here compared the pattern of DNA adducts produced by these two isomers in vitro. The data presented here indicate that N-hydroxy-3AC, generated in situ by the reduction of 3-nitrosochrysene with ascorbic acid, reacts with DNA in vitro to give N-(deoxyguanosin-8-yl)-3-aminochrysene as the major product. Furthermore, the chromatographic profile obtained after 32p_ postlabeling of N-hydroxy-3-AC-modified DNA was consistent with the formation of a single adduct, although the presence of multiple adducts in the single adduct spot detected cannot be ruled out. The apparent susceptibility of the adduct spot to nuclease P1 is consistent with previous results obtained with several C8-deoxyguanosine adducts of aromatic amines [28,29]. HPLC analysis of modified nucleosides derived from the reaction of N-hydroxy-3-aminochrysene with calf thymus DNA indicated a possible minor adduct that was not detected as a chromatographically distinct adduct in the TLC system used for separation of 32p-postlabeled adducts. 1H-NMR analysis of this peak was inconclusive and we were thus unable to determine if this product was a distinct DNA adduct or a degradation product of N-(deoxyguanosin-8-yl)-3-AC. Based on comparisons with the relative retention times of the DNA adducts of other aromatic amines on reversed phase HPLC columns (9,30 - 35), it is likely that this adduct, if it is not derived from N-(deoxyguanosin-8-yl)-3-AC, is an N2-deoxyguanosine or an N6-deoxyadenosine adduct rather than a C8-deoxyadenosine or CS-deoxyinosine
13
adduct, since the latter would be expected to have a greater retention time than a CS-deoxyguanosine adduct. Regardless of the identity of the minor DNA adduct from N-hydroxy-3-AC, these results contrast with what had been observed for the reaction of N-hydroxy-6-AC with calf thymus DNA, where 5(deoxyguanosin-N2-yl)-6-AC, N-(deoxyguanosin-8-yl)-6-AC and N-(deoxyinosin8-yl)-6-AC were all found to be major reaction products [9]. As was discussed in the Introduction, work with several compounds has suggested that the formation of deoxyadenosine adducts from N-hydroxyarylamines can have significant mutagenic consequences. Our results suggest that significant differences in the biological activities of 3-AC and 6-AC may exist under conditions where Noxidation of the compounds is the predominant mode of activation. Many of the commonly studied aromatic amines are preferentially N-oxidized by the 3-methylcholanthrene-inducible forms of cytochromes P-450, CYP1A1 and CYP1A2 [36]. In contrast, 4,4'-methylene-bis(2-chloroaniline) has been shown to be preferentially N-oxidized by phenobarbital-inducible forms, CYP2B1 and CYP2B2 [371 and there is evidence that suggests that 6-AC is also preferentially N-oxidized by phenobarbital-inducible forms of cytochromes P-450 as compared to 3-methylcholanthrene-inducible forms [8]. Although no information on preferential activation was obtained from the experiments conducted, the results presented here suggest that 3-AC is N-oxidized by both 3-MC- and PB-inducible rat liver cytochrome P-450 isozymes. Our data on adduct formation in DNA modified by metabolites of 3-AC generated by 3-methylcholanthrene-induced rat liver microsomes suggest that 3-AC, like 6-AC, may be activated by means other than simple N-oxidation, either ring oxidation or a combination of ring and Noxidation. Previous studies with 6-nitrochrysene and 6-AC have provided indirect evidence that the formation of 1,2-dihydroxy-3,4-epoxy-l,2,3,4tetrahydro-6-aminochrysene is a major activation pathway for these compounds [4,6]. In light of the strong carcinogenic and mutagenic activity associated with the formation of adducts derived from a ring oxidized metabolite of 6-AC [3 - 7] further investigation of the structural determinants of the ring oxidation activation pathways of geometric isomers of amino and nitro derivatives of chrysene, and the biological consequences of the damage produced from these metabolites, will be of interest. ACKNOWLEDGMENTS
The technical assistance of T.A. McRae and L.S. Von Tungeln is gratefully acknowledged. REFERENCES 1 F.A. Beland and F.F. Kadlubar, Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons, Handb. Exp. Pharmacol., 94/I (1990)267-325. 2 P.H. Grantham, N. Ba-Giao, L.C. Mohan, T. Benjamin, P.P. Roller and E.K. Weisburger, The metabolism of 6-aminochrysene in the rat, Eur. J. Cancer, 12 (1976) 227-235. 3 K.B. Delclos, R.P. Walker, K.L. Dooley, P.P. Fu and F.F. Kadlubar, Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of
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4
