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The X-ray crystallographic structure of 7-NDB[a,h]A was determined and indicated that the dihedral angle between the nitro functional group and the ar...
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Chem. Res. Toxicol. 1998, 11, 937-945

937

Structure, Tumorigenicity, Microsomal Metabolism, and DNA Binding of 7-Nitrodibenz[a,h]anthracene Peter P. Fu,*,† Linda S. Von Tungeln,† Li-Hsueh Chiu,† De-Jin Zhan,† Joanna Deck,† Thomas Bucci,† and Ju-Chun Wang‡ National Center for Toxicological Research, Jefferson, Arkansas 72079, and Department of Chemistry, Soochow University, Taipei, Taiwan Received April 16, 1998

It has been previously proposed that a nitropolycyclic aromatic hydrocarbon (nitro-PAH) with its nitro functional group perpendicular or nearly perpendicular to the aromatic moiety exhibits lower tumorigenicity than the corresponding parent aromatic hydrocarbon. We also hypothesized that reduction of the nitro group is not involved, or contributed less significantly in the metabolic activation of this class of nitro-PAHs. To verify this hypothesis, we selected 7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A) for study. The X-ray crystallographic structure of 7-NDB[a,h]A was determined and indicated that the dihedral angle between the nitro functional group and the aromatic dibenz[a,h]anthracenyl moiety was 80.6°, indicating the nitro group preferentially adopts a nearly perpendicular orientation. The tumorigenicity of 7-NDB[a,h]A and dibenz[a,h]anthracene (DB[a,h]A) was determined in the male B6C3F1 neonatal mouse. Mice were administered ip injections of 1/7, 2/7, and 4/7 of the total dose of 7-NDB[a,h]A (400 nmol in 35 µL of DMSO per mouse) within 24 h of birth and at 8 and 15 days of age, respectively, and sacrificed at 12 months of age. DB[a,h]A induced 78 and 96% hepatocellular adenomas and carcinomas, respectively. However, 7-NDB[a,h]A induced only 50 and 8% hepatocellular adenomas and carcinomas compared with the 8 and 4% hepatocellular adenomas and carcinomas induced by the solvent vehicle, DMSO. Aerobic metabolism of 7-NDB[a,h]A by liver microsomes of 15-day old male B6C3F1 neonatal mice resulted in trans3,4-dihydroxy-3,4-dihydro-7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A trans-3,4-dihydrodiol) and trans-10,11-dihydroxy-10,11-dihydro-7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A trans10,11-dihydrodiol) as predominant metabolites. Under anaerobic conditions, 7-NDB[a,h]A was not metabolized (nitroreduced). The DNA adduct levels in liver and lung tissues of male B6C3F1 mice treated with 7-NDB[a,h]A and sacrificed 24 h and 6 days after final dosing were determined by 32P-postlabeling/TLC. In all cases, the DNA adducts derived from 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol were formed. These results suggest that both of the metabolites, 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans10,11-dihydrodiol, are involved in the metabolic activation of 7-NDB[a,h]A, leading to tumor induction in the neonatal mouse. Thus, our results described in this paper support our hypotheses that a nitro-PAH with a perpendicular nitro orientation exhibits lower tumorigenicity than the corresponding parent PAH and that nitroreduction contributes less significantly in the metabolic activation.

Introduction It has been established that nitropolycyclic aromatic hydrocarbons (nitro-PAHs)1 are genotoxic contaminants present in the environment and food chain (1-4). NitroPAHs require metabolic activation in order to exert their biological activities, including mutagenicity and tumorigenicity (2-6). The principal metabolic activation pathways of nitro-PAHs include the following: (i) reduction of the nitro substituent (nitroreduction) to the corresponding N-(hydroxyamino)-PAHs, either with or without the subsequent esterification; (ii) ring oxidation; and (iii) a combination of nitroreduction and ring oxidation (24). Thus, ease of reduction of the nitro group is a factor in determining the biological activities of nitro-PAHs. * To whom correspondence and requests for reprints should be addressed. † National Center for Toxicological Research. ‡ Soochow University.

Studies of metabolic activation of nitro-PAHs on the basis of structure-activity relationships have indicated that several structural and electronic features are important factors in the interpretation and/or prediction of the 1 Abbreviations: PAHs, polycyclic aromatic hydrocarbons; nitroPAHs, nitropolycyclic aromatic hydrocarbons; DB[a,h]A, dibenz[a,h]anthracene; DB[a,h]A trans-3,4-dihydrodiol, trans-3,4-dihydroxy-3,4dihydrodibenz[a,h]anthracene; 7-NDB[a,h]A trans-3,4-dihydrodiol, trans3,4-dihydroxy-3,4-dihydro-7-nitrodibenz[a,h]anthracene; 7-NDB[a,h]A trans-10,11-dihydrodiol, trans-10,11-dihydroxy-10,11-dihydro-7-nitrodibenz[a,h]anthracene; dimethyl sulfoxide, DMSO; DB[a,h]A trans-3,4diol-anti-1,2-epoxide, trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene; 7-NDB[a,h]A trans-3,4-diol-anti-1,2epoxide, trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydro-7-nitrodibenz[a,h]anthracene; 7-NDB[a,h]A trans-10,11-diol-anti-8,9-epoxide, trans-10,11-dihydroxy-anti-8,9-epoxy-8,9,10,11-tetrahydro-7-nitrodibenz[a,h]anthracene; 7-NDB[a,h]A 3,4-DE DNA adduct, DNA adduct derived from 7-NDB[a,h]A trans-3,4-diol-anti-1,2-epoxide; 7-NDB[a,h]A 10,11-DE DNA adduct, DNA adduct derived from 7-NDB[a,h]A trans10,11-diol-anti-8,9-epoxide; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; S9, post mitochondrial supernatant fraction.