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[aH]-6-nitrochrysene, [3H]-6-aminochrysene and [3H]-l,6-dinitropyrene, Cancer Res., 47 (1987) 6272 - 6277. K.B. Delclos, K. El-Bayoumy, S.S. Hecht, R.P. Walker, and F.F. Kadlubar, Metabolism of the carcinogen [~H]6-nitrochrysene in the preweanling mouse: identification of 6-aminochrysene1,2-dihydrodiol as the probable proximate carcinogenic metabolite, Carcinogenesis, 9 (1988) 1875-1884. K. El-Bayoumy, K.B. Delclos, R.H. Hefiich, R.P. Walker, G.-H. Shiue and S.S. Hecht, Mutagenicity, metabolism and DNA adduct formation of 6-nitrochrysene in Salmonella typhimurium, Mutagenesis, 4 (1989) 235-240. K. EI-Bayoumy, G.-H. Shiue and S.S. Hecht, Comparative tumorigenicity of 6-nitrochrysene and its metabolites in newborn mice, Carcinogenesis, 10 (1989) 369-372. K.B. Delclos and R.H. Heflich, Mutation induction and DNA adduct formation in Chinese hamster ovary cells treated with 6-nitrochrysene, 6-aminochrysene and their metabolites, Mutat. Res., 279 (1992) 153-164. R.A. Lubet, C.E. McKinney, J.W. Cameron, F.P. Guengerich and R.W. Nims, Preferential activation of 6-aminochrysene and 2-aminoanthracene to mutagenic moieties by different forms of cytochrome P-450 in hepatic 9000 x g supernatants from the rat, Mutat. Res., 212 (1989) 275 - 284. K.B. Delclos, D.W. Miller, J.O. Lay, Jr., D.A. Casciano, R.P. Walker, P.P. Fu and F.F. Kadlubar, Identification of C8-modified deoxyinosine and N 2- and C8-modified deoxyguanosine as major products of the in vitro reaction of N-hydroxy-6-aminochrysenewith DNA and the formation of these adducts in isolated rat hepatocytes treated with 6-nitrochrysene and 6aminochrysene, Carcinogenesis, 8 (1987) 1703-1709. M.G. Manjnatha, R.A. Mittelstaedt, R.K. Newton, G.R.D. Villani, K.B. Delclos and R.H. Heflich, Molecular characterization of 6-nitroso-chrysene-induced mutations in the hprt gene of Chinese hamster ovary cells, Environ. Mol. Mutagen., 19 (Suppl. 20) (1992) 40. P.P. Fu, D.W. Miller, L.S. Von Tungeln, J.O. Lay, Jr., K. Huang, L. Jones and F.E. Evans, Formation of C8-modified deoxyguanosine and deoxyadenosine as major DNA adducts from anaerobic metabolism of 2-nitropyrene mediated by rat and mouse liver microsomes and cytosols, Carcinogenesis, 12 (1991) 609-616. D.D. Lasko, S.C. Harvey, S.B. Malaika, F.F. Kadlubar and J.M. Essigmann, Specificity of mutagenesis by 4-aminobiphenyl: a possible role for N-(deoxyadensin-8-yl)-4-aminobiphenyl as a premutational lesion, J. Biol. Chem., 263 (1988) 15429-15435. N.A. Silk, J.O. Lay, Jr. and C.N. Martin, Covalent binding of 4,4 '-methylenebis(2-chloroaniline) to rat liver in vivo and of its N-hydroxylated derivative to DNA in vitro, Biochem. Pharmacol., 38 (1989) 279-287. W. Pfau, H.H. Schmeiser and M. Wiessler, Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA, Carcinogenesis, 11 (1990) 313-319. W. Carruthers, Friedel-Crafts acetylation of chrysene, J. Chem. Soc., (1953) 3486- 3489. R.C. Gupta, M.V. Reddy and K. Randerath, 32p-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts, Carcinogenesis, 3 (1982) 1081-1092. D.M. Maron and B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res., 113 (1983) 173-215. M.V. Reddy and K. Randerath, Nuclease Pl-mediated enhancement of sensitivity of 32p. postlabeling test for structurally diverse DNA adducts, Carcinogenesis, 7 (1986) 1543-1551. R.C. Gupta, Enhanced sensitivity of 82p-postlabeling analysis of aromatic carcinogen: DNA adducts, Cancer Res., 45 (1985) 5656-5662. L.-J.W. Lu, R.M. Disher, M.V. Reddy and K. Randerath, 32p-Postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4aminobiphenyl and benzo[a]pyrene, Cancer Res., 46 (1986) 3046-3054. M.W. Chou, R.H. Heflich and P.P. Fu, Metabolism of 1-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites, Carcinogenesis, 7 (1986) 1837-1844. F.F. Kadlubar and F.A. Beland, Chemical properties of ultimate carcinogenic metabolites of arylamines and arylamides, in: R.G. Harvey (Ed.), Polycyclic Hydrocarbons and Carcinogenesis, American Chemical Society, Washington, DC, 1985, pp. 341-370.
15 23 R.H. Heflich, P.C. Howard and F.A. Beland, An examination of the weak mutagenic response of 1-nitropyrene in Chinese hamster ovary cells, Mutat. Res., 161 (1985) 99-108. 24 P.C. Howard, F.A. Beland and C.E. Cerniglia, Reduction of the carcinogen 1-nitropyrene to 1aminopyrene by rat intestinal bacteria, Carcinogenesis, 4 (1983) 985- 990. 25 F.E. Evans, P.P. Fu, T. Cairns and S.K. Yang, Long-range coupling constants for structural analysis of complex polycyclic aromatic hydrocarbons by high-field proton magnetic resonance spectrometry, Anal. Chem., 53 (1981) 558-560. 26 F.E. Evans and N.O. Kaplan, 8-Alkylaminoadenyl nucleosides as probes of dehydrogenase interactions with nucleotide analogs of different glycosyl conformation, J. Biol. Chem., 251 (1976) 6791- 6797. 27 F.E. Evans, D.W. Miller and F.A. Beland, Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene, Carcinogenesis, 1 (1980) 955-959. 28 R.C. Gupta and K. Earley, S2p-Adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures, Carcinogenesis, 9 (1988) 1687-1693. 29 J.A. Gallagher, M.A. Jackson, M.H. George, J. Lewtas and I.G.C. Robertson, Differences in detection of DNA adducts in the ssp-postiabolling assay after either 1-butanol extraction or nuclease P1 treatment, Cancer Lett., 45 (1989) 7-12. 30 F.A. Beland, D.L. Tullis, F.F. Kadlubar, K.M. Strauh and F.E. Evans, Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazo-benzene in vitro and in vivo, Chem.-Bioh Interact., 31 (1980) 1-17. 31 J.H.M. Meerman, F.A. Beland and G.J. Mulder, Role of suifation in the formation of DNA adducts from N-hydroxy-2-acetylaminofluorene in rat liver in ~/vo. Inhibition of N-acetylated aminofluorene adduct formation by pentachlorophenol, Carcinogenesis, 2 (1981) 413-416. 32 F.F. Kadiubar, J.F. Anson, K.L. Dooley and F.A. Beland, Formation of urothelial and hepatic DNA adducts from the carcinogen 2-naphthylamine, Carcinogenesis, 2 (1981) 467-470. 33 J.G. Westra, T.J. Flammang, N.F. Fullerton, F.A. Beland, C.C. Weis and F.F. Kadiubar, Formation of DNA adducts in v/vo in rat liver and intestinal epithelium after administration of the carcinogen 3,2'-dimethyl-4-aminobiphenyl and its hydroxamic acid, Carcinogenesis, 6 (1985) 37 - 44. 34 T.J. Flammang, J.G. Westra, F.F. Kadiubar and F.A. Beland, DNA adducts formed from the probable proxhnate carcinogen N-hydroxy-3,2'-dimethyl-4-aminobiphenyl,by acid catalysis or S-acetyl coenzyme A-dependent enzymatic esterification, Carcinogenesis, 6 (1985) 251-258. 35 R.C. Gupta, K. Earley, N.F. Fullerton and F.A. Beland, Formation and removal of DNA adducts in target and nontarget tissues of rats administered multiple doses of 2acetylaminophenanthrene, Carcinogenesis, 11 (1989) 2025-2033. 36 F.F. Kadlubar and G.J. Hammons, The role of cytochrome P-450 in the metabolism of chemical carcinogens, in: F.P. Guengerich (Ed.), Mammalian Cytochromes P-450, Vol. II, CRC Press, Boca Raten, 1987, pp. 81-130. 37 M.A. Butler, F.P. Guengerich and F.F. Kadiubar, Metabolic oxidation of the carcinogens 4aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline)by human hepatic rnicrosomes and by purified rat hepatic cytochrome P-450 monooxygenases, Cancer Res., 49 (1989) 25- 31.
1
Elsevier ScientificPublishers IrelandLtd.
PRODUCTS FORMED FROM THE IN VITRO REACTION OF METABOLITES OF 3-AMINOCHRYSENE WITH CALF THYMUS DNA
DIOGENES HERRENO-SAENZ, FREDERICK E. EVANS, CHING-CHENGLAI, JOAQUIN ABIAN, PETER P. FU and K. BARRYDELCLOS National Center for Toxicological Research, Jefferson, AR 75079 (USA)
(Received April 13th, 1992) (Revision received October 20th, 1992) (Accepted October 20th, 1992)
SUMMARY
3-Aminochrysene, a mutagenic geometric isomer of the mutagenic and carcinogenic aromatic amine 6-aminochrysene, has been synthesized and its metabolic activation studied by characterization of the products formed from the reaction of metabolites with calf thymus DNA. DNA adducts produced by 3aminochrysene via N-oxidation were examined by preparing 3-nitrosochrysene and incubating the nitroso derivative with calf thymus DNA in the presence of ascorbic acid (to generate the N-hydroxy derivative) at pH 5. The major adduct, as determined by 1H-NMR and thermospray-mass spectrometry of the modified nucleoside obtained after enzymatic hydrolysis of the modified DNA, was N(deoxyguanosin-8-yl)-3-aminochrysene. Thus, the reaction of N-hydroxy-3aminochrysene with DNA differs from that of N-hydroxy-6-aminochrysene, which had previously been shown to generate N-(deoxyguanosin-8-yl)-6aminochrysene, 5~deoxyguanosin-N2-yl)-6-aminochrysene and N-(deoxyinosin-8yl)-6-aminochryseneas major adducts. 32p-Postlabeling analysis of DNA treated with 3-aminochrysene in the presence of liver microsomes from rats pretreated with phenobarbital indicated an adduct pattern identical to that seen with DNA that had been treated with 3-nitrosochrysene and ascorbic acid. However, DNA treated with 3-aminochrysene (3-AC) in the presence of liver microsomes from rats pretreated with 3-methylcholanthrene contained a major adduct that was chromatographically distinct from N-(deoxyguanosin-8-yl)-3-aminochrysene.
Correspondence to: K. BarryDelclos,Divisionof BiochemicalToxicology,HFT-110NationalCenter for ToxicologicalResearch,Jefferson,AR 72079, USA. Ab~eviations: 3-AC,3-aminochrysene;6-AC,6-aminochrysene;3-MC-microsomes,livermicrosomes from rats pretreatedwith 3-methylcholanthrene;6-NC, 6-nitrochrysene;PAH, polycyclicaromatic hydrocarbon;PB-microsomes,livermicrosomesfromrats pretreatedwith phenobarbital;TSP-MS, thermospray-massspectrometry.