S0893-228x(98)00079-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/29/1998

938 Chem. Res. Toxicol., Vol. 11, No. 8, 1998

Figure 1. Structures of dibenz[a,h]anthracene and 7-nitrodibenz[a,h]anthracene.

bacterial mutagenicity of nitro-PAHs. These include (i) the first half-wave reduction potential (7-12), (ii) the molecular dimensions and degree of aromaticity (12, 13), (iii) the geometric location and the orientation of the nitro substituent (13-18), and (iv) the number of electrons involved in the first step of nitroreduction (19, 20). On the basis of structure-activity relationships, we have found that orientation of the nitro substituent relative to the aromatic moiety can be used to correlate with direct-acting bacterial mutagenicity, reductive metabolism (nitroreduction) of nitro-PAHs, and the capability to bind to DNA (14-18, 21-23). It has been demonstrated that a nitro-PAH with its nitro substituent perpendicular or nearly perpendicular to the plane of the aromatic moiety will be a weak or nondirect-acting mutagen, exhibit low DNA binding capability, and will be nitroreduced to a lesser extent. Naturally, it is relevant to determine whether orientation of the nitro group is also an important factor in correlation with the tumorigenic activity of a nitro-PAH. On the basis of the lower tumorigenic activity of 6-nitrobenzo[a]pyrene and 7-nitrobenz[a]anthracene than their parent compounds, benzo[a]pyrene and benz[a]anthracene, respectively (24), we have hypothesized that nitro-PAHs with a perpendicular or nearly perpendicular orientation of the nitro substituent exhibit lower tumorigenic potency than the parent PAH (3). This hypothesis requires confirmation. 7-Nitrodibenz[a,h]anthracene (7-NDB[a,h]A) is a nitroPAH derived from the potent carcinogen, dibenz[a,h]anthracene (DB[a,h]A) (Figure 1). The structure of 7-NDB[a,h]A is unique because it has two, but unequal, bay regions. The nitro group resides in one of the bay regions and should possess an orientation preferentially perpendicular to the aromatic moiety. In this study of 7-NDB[a,h]A, we have (i) determined its molecular structure by X-ray crystallographic analysis, (ii) determined its tumorigenicity in the neonatal male B6C3F1 mouse, (iii) studied the aerobic and anaerobic metabolism by liver microsomes of 15-day old male B6C3F1 mice, and (iv) analyzed the 7-NDB[a,h]A-modified DNA adduct formation in vivo by 32P-postlabeling/TLC. The results of this study support our hypothesis.

Materials and Methods Caution: DB[a,h]A and 7-NDB[a,h]A have been determined to be carcinogenic in animal bioassays. Therefore, appropriate safety procedures should be followed when working with these compounds. Materials. DB[a,h]A, triethylamine, trifluoroacetic acid, and dimethyl sulfoxide (DMSO) were purchased from Aldrich Chemical Co. (Milwaukee, WI). All solvents used were HPLC grade. The liver microsomes of 15-day-old male B6C3F1 mice (5-7 g body weight), obtained from the National Center for Toxicological Research (NCTR) breeding colony, were prepared as previ-

Fu et al. ously described (25). The protein content was assayed according to established procedures (26). Synthesis of 7-NDB[a,h]A. DB[a,h]A (278 mg, 1 mmol) in acetic anhydride (35 mL) was added to a solution of sodium nitrate (85 mg, 1 mmol) in trifluoroacetic acid (30 mL). The resulting solution was stirred at ambient temperature under argon for 17 h. The reaction products were partitioned between 300 mL of ethyl acetate and 300 mL of water containing 2 mL of concentrated sulfuric acid. The organic layer was collected, washed with water, and dried over anhydrous magnesium sulfate and the solvent removed under reduced pressure. The yellowish residue was column chromatographed over silica gel. Elution with hexane yielded the unreacted DB[a,h]A (80 mg). Elution with benzene gave 102 mg of 7-NDB[a,h]A: mass spectrum (70 eV) m/z 323 (M+); 500 MHz NMR (acetone-d6) δ 7.70 (d, J5,6 ) 10.0 Hz, 1, H6), 7.81 (m, 3, H2,3,10), 7.90 (t, J8,9 ) 9.5 Hz, 1, H9), 8.02 (d, J5,6 ) 10.0 Hz, 1, H5), 8.13 (m, 3, H4,11,12), 8.24 (d, J12,13 ) 10.0 Hz, 1, H13), 8.42 (d, J8,9 ) 9.5 Hz, 1, H8), 9.16 (d, J1,2 ) 9.5 Hz, 1, H1), and 9.74 (s, 1, H14). X-ray Crystallographic Determination of 7-NDB[a,h]A (C22H12NO2). A thin plate crystal with dimensions approximately 0.01 × 0.20 × 0.60 mm was used for X-ray structural analysis. Cell constants were derived from least-squares refinement of 25 reflections having 24 < 2θ < 40. Intensity data were collected using the θ - 2θ scan mode on a Siemens P4 diffractometer equipped with graphite-monochromatized Cu KR radiation (λ ) 1.541 78 Å). A total of 1178 reflections was measured, which was averaged to 949 unique reflections (Rint ) 0.0685). An empirical absorption correction based on a series of Ψ-scans was applied to the data. Three standard reflections were measured every 100 reflections, and only small ( 2σ(F)]. Crystal data: C22H12NO2, Mr ) 322.3, monoclinic, P21/n, a ) 14.118(4) Å, b ) 3.824(2) Å, c ) 14.479(3) Å, b ) 103.67(2)°, V ) 759.5(3) Å3, Z ) 2, Dx ) 1.409, T ) 298 K, µ ) 0.729 mm-1, 2θmax ) 110°, R ) 0.0676, wR ) 0.0641, S ) 2.59. Neonatal Mouse Tumorigenicity Bioassay. Male B6C3F1 mice were bred and maintained at the National Center for Toxicological Research in a specific pathogen-free environment at 23 °C. The neonatal mice were administered ip injections of 1/7, 2/7, and 4/7 of the total dose of DB[a,h]A or 7-NDB[a,h]A (400 nmol in 35 µL of DMSO per mouse) within 24 h of birth and at 8 and 15 days of age, respectively. Animals were housed four per cage after weaning, fed ad libitum, receiving a standard NIH-31 diet, and maintained on a 12 h light-dark cycle. Mortality was determined daily. Animal weights were monitored monthly, beginning at 28 days of age, until final sacrifice. Animals were sacrificed at 12 months of age and subjected to necropsy for gross tumor count and microscopic observation and histopathological diagnosis. Liver and lung masses were classified as either adenomas or carcinomas. Tumor rates between the treated and the control groups and between the DB[a,h]Atreated and 7-NDB[a,h]A-treated groups were compared by Fisher’s Exact test. Metabolism of 7-NDB[a,h]A by Liver Microsomes of 15Day-Old Male B6C3F1 Mice. The aerobic metabolism of 7-NDB[a,h]A was conducted in a 200 mL reaction mixture