2
Key words: Aromatic amines; Metabolic activation; DNA adducts; 32p_ postlabeling
INTRODUCTION
A substantial body of evidence indicates that the primary mode of activation of carcinogenic aromatic amines involves N-oxidation as a critical metabolic transformation. The predominant lesions produced in DNA from activated Nhydroxyarylamines and the lesions that are believed to play major roles in the initiation of tumorigenesis by this class of compounds, have generally been found to be adducts formed by reaction of the ultimate carcinogen with the C8 position of deoxyguanosine [1]. There are, however, other possible activation pathways and other sites of modification on DNA that have been identified for several activated N-hydroxyarylamines [1]. 6-AC is an example of an aromatic amine that is activated in a manner that is atypical for this class of compounds. Grantham et al. [2] were unable to find N-oxidized metabolites of 6-AC in rat excreta and suggested that this could be due to either the dosing regimen used or the fact that this compound was metabolized in a manner analogous to PAHs. More recent work on the metabolic activation of 6-NC and 6-AC has indicated that 6-AC can be activated by N-oxidation or by ring oxidation and that both pathways can lead to toxicity as measured by the induction of mutations or tumors [3- 7]. In contrast to the majority of aromatic amines that have been studied, 6-AC appears to be preferentially N-oxidized by phenobarbital-inducible cytochromes P-450 IIB1 and IIB2 [8]. Furthermore, three major DNA adducts are produced on reaction of N-hydroxy-6-aminochrysene with calf t h y m u s DNA: 5(deoxyguanosin-N2-yl)-6-aminochrysene, N-(deoxyguanosin-8-yl)-6-aminochrysene and N-(deoxyinosin-8-yl)-6-aminochrysene [9]. The latter adduct has been hypothesized to result from the oxidative deamination of the corresponding deoxyadenosine adduct [9]. While the roles of each of these adducts have not been determined, recent analyses of point mutations induced in the hprt gene of CHO cells by 6-nitrosochrysene, under conditions where the adduct pattern produced was identical to that produced from the reaction of N-hydroxy-6-AC with DNA, have indicated that the mutations in 14 of 17 independently isolated mutants with single base pair substitutions had sequence changes that were consistent with mutagen-induced damage at deoxyadenosine residues [10]. Indications of the potential biological significance of the modification of deoxyadenosine residues by arylamine metabolites have also been reported for 2-nitropyrene [11], 4-aminobiphenyl [12], 4,4'-methylenebis(2-chloroaniline) [13] and aristolochic acid [14]. We are interested in determining the structural features that determine both the reactivity of N-hydroxyamino PAHs with DNA and the activation of these compounds by ring oxidative pathways as opposed to N-oxidation. These differences in metabolic activation can then be related to differences in biological activity. As a first step toward this goal, we report here the synthesis of 3-AC
and 3-nitrosochrysene, the chemical preparation of DNA adducts from Nhydroxy-3-AC by reaction of 3-nitrosochrysene with calf thymus DNA in the presence of ascorbic acid and in vitro formation of DNA adducts from 3-AC mediated by PB- and 3-MC-microsomes from rat liver. These results are compared with the results that have previously been reported for the geometric isomer, 6-aminochrysene. MATERIALS AND METHODS
Materials 3-Aminochrysene was synthesized as follows: Friedel-Crafts acetylation of chrysene with acetyl chloride in carbon disulfide [15] yielded 6-acetylchrysene as the major product (approx. 60% yield) and 3-acetylchrysene as a minor product (10% yield). 3-Acetylchrysene was reacted with hydroxylamine hydrochloride in pyridine and absolute ethanol at 80°C to give 3-acetylchrysene oxime in a 90% yield. Beckmann rearrangement of the oxime to 3-acetylaminochrysene followed by hydrolysis to 3-aminochrysene (38% yield) was achieved by heating at 120°C in acetic anhydride and acetic acid for 8 h. The 1H-NMR data for 3aminochrysene are given in Table I. For the synthesis of 3-nitrosochrysene, 3-AC (0.359 mmol) was dissolved in 25 ml of acetone and treated under argon with 2 M equivalents of m-chloroperoxybenzoic acid at 0°C for 2 h. Ice-cold ethyl acetate (100 ml) was added to the reaction mixture and the solution was washed three times with saturated sodium bicarbonate and three times with water. The solution was dried over anhydrous magnesium sulfate and solvent was removed under reduced pressure. Purification by open-column silica gel chromatography with benzene as the eluting solvent gave 3-nitrosochrysene in 50% yield. The identity of the product as 3-nitrosochrysene was confirmed by mass spectrometry [m/z (relative intensity): 257 (60%); 227 (100%)]. All enzymes used in the hydrolysis of DNA for adduct analysis by HPLC or by 32P-postlabeling were obtained from Sigma Chemical Co., St. Louis, MO. T4 polynucleotide kinase was from United States Biochemical Corporation, Cleveland, OH. [7-S2p]ATP (-> 3000 Ci]mmol) was synthesized from carrier-free [32P]phosphate (ICN Biomedicals, Costa Mesa, CA) as described by Gupta et al. [16].
Mutagenicity assays Reversion to prototrophy was measured using Salmonella typhimurium histidine auxotrophic strain TA98 by the method outlined by Maron and Ames [17]. Assays were carried out in quadruplicate using 1 - 20 nmol/plate. The postmitochondrial supernatant fraction ($9) was prepared from a liver homogenate of Aroclor 1254-pretreated male Sprague- Dawley rats and used at a concentration of 50 ~l/plate. The number of revertants per plate observed in solvent controls ranged from 25 to 40. The number of revertants per nmol was calculated from linear regression analysis of the increasing portion of the dose-response curve.
4 TABLE I 1H-NMR C H E M I C A L S H I F T S O F 3 - A M I N O C H R Y S E N E A N D N-(DEOXYGUANOSIN-8-YL)-3A M I N O C H R Y S E N E IN M E T H A N O L - d 4 A N D DMSO-d6 a Assignment
3-AC 1 2 3 (NHfNH2) 4 5 6 7 8 9 10 11 12
3-Aminochrysene
N-(deoxyguanosin-8-yl)-3-AC
Methanol-d 4
DMSO-d 6
Methanol-d 4
7.76 7.12 -8.01 8.62 7.93 7.96 7.59 7.66 8.78 8.46 7.85
7.75 7.05 5.56 7.85 8.53 7.99 8.02 7.63 7.69 8.83 8.44 7.86
7.90 7.90 -9.23 8.80 8.01 7.99 7.61 7.68 8.81 8.62 7.94
7.99 8.09 9.05 9.25 8.63 8.11 8.08 7.68 7.74 8.91 8.69 8.02
----------
-----------
6.53 2.78 2.23 4.64 4.06 4.03 3.96 -----
6.40 2.62 2.08 4.49 3.98 3.86 3.86 10.61 6.38 5.35 6.13
DMSO-d 6
dG 1' 2' 2" 3' 4' 5' 5" 1-NH 2-NH2 3'-OH 5'-OH
a Chemical shifts a r e given in ppm. A s s i g n m e n t s for 5 ' a n d 5 " p r o t o n s of t h e 2 '-deoxyribose moiety of t h e adduct m a y be reversed. Coupling c o n s t a n t s (J, Hz) for 3-aminochrysene in methanol-d 4 are as follows: J1-2, 8.5; J2-4, 2.2; J5-6, 9.0; J7-8, 8.0; J7-9, 1.4; J8-9, 6.9; Jg-lo, 8.4; Jll-12, 9.0. Coupling c o n s t a n t s for N-(deoxyguanosin-8-yl)-3-AC in methanol-d 4 are as follows: J1-2, not detected due to m a g n e t i c equivalence; J5-6, 9.2; J7-8, 8.0; J8-9, 6.9; J9-1o, 8.4; Jl1-12, 9.0; J l ' - 2 ' , 9.3; J 1 ' - 2 " , 6.0; J 2 ' - 2 " , - 1 3 . 3 ; J 2 ' - 3 ' , 6.5; J 2 " - 3 ' , 2.0; J 3 ' - 4 ' , 2.6; J 4 ' - 5 ' , 2.4; J 4 ' - 5 " , 1.8; J 5 ' - 5 " , - 11.7. First- order m e a s u r e m e n t s indicate t h a t t h e coupling c o n s t a n t s are the s a m e within experim e n t a l error for both solvents, m e t h a n o l - d 4 and DMSO-d 6.
In vitro DNA binding and analysis of modified DNA Reaction of 3-nitrosochrysene with DNA in the presence of ascorbic acid. Calf thymus DNA (1 mg/ml) was dissolved in argon-purged 10 mM sodium citrate buffer (pH 5) and treated overnight at 37°C with 3-nitrosochrysene (0.1 mg/ml) and ascorbic acid (5-fold molar excess over 3-nitrosochrysene). The incubation mixture was extracted with equal volumes of solvents as follows: 3 times with ethyl acetate, 3 times with phenol, 2 times with phenol/chloroform/isoamyl alcohol
(25:24:1 by vol.) and 1 time with chloroform/isoamyl alcohol (24:1 v/v). The phenol-containing solutions were saturated with 50 mM Tris-HCI (pH 8) prior to use. DNA was precipitated with 2 vols. of cold ethanol after addition of NaC1 to a final concentration of 0.5 M. The modified DNA was dissolved in 5 mM BisTris, 0.1 mM EDTA (pH 7.1), and its concentration was determined by scanning with a Beckman Model DU-65 spectrophotometer (1 mg/ml DNA = 20 absorbance units at 260 nm). For HPLC analysis and collection of 3-aminochrysenenucleoside adducts, modified DNA was treated with DNase I (Type VII, 0.1 mg/mg DNA) in the presence of magnesium chloride (10 ~l of a 1-M solution/mg DNA) for 3 h at 37°C. Snake venom phosphodiesterase (Type VII, 0.01 U/mg DNA) and bacterial alkaline phosphatase (Type III-S, 0.6 U/mg DNA) were added and the incubation was continued overnight. All DNA hydrolysis incubations were carried out under an argon atmosphere. The hydrolysis mixture was extracted three times with equal volumes of water-saturated n-butanol, the pooled n-butanol extracts were washed once with n-butanol-saturated water and the solvent was evaporated under reduced pressure. The residue was dissolved in methanol and separations were carried out on a 5-~m Vydac C18 analytical column (Separation Group, Hesperia, CA) using a 40-min linear gradient from 40% methanol in water to 100% methanol at a flow rate of 1.5 ml/min. To prepare samples for NMR spectroscopy, additional purification of the peaks was carried out isocratically (55% or 70% methanol in water) using the same analytical column described above and a flow rate of 1.5 ml/min. HPLC analyses were carried out with instrumentation from Waters Associates, Inc., Milford, MA (two model 510 pumps, a model 660 solvent programmer, a model U6K injector and a model 440 absorbance monitor). In some cases, ultraviolet spectra were taken during elution with a Hewlett-Packard 1040B photodiode array detector. For analysis by s2p-postlabeling, the modified DNA was hydrolyzed by incubating with micrococcal nuclease (0.012 U/#g) and spleen phosphodiesterase (0.001 U/~g) in 20 mM sodium succinate, 10 mM CaC12 (pH 6.0) at 37°C for 3.5 h as described by Gupta et al. [16]. The DNA digest was then treated with nuclease P1 (0.54 U/~g DNA) for 1 h at 37°C [18l or extracted with n-butanol [19], [5'-S2p]phosphorylated at 37°C for 40 min with T4 polynucleotide kinase (1 U/~g DNA) and 200 ~Ci [7-S2P]ATP and treated for 30 rain with 30 mU apyrase [16]. 32p-Labeled mutagen-nucleotide adducts were separated from unmodified nucleotides (D1) using Machery-Nagel PEI cellulose plates from Alltech Associates, Inc., Deerfield, IL with 0.65 M sodium phosphate buffer (pH 6.0) as the developing solvent. The adducts, which remained as a compact spot at the origin of the D1 plate, were excised and placed in contact [20] with the origin (1.5 cm from bottom, 1.5 cm from left border) of a 10 × 10 cm E. Merck PEI cellulose plates (Alltech Associates, Inc.) for transfer and development in the D3 (3.3 M lithium formate, 7.7 M urea, pH 8.2) and D4 (0.72 M sodium phosphate, 0.45 M Tris, 7.6 M urea, pH 8.2) directions. Following development in the D4 direction, a wick (Whatman 1 filter paper, 1.2 cm in width) was attached to the top of the plate (top with respect to D4 development direction) and plates were washed with 0.9 M sodium phosphate buffer (pH 6.8) until the solvent reached the top of the wick. The wicks were removed and the corners of the plate marked
with radioactive ink spots. Adduct spots were visualized by placing the plates in contact with Cronex X-ray film (DuPont, Wilmington, DE) for a length of time determined by scanning the plate with a survey meter equipped with a GeigerMueller detector. In some cases, the adduct spots were cut from the plates, placed in 20-ml scintillation vials with 10 ml Ultima Gold (Packard, Downers Grove, IL) and counts were determined in a scintillation counter. Binding of 3-AC to DNA mediated by PB- or 3-MC-microsomes from rat liver. DNA modified by microsomal metabolites of 3-aminochrysene was prepared by incubating for 1 h at 37°C a mixture containing, 50 nmol/ml 3-AC, 50 #mol/ml Tris-HC1 buffer (pH 7.5), 3 #mol/ml magnesium chloride, 1 U/ml glucosephosphate-dehydrogenase (Type XII, Sigma Chemical Co., St. Louis, MO), 2 ~mol glucose 6-phosphate, 125 nmol NADP +, 1 mg rat liver microsomal protein and 2 mg calf thymus DNA. In some cases, 50 nmol of 6-AC was used in place of 3-AC. The rat liver microsomes were prepared as described previously from rats that had been pretreated for 3 consecutive days prior to sacrifice with intraperitoneal injections of 3-methylcholanthrene (25 mg/kg) or phenobarbital (80 mgfkg) [21]. DNA was purified from the microsomal incubations by solvent extractions and precipitation (approx. 