Tumorigenicity of 7-Nitrodibenz[a,h]anthracene containing 10 mmol of Tris-HCl, pH 7.4, 600 µmol of MgCl2, 200 µmol of NADP+, 400 µmol of glucose 6-phosphate, 20 units of glucose 6-phosphate dehydrogenase, 200 mg of mouse liver microsomal protein, and 8 µmol of 7-NDB[a,h]A (dissolved in 8 mL of acetone). After being shaken at 37 °C for 60 min, the reaction was quenched with an equal volume of acetone, and the metabolites and residual substrate were extracted twice with ethyl acetate (2 × 1.5 vol). To stabilize the metabolites, 1% triethylamine was added to the ethyl acetate fraction. The organic phase was collected and the solvent evaporated under reduced pressure. The residue was dissolved in methanol for analysis by HPLC. Repeated incubations under identical conditions were conducted to obtain sufficient metabolites for structural identification and for the synthesis of 7-NDB[a,h]Amodified DNA adduct standards described in the following section. A hypoxic incubation (in a 200 mL reaction mixture) was conducted as described above for aerobic conditions except that the reaction mixture was argon-purged for 5 min in an ice bath prior to incubation in a closed system filled with argon. 7-NDB[a,h]A-Induced DNA Adducts in Vivo. To determine 7-NDB[a,h]A-induced DNA adducts in vivo, the synthetic DNA adduct standards were first prepared. 7-NDB[a,h]A trans3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol (50100 µg) obtained from metabolism of 7-NDB[a,h]A by liver microsomes of 15-day-old male B6C3F1 mice were dissolved in THF (100 µL) and oxidized by m-chloroperbenzoic acid to the corresponding anti-diol epoxides by procedures previously described for the synthesis of PAH diol epoxides (28). The antidiol epoxides formed in situ were reacted with calf thymus DNA, and the resulting DNA adducts were purified and isolated following the previously described procedures (28). Seven male B6C3F1 neonatal mice were dosed with 7-NDB[a,h]A as described for the neonatal mouse tumorigenicity assay. Three or four mice were sacrificed 24 h or 6 days after the final dosing, respectively. To assess in vivo DNA adduct formation, nuclei were isolated from the liver and lung by the method of Basler et al. (29), and DNA was prepared from the nuclei by slight modification of the method described by Beland et al. (30). The modified DNA adducts were analyzed by 32P-postlabeling/ TLC. 32P-Postlabeling/TLC Identification of 3′5′-Bisphosphate Deoxyribonucleotides. Following the procedure of Reddy and Randerath (31), the modified DNA (10 µg dissolved in 20 µL of 5 mM Bis-Tris, 0.1 mM EDTA buffer, pH 7.1) was hydrolyzed to deoxyribonucleoside 3′-monophosphates by micrococcal nuclease (125 U/mL) and spleen phosphodiesterase (12.5 U/mL). DNA adducts were enriched by the nuclease P1 method (31). The resulting deoxyribonucleoside 3′-monophosphates were then [5′-32P]phosphorylated to the corresponding 3′,5′-bisphosphate deoxyribonucleotides that were applied in a 5 µL volume to 10 × 10 cm PEI-cellulose plates (MachereyNagel, Alltech Associates, Deerfield, IL) for chromatography. The solvent systems used were as follows: D1 (with wick), 0.65 M sodium phosphate buffer (pH 6.8), overnight; D2 (run twice), 3.5 M lithium formate, 8.5 M urea (pH 3.5); D3 (run twice), 1.2 M lithium chloride, 8.5 M urea, 0.5 M Tris-HCl (pH 8.0) and D4, 1.7 M sodium phosphate buffer (pH 6.8). The adduct spots were located by autoradiography at -80 °C using intensifying screens. Adduct spots were excised for determining radioactivity by liquid scintillation counting. Physiochemical Properties of Metabolites. Reversedphase HPLC was performed with a Waters system consisting of two model 510 pumps, a model U6K injector, a model 440 solvent programmer, and a Hewlett-Packard 1040A photodiode array detection system. The crude metabolites were separated on a DuPont Zorbax ODS column (9.4 × 250 nm) using a 40min linear gradient of 60-100% methanol in water at a flow rate of 1.0 mL/min. UV/vis absorption spectra were measured in methanol on a Beckman model DU65 spectrophotometer or measured directly upon elution with the diode array detector. Mass spectral analyses were performed on a Finnigan 1015

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Figure 2. Atom labeling of 7-nitrodibenz[a,h]anthracene. Due to the necessity of numbering all the atoms to describe the X-ray crystallographic data of a molecule, the numbering system employed in this figure and in the discussion of the X-ray crystallographic data is different than in Figure 1 and the remainder of the text. mass spectrometer by electron impact with a solid probe at 70 eV and 250 °C ionizer temperature. Fourier transform proton nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AM500 NMR spectrometer operating at 500 MHz. Samples were dissolved in acetone-d6. The chemical shifts are reported in ppm downfield from tetramethylsilane by assigning the residual solvent signal to 2.04 ppm. The coupling constants (J) are first-order measurements.