70% recovery of DNA added to reaction), hydrolyzed and analyzed by 32p-postlabeling as described above. To ensure that any adduct spots observed were due to chemical treatment, DNA was incubated under identical conditions in the absence of test compound, prior to isolation, hydrolysis and analysis. Characterization of the major DNA adduct derived from N-hydroxy-3-A C by mass and nuclear magnetic resonance spectroscopy. For analysis of the products of DNA hydrolysis by thermospray (TSP) HPLC-mass spectrometry, a Finnigan (San Jose, CA) TSQ 70 triple quadrupole mass spectrometer equipped with a Finnigan TSP source and interface was used in conjunction with a Spectra-Physics SP8700XR programmer and pumping system with a 5-m Spherisorb ODS-2 reverse phase column (0.46 x 15 cm, Phase Separations Inc., Norwalk, CT). Samples were dissolved in methanol for injection and were separated with a linear gradient from 70% solvent B: 30% solvent A to 100% solvent B in 5 min at a flow rate of 1 ml/min. (Solvent A: 0.05 M ammonium acetate buffer (1% formic acid); solvent B: 70% methanol/30% 0.05 M ammonium acetate buffer (1% formic acid)). It was determined from the HPLC-UV data, using the molar absortivity of 3-aminochrysene at 254 nm as the reference, that the quantities of adduct injected were in the range of 100- 500 ng. Interface and jet temperatures were set at 80°C and 230°C, respectively. Mass spectrometric analyses were carried out by scanning the third quadrupole of the instrument (Q3) between 130 and 600 u at a rate of 0.5 scans/s. To enhance the signal, spectra were obtained in the filament on mode. 1H-NMR measurements were carried out on a Bruker AM500 spectrometer. Samples of about 400 ug of adduct were dissolved in 0.6 ml of methanol-dt or DMSO-d6 and NMR spectra were recorded at 29°C. The chemical shifts are reported in ppm by assigning the methanol resonance to 3.30 ppm or the DMSO resonance to 2.49 ppm. Resonance assignments are based on chemical shift and coupling constant measurements, integrations, selective decoupling experi-
ments, long-range coupling constants, nuclear Overhauser effect (NOE) measurements and comparison to model compounds. RESULTS
Mutagenicity of S-aminochrysene The mutagenic activity of 3-AC was compared to that of 6-AC in Salmonella typhimurium strain TA98 in the absence and presence of liver $9 from Aroclorpretreated rats. In the absence of $9, 3-AC treatment resulted in 183 revertants/nmol while 6-AC treatment gave 115 revertants/nmol. The direct-acting mutagenicity of 6-AC has been previously reported [5]. In the presence of $9, 3-AC treatment gave 170 revertants/nmol in comparison to 710 revertants/nmol for 6-AC treatment.
Characterization of the major DNA adduct formed from the ascorbic acidenhanced binding of 3-nitrosochrysene to calf thymus DNA Nitrosoarenes have generally been found to react poorly with DNA relative to N-hydroxyarylamines [22,23]. However, ascorbic acid has been shown to be an effective reducing agent for generating N-hydroxyarylamines from nitroso derivatives of nitro PAHs [9,24]. In order to determine the profile of DNA adducts formed from N-hydroxy-3-AC, 3-nitrosochrysene was incubated with calf thymus DNA at pH 5 in the presence of ascorbic acid (see Materials and Methods). HPLC analyses of enzymatic hydrolysates of DNA recovered from this reaction revealed two potential modified nucleosides with retention times of
%_ MINUTES Fig, 1. Reversed-phase HPLC profile of an n-butanol extract of an enzymatic hydrolysate of calf thymus DNA modified by reaction with 3-nitrosochrysene in the presence of ascorbic acid. The conditions for the reaction, enzymatic digestion and HPLC separation are described in Materials and Methods.
A rG
11
2
s
3
o 8 N
~H
8
N/~ 5H
o
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a °2 ,
1
o~2~
1,2,6.? 12
,'~.o
4.°
olo
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T s
~,'o
2 ~
--
~:o
12
'r
i:o
I
I
~:o
,:o
3:o
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B CH0200
~z
x
I. . . . . . . .
110
'
10.0
~Yo
8:0
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,'o
ppm
Fig. 2. 500 MHz 1H-NMR spectra with resonance assignments of N-(deoxyguanosin-8-yl)-3aminochrysene. (A) Spectrum recorded in methanol-d 4. (B) Spectrum recorded in DMSO-d6. Insets show an expanded view of the region of the spectra containing the resonances of chrysene protons H1, H2, H6, H7 and H12.