Results X-ray Crystallographic Determination of 7-NDB[a,h]A. Ring A of the 7-NDB[a,h]A (Figure 2) has normal aromatic carbon-carbon bonds ranging from 1.360(12) to 1.418(10) Å, with an average of 1.39 Å. The shortest C-C distance, which is 1.346(11) Å, is observed between C3 and C4 (on the B-ring), indicating that more double character is localized on this C-C bond. Because a crystallographic center of symmetry is imposed on the center of the molecule, the nitro group is disordered. If two adjacent molecules were occupied in the same direction with respect to the nitro group, an unreasonable short O-O contact [O1-O2A ) 1.72(1) Å] would exist. To avoid this unsuitable short contact, the molecules must be packed in an alternate way (one molecule lies in one direction and the other must be oriented in the direction such that two nitro groups are pointed in opposite directions). Because of the disorder, the C-C distances of the C-ring on the nitro-group side and on the other side are averaged to the same value. The largest deviation of the least-squares plane formed by all atoms on the aromatic moiety is only 0.03 Å. The aromatic moiety basically is planar. The plane containing the nitro group is approximately perpendicular to that aromatic moiety plane with the angle of these two planes equal to 80.6°, which indicates that the nitro functional group of this compound preferentially adopts a nearly perpendicular orientation. Table 1 lists the atomic positional and isotropic displacement parameters. Table 2 summarizes bond distances and angles. Neonatal Mouse Tumorigenicity Bioassay. The tumorigenicity of 7-NDB[a,h]A, DB[a,h]A, and the sol-

940 Chem. Res. Toxicol., Vol. 11, No. 8, 1998

Fu et al.

Table 1. Atomic Coordinates and Equivalent Isotropic Displacement Coefficients (Å2) O(1) O(2) N(1) C(1) C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(11) H(3) H(4) H(5) H(6) H(7) H(8) H(11)

x

y

z

Ueq

0.9946(7) 0.9848(7) 0.9883(7) 0.9051(5) 0.9196(5) 0.8391(5) 0.7502(5) 0.6383(5) 0.6189(5) 0.6918(6) 0.7842(5) 0.8084(5) 0.7325(5) 0.9877(5) 0.849(4) 0.684(3) 0.578(4) 0.559(4) 0.676(4) 0.841(4) 1.0197

0.116(3) -0.443(3) -0.144(3) 0.035(1) 0.134(1) 0.272(2) 0.312(2) 0.245(2) 0.146(2) -0.001(2) -0.038(2) 0.068(1) 0.209(1) -0.094(1) 0.33(1) 0.40(1) 0.36(1) 0.21(1) -0.01(2) -0.17(1) 0.1581

0.2457(6) 0.2232(5) 0.1945(7) 0.0217(4) -0.0685(4) -0.1381(5) -0.1207(5) -0.0161(5) 0.0677(6) 0.1393(6) 0.1264(5) 0.0409(4) -0.0310(5) 0.0871(4) -0.20(4) -0.15(3) -0.07(3) 0.09(4) 0.20(4) 0.18(3) -0.1491

0.082(4)a 0.079(4)a 0.059(4)a 0.046(2)a 0.048(2)a 0.057(3)a 0.057(3)a 0.062(3)a 0.067(3)a 0.064(3)a 0.058(3)a 0.049(2)a 0.052(3)a 0.049(2)a 0.08(2) 0.06(2) 0.08(2) 0.07(2) 0.09(2) 0.07(2) 0.08

a Equivalent isotropic U defined as one-third of the trace of the orthogonalized Uij tensor. Parameters without standard deviation were not refined in the least-squares refinement.

vent vehicle, DMSO, was determined in the male B6C3F1 neonatal mouse. Mice were administered ip injections of 7-NDB[a,h]A, DB[a,h]A (at a total dose of 400 nmol in 35 µL of DMSO), or DMSO (35 µL) per mouse on 1, 8, and 15 days of age. The mortality of 7-NDB[a,h]A, DB[a,h]A, and DMSO-treated animals before allocation (28 days of age) was 23, 19, and 30%, respectively. Twentyfour animals were allocated to each treatment group at 28 days of age and maintained until 12 months of age. During this period, only one of the 24 DB[a,h]A-treated animals died, while all the 7-NDB[a,h]A and DMSOtreated animals survived. Animal weights and clinical observations for all treatment groups were monitored monthly beginning at 28 days of age until final sacrifice at 12 months of age. In all cases, the mean body weight of mice increased with age until 9 months of age. No significant mean body weight differences were observed between the chemical-treated animals and those dosed with DMSO (data not shown). Liver and lung nodules were observed at necropsy and then diagnosed by microscopic examination and summarized in Table 3. By microscopic observation, while animals receiving DMSO developed 8 and 4% hepatocellular adenomas and carcinomas, those receiving DB[a,h]A induced 78 and 96% hepatocellular adenomas and carcinomas, respectively. The incidence of both hepatocellular adenomas and carcinomas are significantly different from the DMSO control (P < 0.0001). 7-NDB[a,h]A induced 50% hepatocellular adenomas, which is significantly different from the DMSO control (P ) 0.0034), but only 8% hepatocellular carcinomas, which is not different from DMSO (Table 3). While there is no significant difference between the 7-NDB[a,h]A-treated and DB[a,h]A-treated groups in development of hepatocellular adenomas, the incidence of hepatocellular carcinomas in the DB[a,h]A-treated group is significantly higher than the 7-NDB[a,h]A-treated group (P < 0.0001) (Table 3). Mice receiving the solvent control, DMSO, had 1.7 liver tumors per tumor-bearing mouse observed microscopi-

cally (Table 3) and exhibited a tumor rate of 13% for liver (two out of 24 mice had adenomas and one mouse had a carcinoma). 7-NDB[a,h]A-treated mice had 1.5 liver tumors per tumor-bearing mouse and exhibited a tumor rate of 50% for liver (12 out of 24 mice had adenomas; however, two of the 12 mice also had carcinomas). DB[a,h]A-treated mice had too many tumors to quantify due to the tumors being large and coalescent. Furthermore, DB[a,h]A-treated mice had a 100% liver tumor rate (one out of 23 mice had only adenomas, five had only carcinomas, and 17 had a combination of both adenomas and carcinomas), which is significantly different (P ) 0.0001) from the 7-NDB[a,h]A-treated group. In general, no correlation was observed between body weight and tumorigenicity of the treatment groups. DMSO-treated animals did not develop any bronchiolar-alveolar adenomas or carcinomas. The one lung nodule observed grossly at necropsy on a DMSO-treated animal was not confirmed microscopically. Only one 7-NDB[a,h]A-treated mouse developed a bronchiolaralveolar adenoma. Forty-three percent of the DB[a,h]Atreated mice developed bronchiolar-alveolar adenomas, which is significantly different from the DMSO control (P ) 0.0002), and 4% developed lung adenocarcinoma (primary), which is not significantly different from the DMSO control (Table 3). Metabolism of 7-NDB[a,h]A by Liver Microsomes of 15-Day-Old Male B6C3F1 Mice. 7-NDB[a,h]A was incubated aerobically with liver microsomes of 15-dayold male B6C3F1 neonatal mice. The reversed-phase HPLC profile of the ethyl acetate extractable metabolites is shown in Figure 3. The chromatographic peak eluting at 62 min contained the recovered substrate, 7-NDB[a,h]A. The metabolite contained in the chromatographic peak eluting at 41 min had a mass spectrum that showed molecular ions at m/z 357 (data not shown), suggesting that it is a 7-NDB[a,h]A dihydrodiol. Its structure was then fully characterized by proton NMR analysis (data not shown). Assignments were made by integration, homonuclear decoupling experiments, and nuclear Overhauser enhancement measurements. On the basis of the mass and proton NMR spectral analyses, the material contained in the chromatographic peak at 41 min was identified as 7-NDB[a,h]A trans-3,4-dihydrodiol. NMR spectral parameters of 7-NDB[a,h]A trans-3,4-dihydrodiol are as follows: δ 4.60 (dt, J1,3 ) 2.4 Hz, J2,3 ) 10.1 Hz, 1, H3), 5.00 (d, J3,4 ) 12.0 Hz, 1, H4), 6.37 (dd, J1,2 ) 10.1 Hz, J2,4 ) 2.4 Hz, 1, H2), 7.49 (dd, J1,2 ) 10.1 Hz, J1,3 ) 2.4 Hz, 1, H1), 7.73 (t, J8,10 ) 1.7 Hz, J9,10 ) 7.7 Hz, J10,11 ) 8.6 Hz, 1, H10), 7.77 (t, J8,9 ) 7.7 Hz, J9,10 ) 7.7 Hz, 1, H9), 7.78 (d, J5,6 ) 9.0 Hz, 1, H6), 7.85 (d, J12,13 ) 9.0 Hz, 1, H12), 8.05 (dd, J8,9 ) 7.7 Hz, J8,10 ) 1.7 Hz, 1, H8), 8.06 (d, J12,13 ) 9.0 Hz, 1, H13), 8.18 (d, J5,6 ) 9.0 Hz, 1, H5), 8.35 (d, J10,11 ) 8.6 Hz, 1, H11), and 9.14 (s, 1, H14). Previously, we identified 7-NDB[a,h]A trans-3,4-dihydrodiol as a metabolite from metabolism of 7-NDB[a,h]A by rat liver microsomes (32). The mass, UV/vis absorption, and NMR spectra are all identical, with the exception that more accurate NMR chemical shifts and coupling constants were obtained from this study. The metabolite contained in the chromatographic peak eluting at 43 min was similarly characterized. It also had a mass spectrum with the molecular ions at m/z 357 (data not shown) and a UV/vis absorption spectrum similar to that of 7-NDB[a,h]A trans-3,4-dihydrodiol. On the basis of analysis of its NMR data, UV/vis absorption

Tumorigenicity of 7-Nitrodibenz[a,h]anthracene

Chem. Res. Toxicol., Vol. 11, No. 8, 1998 941

Table 2. Bond Lengths (Å) and Interbond Angles for 7-NDB[a,h]A atom

bond length

atom

bond length

O(1)-N(1) N(1)-C(11) C(1)-C(11) C(2)-C(11) C(2)-C(3) C(4)-C(10) C(5)-C(10) C(7)-C(8) C(9)-C(10) C(3)-H(3) C(5)-H(5) C(7)-H(7)