17.6 and 27.9 min (Fig. 1). Attempts to definitively identify the material eluting at 17.6 min were unsuccessful due to the small quantity of material available, the impurity of the material collected, which was indicated by the electon impact mass spectrum and unusual broadening of resonances in the ;H-NMR spectrum (data not shown). 1H-NMR spectra of the adduct eluting at 27.9 min were recorded at 500 MHz in both methanol-d4 (Table I and Fig. 2A) and DMSO-d6 (Table I and Fig. 2B) under various experimental conditions to identify the adduct. Decoupling experiments and coupling constant measurements, as well as an NOE observed for H5 as a result of saturation of H4, confirmed the presence of all expected chrysene protons and established the C3 position of the chrysene ring as the site of substitution. Fine structure observed in resolution enhanced spectra due to small long-range coupling (JH-H = 0.5 - 1.5 Hz) from H4 to H1, H2 and H12 also establish that the apparent singlet at 7.90 arises from the chrysene ring and not from a nucleic acid base [25]. Both chemical shift and coupling constant data (Table I) establish the presence of a 2'-deoxyribose ring. Moreover, the magnitude of the coupling constants of the sugar ring are indicative of an unusually high population of C2 '-endo sugar ring conformation and a predominant gauche+ (~ = 60 °) conformation about the C4' - C5' bond. This is a characteristic of CS-amino-substituted purine nucleosides and nucleotides [26] and the results are nearly identical to those reported for the closely related adduct N-(deoxyguanosin-8-yl)-2-aminofluorene [27]. Thus, the coupling constant data alone strongly suggest a substitution via nitrogen at the C8 position of a
100-
I x5
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00|MH-116] 393.2
00-166.0
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.
--
c .... & . . . . i. . . . . I .... 150
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-
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4~19.0
I
I
[MH+ ~509.3
I.L.o.L..LL . . . . .' .. . .. . . . .I .. .. . .. . . ' .". ..... ,. . . . ' I. . . . II . ._.L ' .... I .... . I .... .. ' ....
l .... 200
.
250
300
350
400
f .
.
.
.
I .... 450
.
.
i ....
l _ .
i .... 500
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.
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Fig. 3. Mass spectrum of N-(deoxyguanosin-8-yl)-3-aminochyrsene. DNA was treated with 3nitrosochrysene and ascorbic acid, enzymatically hydrolyzed to nucleosides and analyzed by thermospray HPLC-mass spectrometry as described in Materials and Methods.
10
purine ring. The absence of any singlet that can be attributed to a nucleic acid base is evidence that the substitution is to a guanine base. This was confirmed by recording spectra in DMSO-d6 (Fig. 2B) where the expected exchangeable protons at N1 (10.62 ppm) and NH2 (6.38 ppm) were found as well as that of the amine nitrogen at C8 of the substituted guanine ring (9.05 ppm). The exchangeable protons for the 3' (5.35 ppm) and 5' (6.13 ppm) hydroxyl groups were also identified in the spectrum. It is therefore concluded that the major adduct produced from the reaction of N-hydroxy-3-aminochrysenewith calf thymus DNA is N-(deoxyguanosin-8-yl)-3-aminochrysene. Further supporting evidence for the structure of the adduct and confirmation of its molecular weight were obtained from HPLC-MS analysis. Under the chromatographic conditions used for the analysis (see Materials and Methods), a single peak with a retention time of 12.5 min was obtained. The TSP spectrum of the adduct is shown in Fig. 3. The ion observed at m/z 509 is that expected for the [M + H] ÷ ion of N-(deoxyguanosin-8-yl)-3-aminochrysene(tool. wt. 508). The signal at m/z 393 can be assigned to the loss of deoxyribose (116 amu) from the molecular ion. The signals at m/z 134 and m/z 166 also support the presence of deoxyribose (S) in the adduct ([S + NHJ ÷ and [S + NH4 + CH3OH] ÷, respec-
Fig. 4. Autoradiogram of 32p-postlabeled n-butanol extract of an enzymatic digest of calf thymus DNA treated with 3-nitrosochrysene and ascorbic acid. DNA was hydrolyzed, extracted and labeled as described in Materials and Methods. 'O' marks the origin of the chromatogram. D3 was run from bottom to top and D4 and D5 were run from left to right.
11
tively). Other minor signals at m/z 116, 117 and 419 are also due to the deoxyribose moiety in the structure ([S + NH4-H20] +, [S + H] ÷ and [MH - 90] +, respectively). The ion at m/z 152 is indicative of the guanine moiety ([BH21 +). Except for a very low intensity signal at m/z 244, which may correspond to the [chrysene-NHs] ÷ ion, no other signals arising from the breakdown of the guanine-chrysene bonds are observed.