1.228(15) 1.564(12) 1.406(8) 1.406(10) 1.430(8) 1.435(10) 1.402(10) 1.367(11) 1.414(8) 0.91(6) 1.11(5) 1.02(6)

O(2)-N(1) C(1)-C(9) C(1)-C(2) C(2)-C(1) C(3)-C(4) C(5)-C(6) C(6)-C(7) C(8)-C(9)

1.224(15) 1.462(10) 1.420(9) 1.420(9) 1.346(11) 1.360(12) 1.394(10) 1.418(10)

C(4)-H(4) C(6)-H(6) C(8)-H(8)

1.15(4) 0.99(6) 1.10(4)

atom

bond angles (deg)

atom

bond angles (deg)

O(1)-N(1)-O(2) O(2)-N(1)-C(11) C(9)-C(1)-C(2) C(11)-C(2A)-C(1A) C(1)-C(2)-C(3) C(3)-C(4)-C(10) C(5)-C(6)-C(7) C(7)-C(8)-C(9) C(1)-C(9)-C(10) C(4)-C(10)-C(5) C(5)-C(10)-C(9) N(1)-C(11)-C(2A) H(3)-C(3)-C(4) H(4)-C(4)-C(3) H(5)-C(5)-C(6) H(6)-C(6)-C(5) H(7)-C(7)-C(6) H(8)-C(8)-C(7)

123.5(10) 117.5(9) 119.8(5) 119.4(5) 118.9(6) 120.6(6) 120.4(7) 121.6(6) 118.2(6) 119.2(6) 120.4(6) 113.0(6) 120(3) 122(2) 118(3) 125(3) 115(3) 121(3)

O(1)-N(1)-C(11) C(9)-C(1)-C(11) C(11)-C(1)-C(2) C(11)-C(2A)-C(3A) C(4)-C(3)-C(2) C(6)-C(5)-C(10) C(6)-C(7)-C(8) C(1)-C(9)-C(8) C(8)-C(9)-C(10) C(4)-C(10)-C(9) N(1)-C(11)-C(1) C(1)-C(11)-C(2A) H(3)-C(3)-C(2) H(4)-C(4)-C(10) H(5)-C(5)-C(10) H(6)-C(6)-C(7) H(7)-C(7)-C(8) H(8)-C(8)-C(9)

119.0(10) 124.4(6) 115.9(6) 121.7(6) 122.0(7) 120.6(6) 120.1(7) 124.8(5) 117.0(6) 120.5(6) 121.4(7) 124.8(6) 118(3) 117(2) 122(3) 114(3) 123(3) 117(3)

Table 3. Tumor Incidence in Male B6C3F1 Mice Treated with DB[a,h]A, 7-NDB[a,h]A, or DMSOa seen grossly at necropsy no. of mice

no. (%) with liver nodules

mean no. of liver nodules in nodule-bearing mice

no. (%) with lung nodules

mean no. of lung nodules in nodule-bearing mice

24 24 23

2 (8) 10 (42)b 23 (100)c

1.0 1.7 17.0

1 (4) 0 (0) 5 (22)

1.0 0 1.6

DMSO 7-NDB[a,h]A DB[a,h]A

seen microscopically

DMSO 7-NDB[a,h]A DB[a,h]A

carcinomas

mean no. of tumors per liver section

adenomas

carcinomas

mean no. of tumors per lung section

1 (4) 2 (8) 22 (96)c,e

1.7 1.5 multiplef

0 (0) 1 (4) 10 (43)g,h

0 (0) 0 (0) 1 (4)

0 1.0 >1.5

no. (%) with liver tumors

no. of mice

adenomas

24 24 23

2 (8) 12 (50)d 18 (78)c

no. (%) with lung tumors

a The neonatal mice were administered ip injections of 1/7, 2/7, and 4/7 of the total dose (400 nmol per mouse) within 24 h of birth and at 8 and 15 days of age, respectively. The tumor incidence is that observed 1 year posttreatment. b Significantly different from DMSO control (P < 0.0173) by Fisher Exact test. c Significantly different from DMSO control (P < 0.0001). d Significantly different from DMSO control (P ) 0.0034). e Significantly different from 7-NDB[a,h]A-treated group (P < 0.0001). f When the number of tumors exceed five to eight per section, they became confluent, could no longer be quantified, and were designated as “multiple”. g Significantly different from DMSO control (P ) 0.0002). h Significantly different from 7-NDB[a,h]A-treated group (P ) 0.0018).

spectrum, and mass spectrum, this metabolite was identified as 7-NDB[a,h]A trans-10,11-dihydrodiol. Its NMR spectral parameters are as follows: δ 4.51 (dt, J8,10 ) 2.6 Hz, J9,10 ) 10.3 Hz, J10,11 ) 12.5 Hz, 1, H10), 4.81 (d, J10,11 ) 12.5 Hz, 1, H11), 6.26 (dd, J8,9 ) 10.5 Hz, J9,10 ) 10.3 Hz, 1, H9), 6.72 (dd, J8,9 ) 10.5 Hz, J8,10 ) 2.6 Hz, 1, H8), 7.56 (d, J5,6 ) 9.5 Hz, 1, H6), 7.76 (t, J1,3 ) 1.3 Hz, J2,3 ) 7.7 Hz, J3,4 ) 7.7 Hz, 1, H3), 7.83 (t, J1,2 ) 8.4 Hz, J2,3 ) 7.7 Hz, 1, H2), 8.00 (d, J5,6 ) 9.5 Hz, 1, H5), 8.03 (dd, J2,4 ) 1.3 Hz, J3,4 ) 7.7 Hz, 1, H4), 8.14 (d, J12,13 ) 8.4 Hz, 1, H12), 8.37 (d, J12,13 ) 8.4 Hz, 1, H13), 9.03 (d, J1,2 ) 8.4 Hz, 1, H1), and 9.67 (s, 1, H14). The NMR coupling constant between the carbinol protons of 7-NDB[a,h]A trans-3,4-dihydrodiol is 12.0 Hz (J10,11), and the coupling constant between the carbinol protons of 7-NDB-

[a,h]A trans-10,11-dihydrodiol is 12.5 Hz (J10,11). The magnitude of these carbinol constants indicates that both of these dihydrodiol metabolites have a trans configuration and preferentially adopt a quasidiequatorial conformation (33-35). On the basis of the NMR measurements with an internal standard (1,4-dioxane) of known quantity, it was estimated that the ratio of 7-NDB[a,h]A trans3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol is about 1:3. Due to the small quantity formed and the close proximity of elution from the HPLC column, the minor metabolites eluted between 48 and 52 min were not identified. When 7-NDB[a,h]A was incubated under hypoxic conditions, no metabolism was detected.

942 Chem. Res. Toxicol., Vol. 11, No. 8, 1998

Fu et al.

Figure 3. Reversed-phase HPLC profiles of ethyl acetate-extractable metabolites obtained from aerobic incubation of 7-NDB[a,h]A with liver microsomes from 15-day old male B6C3F1 mice.

Chemical Synthesis of DNA Adducts Derived from 7-NDB[a,h]A trans-3,4-Dihydrodiol and 7-NDB[a,h]A trans-10,11-Dihydrodiol. DNA adducts derived from the trans-3,4- and -10,11-dihydrodiols of 7-NDB[a,h]A were prepared for use as standards for identification of 7-NDB[a,h]A-modified DNA adducts in vivo. Following the general procedures previously described for the synthesis of PAH diol epoxides as well as nitro-PAH diol epoxides (28), 7-NDB[a,h]A trans-3,4dihydrodiol and 7-NDB[a,h]A 10,11-dihydrodiol (ca. 50100 µg) obtained from microsomal metabolism described above were converted to the corresponding anti-diol epoxides by reaction with m-chloroperbenzoic acid. The resulting anti-diol epoxides, without purification and characterization (due to small quantity), were subsequently reacted with calf thymus DNA. 32P-Postlabeling/ TLC analysis at TLC conditions determined to be optimal showed the DNA adducts derived from 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol each exhibited one clean TLC spot (Figure 4A,B). 32 P-Postlabeling/TLC Identification of 3′5′-Bisphosphate Deoxyribonucleotides. 7-NDB[a,h]AInduced DNA Adducts in Vivo. Under identical conditions described in the neonatal mouse tumorigenicity assay, seven male B6C3F1 neonatal mice were administered ip injections of 1/7, 2/7, and 4/7 of the total dose of 7-NDB[a,h]A within 24 h of birth and at 8 and 15 days of age, respectively. Three mice at 24 h and four mice at 6 days after the final dosing were sacrificed, and the liver and lung DNA of each animal were analyzed by 32P-postlabeling/TLC analysis. The 32P-postlabeling autoradiograms of DNA adducts formed in the liver and lung DNA of a 7-NDB[a,h]A-treated mouse that was sacrified 24 h after the final dose are shown in Figure 4C,D, respectively. Comparison of these TLC autoradiograms with those of the synthetic standards (shown in Figure 4A,B) clearly indicate that DNA adducts derived

Figure 4. 32P-Postlabeling autoradiograms of DNA adducts formed from (A) reaction of 7-NDB[a,h]A trans-3,4-diol-1,2epoxide with calf thymus DNA; (B) reaction of 7-NDB[a,h]A trans-10,11-diol-8,9-epoxide with calf thymus DNA; (C) liver DNA from a mouse treated with 7-NDB[a,h]A at 8 and 15 days of age and sacrificed 24 h after final dosing; and (D) lung DNA from a mouse treated with 7-NDB[a,h]A at 8 and 15 days of age and sacrificed 24 h after final dosing.

from both 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol were formed. These data and those from results of the DNA derived from mice sacrificed 6 days after the final dosing are summarized in Table 4. No spots corresponding to those derived from 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans10,11-dihydrodiol were formed in the liver and lung DNA of DMSO-treated mice (data not shown). On the basis

Tumorigenicity of 7-Nitrodibenz[a,h]anthracene

Chem. Res. Toxicol., Vol. 11, No. 8, 1998 943

Scheme 1. Proposed Metabolic Activation of 7-NDB[a,h]A Leading to Tumor Initiation

Table 4. DNA Adduct Levels in Liver and Lung Tissues of Male B6C3F1 Mice Treated with 7-NDB[a,h]Aa 7-NDB[a,h]A-modified-DNA adduct (RAL/109 dNTP) tissue

sacrifice timeb

spot 1c

spot 2d

spot 3e

liverf

24 h 6 days 24 h 6 days

16.5 ( 5.3 7.6 ( 2.5 8.7 ( 1.13 5.9 ( 2.6

3.30 ( 0.63 1.17 ( 0.09 2.59 ( 0.13 1.81 ( 1.30

1.44 ( 0.21 0.36 ( 0.29 0.72 ( 0.35 0.61 ( 0.57

liverf lungg lungg

a The neonatal mice were adminstered ip injections of 1/7, 2/7, and 4/7 of the total dose of 7-NDB[a,h]A (400 nmol in 35 µL of DMSO per mouse) within 24 h of birth and at 8 and 15 days of age, respectively. b The animals were sacrificed 24 h and 6 days after the final dose. c The adduct contained in this spot has not been identified. d On the basis of comparison with the authentic standard, the adduct contained in this spot has been identified as 7-NDB[a,h]A trans-3,4-dihydrodiol-modified DNA adduct. e On the basis of comparison with the authentic standard, the adduct contained in this spot has been identified as 7-NDB[a,h]A trans10,11-dihydrodiol-modified DNA adduct. f Mean ( standard deviation from three animals per group. g Mean ( standard deviation from four animals per group.