Fig. 5. Autoradiograms of 32p-postlabeled n-butanol extracts (Panel A) or nuclease Fl-treatec (Pane|s B - D) enzymatic digests of DNA from microsomal incubations conducted in the presence oJ absence of 3-AC. (A and B) Calf thymus DNA treated with 3-AC in the presence of PB microsomes (C) Calf thymus DNA from microsomal incubations with 3-AC in the presence of 3-MC microsomes (D) Calf thymus DNA from microsomal incubations with DMSO in the presence of 3-MC microsomes
12
s2p-Postlabeling analysis of calf thymus DNA modified by 3-nitrosochrysene in the presence of ascorbic acid or by microsomal metabolites of 3-aminochrysene 32P-Postlabeling analysis of calf thymus DNA modified by 3-nitrosochrysene in the presence of ascorbic acid, confirmed the presence of a single major adduct spot (Fig. 4, Spot 1; RfD3 = 0.45, RfD4 = 0.35) that was not present in control DNA. Only 30% of the N-(deoxyguanosin-8-yl)-3-AC-3 ',5 '-bisphosphate detected by the n-butanol method was recovered in the nuclease Pl-treated samples when equivalent amounts of N-hydroxy-3-AC-modified DNA were analyzed by these two enhancement techniques. 82P-Postlabeling analysis of calf thymus DNA that had been modified by incubation with 3-AC in the presence of PB-microsomes (Fig. 5A, Spot 1) indicated a single adduct spot that was chromatographically identical to that described above for DNA modified by N-hydroxy-3-AC and exhibited low recovery when the nuclease P1 enhancement procedure was used (Fig. 5B). This adduct was also observed in DNA recovered from an incubation of 3-AC with 3-MC-microsomes when the n-butanol enhancement method [20] was used prior to 32p_ postlabeling (not shown). However, when the nuclease P1 enhancement procedure was used, a second, chromatographically distinct, adduct spot (D3 Rf = 0.64, D4 Rf = 0.53) was the major product observed (Fig. 5C, Spot 2). This adduct spot was not present in DNA that was incubated with 3-MC microsomes with solvent alone (Fig. 5D). DISCUSSION
Like its geometric isomer, 6-AC, 3-AC is mutagenic in Salmonella strain TA98 both in the absence and in the presence of an exogenous metabolic activation system and we have here compared the pattern of DNA adducts produced by these two isomers in vitro. The data presented here indicate that N-hydroxy-3AC, generated in situ by the reduction of 3-nitrosochrysene with ascorbic acid, reacts with DNA in vitro to give N-(deoxyguanosin-8-yl)-3-aminochrysene as the major product. Furthermore, the chromatographic profile obtained after 32p_ postlabeling of N-hydroxy-3-AC-modified DNA was consistent with the formation of a single adduct, although the presence of multiple adducts in the single adduct spot detected cannot be ruled out. The apparent susceptibility of the adduct spot to nuclease P1 is consistent with previous results obtained with several C8-deoxyguanosine adducts of aromatic amines [28,29]. HPLC analysis of modified nucleosides derived from the reaction of N-hydroxy-3-aminochrysene with calf thymus DNA indicated a possible minor adduct that was not detected as a chromatographically distinct adduct in the TLC system used for separation of 32p-postlabeled adducts. 1H-NMR analysis of this peak was inconclusive and we were thus unable to determine if this product was a distinct DNA adduct or a degradation product of N-(deoxyguanosin-8-yl)-3-AC. Based on comparisons with the relative retention times of the DNA adducts of other aromatic amines on reversed phase HPLC columns (9,30 - 35), it is likely that this adduct, if it is not derived from N-(deoxyguanosin-8-yl)-3-AC, is an N2-deoxyguanosine or an N6-deoxyadenosine adduct rather than a C8-deoxyadenosine or CS-deoxyinosine
13
adduct, since the latter would be expected to have a greater retention time than a CS-deoxyguanosine adduct. Regardless of the identity of the minor DNA adduct from N-hydroxy-3-AC, these results contrast with what had been observed for the reaction of N-hydroxy-6-AC with calf thymus DNA, where 5(deoxyguanosin-N2-yl)-6-AC, N-(deoxyguanosin-8-yl)-6-AC and N-(deoxyinosin8-yl)-6-AC were all found to be major reaction products [9]. As was discussed in the Introduction, work with several compounds has suggested that the formation of deoxyadenosine adducts from N-hydroxyarylamines can have significant mutagenic consequences. Our results suggest that significant differences in the biological activities of 3-AC and 6-AC may exist under conditions where Noxidation of the compounds is the predominant mode of activation. Many of the commonly studied aromatic amines are preferentially N-oxidized by the 3-methylcholanthrene-inducible forms of cytochromes P-450, CYP1A1 and CYP1A2 [36]. In contrast, 4,4'-methylene-bis(2-chloroaniline) has been shown to be preferentially N-oxidized by phenobarbital-inducible forms, CYP2B1 and CYP2B2 [371 and there is evidence that suggests that 6-AC is also preferentially N-oxidized by phenobarbital-inducible forms of cytochromes P-450 as compared to 3-methylcholanthrene-inducible forms [8]. Although no information on preferential activation was obtained from the experiments conducted, the results presented here suggest that 3-AC is N-oxidized by both 3-MC- and PB-inducible rat liver cytochrome P-450 isozymes. Our data on adduct formation in DNA modified by metabolites of 3-AC generated by 3-methylcholanthrene-induced rat liver microsomes suggest that 3-AC, like 6-AC, may be activated by means other than simple N-oxidation, either ring oxidation or a combination of ring and Noxidation. Previous studies with 6-nitrochrysene and 6-AC have provided indirect evidence that the formation of 1,2-dihydroxy-3,4-epoxy-l,2,3,4tetrahydro-6-aminochrysene is a major activation pathway for these compounds [4,6]. In light of the strong carcinogenic and mutagenic activity associated with the formation of adducts derived from a ring oxidized metabolite of 6-AC [3 - 7] further investigation of the structural determinants of the ring oxidation activation pathways of geometric isomers of amino and nitro derivatives of chrysene, and the biological consequences of the damage produced from these metabolites, will be of interest. ACKNOWLEDGMENTS
The technical assistance of T.A. McRae and L.S. Von Tungeln is gratefully acknowledged. REFERENCES 1 F.A. Beland and F.F. Kadlubar, Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons, Handb. Exp. Pharmacol., 94/I (1990)267-325. 2 P.H. Grantham, N. Ba-Giao, L.C. Mohan, T. Benjamin, P.P. Roller and E.K. Weisburger, The metabolism of 6-aminochrysene in the rat, Eur. J. Cancer, 12 (1976) 227-235. 3 K.B. Delclos, R.P. Walker, K.L. Dooley, P.P. Fu and F.F. Kadlubar, Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of
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4
5 6 7 8
9
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12 13
14 15 16 17 18 19 20
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