of the assumption that these adducts have the same efficiency in labeling, these overall results indicate that (i) in all cases the DNA adduct derived from 7-NDB[a,h]A trans-3,4-dihydrodiol was formed in a quantity about 3-fold higher than that derived from 7-NDB[a,h]A trans10,11-dihydrodiol, (ii) the total DNA adduct contained in the liver of mice sacrificed 24 h after final dosing is higher than that of animals sacrificed 6 days after final dosing, and (iii) an unknown DNA adduct, labeled as spot 1, had the highest quantity.

Discussion We have previously observed that nitro-PAHs with their nitro functional group perpendicular or nearly perpendicular to the aromatic moiety exhibit weak or nondirect-acting mutagenicity (11, 12, 14-18, 21-23). On the basis of these observations, we have proposed that nitro-PAHs of this type also exhibit lower tumorigenicity than the corresponding parent PAHs and that nitrore-

duction is not involved in the metabolic activation leading to tumor induction. In this paper, we first employed X-ray crystallographic measurement to determine that 7-NDB[a,h]A had its nitro group nearly perpendicular to the aromatic moiety (dihedral angle 80.6 °C). This orientation confirmed our previous prediction based on the NMR spectral analysis (36). The tumorigenicity of 7-NDB[a,h]A and its parent compound, DB[a,h]A, was then compared in the male B6C3F1 neonatal mouse. It was found that 7-NDB[a,h]A induced much lower hepatocellular carcinoma (P < 0.0001) and a combination of hepatocellular adenoma and carcinoma (P < 0.0001) incidence than DB[a,h]A. Furthermore, 7-NDB[a,h]A also induced a much lower incidence of lung adenoma than DB[a,h]A. The overall tumor rate in 7-NDB[a,h]Atreated animals was drastically decreased, compared to that of DB[a,h]A-treated animals. Consequently, the tumorigenicity of 7-NDB[a,h]A compared with that of DB[a,h]A supports our hypothesis that a nitro-PAH with its nitro group adopting a perpendicular or nearly perpendicular orientation exhibits lower tumorigenicity than the parent PAH. Furthermore, no nitroreduction occurred in the anaerobic liver microsomal metabolism of 7-NDB[a,h]A in vitro. We have previously found that 6-nitrobenzo[a]pyrene and 7-nitrobenz[a]anthracene exhibited lower tumorigenicity than benzo[a]pyrene and benz[a]anthracene, respectively, in the neonatal tumorigenicity bioassay in the CD-1 mouse strain (24). Under anaerobic conditions, liver microsomal metabolism of these two compounds also did not produce nitroreduction products. Thus, the results presented in this paper are consistent with these findings and further support that this hypothesis is applicable when the B6C3F1 strain is employed for study. To determine the mechanism by which 7-NDB[a,h]A induces tumors in the neonatal mouse, aerobic metabolism of 7-NDB[a,h]A by liver microsomes of 15-day-old male B6C3F1 mice in vitro and the DNA adduct formation in vivo were studied. Aerobic metabolism resulted in the formation of 7-NDB[a,h]A trans-10,11-dihydrodiol

944 Chem. Res. Toxicol., Vol. 11, No. 8, 1998

as the predominant metabolite and 7-DB[a,h]A trans-3,4dihydrodiol as the second most abundant metabolite. Under anaerobic conditions, 7-NDB[a,h]A was not metabolized (nitroreduced). In all cases, the DNA adducts derived from both 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol were detected in vivo, with the yield of the former adduct approximately 3-fold higher. As shown in Scheme 1, these results indicate that both 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol are involved in the metabolic activation of 7-NDB[a,h]A leading to tumor initiation in the neonatal mouse and that nitroreduction is not involved. The neonatal mouse tumorigenicity bioassay has been found to be highly sensitive to genotoxic chemical carcinogens (37, 38). Results presented in this paper further illustrate this general phenomenon. Comparison of the total DNA adducts observed at 24 h and 6 days after final dosing indicates that DNA adducts are lost at a higher rate from the liver than the lung (Table 4). This could occur if repair of the 7-NDB[a,h]A-modified DNA is higher in the liver than in the lung, or alternatively, may be due to a more rapid cell turnover in liver than lung. If the latter is the case, the higher initial DNA binding coupled with the higher rate of cell division in the liver may result in a greater mutation frequency in this tissue and may partially explain the targeting of the liver for 7-NDB[a,h]Ainduced carcinogenicity. There were three DNA adducts formed in vivo in the neonatal mice treated with 7-NDB[a,h]A (Table 4). The DNA adducts in spots 2 and 3 are derived from 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol, respectively. Because these adducts are identical to the synthetic standards formed from reaction of these two trans-dihydrodiols with m-chloroperbenzoic acid, respectively, their formation in vivo are believed to be mediated through the interaction of the corresponding anti-diol epoxides with cellular DNA. Due to an insufficient amount of these two 7-NDB[a,h]A trans-dihydrodiols from metabolism, neither the structures of the anti-diol epoxides nor the structures of the resulting DNA adducts have been fully characterized. On the other hand, the structure of the DNA adduct labeled as spot 1 in Table 4 has not been determined.

Acknowledgment. We thank Gail Wagoner, Howard Durrett, and Bob Harmon for animal care, Gene White for necropsy supervision, and Amber Dedman for computer support.

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