Oxidative DNA Damage Induced by Benz[a]anthracene Dihydrodiols

Oxidative DNA Damage Induced by Benz[a]anthracene. Dihydrodiols in the Presence of Dihydrodiol. Dehydrogenase. Kazuharu Seike,†,‡ Mariko Murata,â€...
0 downloads 0 Views 332KB Size
Download PDF
Chem. Res. Toxicol. 2004, 17, 1445-1451

1445

Oxidative DNA Damage Induced by Benz[a]anthracene Dihydrodiols in the Presence of Dihydrodiol Dehydrogenase Kazuharu Seike,†,‡ Mariko Murata,† Kazutaka Hirakawa,§,| Yoshihiro Deyashiki,⊥ and Shosuke Kawanishi*,† Department of Environmental and Molecular Medicine, Mie University School of Medicine, and Department of Radiation Chemistry, Life Science Research Center, Mie University, Tsu, Mie 514-8507, Japan, and Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 502-8585, Japan Received April 29, 2004

Tobacco smoke and polluted air are risk factors for lung cancer and contain many kinds of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) and benz[a]anthracene (BA). BA, as well as B[a]P, is assessed as probably carcinogenic to humans (IARC group 2A). BA is metabolized to several dihydrodiols. Dihydrodiol dehydrogenase (DD), a member of the aldo-keto reductase superfamily, catalyzes NAD(P)+-linked oxidation of dihydrodiols of aromatic hydrocarbons to corresponding catechols. To clarify the role of DD on PAH carcinogenesis, we examined oxidative DNA damage induced by trans-dihydrodiols of BA and B[a]P treated with DD using 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene. In addition, we investigated the formation of 8-oxo-7,8dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA by using HPLC with an electrochemical detector. DD-catalyzed BA-1,2-dihydrodiol caused Cu(II)-mediated DNA damage including 8-oxodG formation in the presence of NAD+. BA1,2-dihydrodiol induced a Fpg sensitive and piperidine labile G lesion at the 5′-ACG-3′ sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The observation of NADH production by UV-visible spectroscopy suggested that DD catalyzed BA-1,2-dihydrodiol most efficiently to the corresponding catechol among the PAH-dihydrodiols tested. A time-of-flight mass spectroscopic study showed that the catechol form of BA-1,2-dihydrodiol formed after DD treatment. In conclusion, BA-1,2dihydrodiol can induce DNA damage more efficiently than B[a]P-7,8-dihydrodiol and other BA-dihydrodiols in the presence of DD. The reaction mechanism on oxidative DNA damage may be explained by theoretical calculations with an enthalpy change of dihydrodiols and oxidation potential of their catechol forms. DD may play an important role in BA carcinogenesis via oxidative DNA damage.

Introduction Exposure to tobacco smoke and pollutants has long been known as the major risk factor for the development of lung cancer (1). Tobacco smoke and polluted air contain many kinds of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) and benz[a]anthracene (BA). PAHs exert mutagenicity and carcinogenicity after metabolic activation (2-4). BA, as well as B[a]P, has been classified as a group 2A carcinogen, which is probably carcinogenic to humans, by the International Agency for Research on Cancer (IARC) (2, 5). BA produced hepatomas and lung adenomas following repeated oral admin* To whom correspondence should be addressed. Tel. and Fax: (+81) (59) 231 5011. E-mail: [email protected]. † Mie University School of Medicine. ‡ Present address: National Institute of Advanced Industrial Science and Technology, Research Center for Glycoscience, Cell Regulation Analysis Team, Tsukuba, Ibaraki 305-8562, Japan. § Mie University. | Present address: Division of Applied Science and Basic Engineering, Faculty of Engineering, Shizuoka University, Hamamatsu, Shizuoka 432-8561, Japan. ⊥ Gifu Pharmaceutical University.

istration to mice (5). In addition, BA is a complete skin carcinogen (5). BA is metabolized to the 1,2-, 3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols (2, 6). Dihydrodiol dehydrogenase (DD; EC 1.3.1.20), a member of the aldo-keto reductase (AKR) superfamily, catalyzes NAD(P)+-linked oxidation of trans-dihydrodiols of aromatic hydrocarbons to corresponding catechols. The enzyme is involved in the production of genotoxic o-quinone, which can induce DNA damage through autoxidation of its catechol intermediates (7). Relevantly, it is suggested that overexpression of DD is assosiated with lung cancer and hepatocellular carcinoma (8, 9). These studies on DD let us consider the significance of oxidative DNA damage in PAH carcinogenesis, although DNA adduct formation is the most widely accepted mechanism. To clarify the role of DD on PAH carcinogensis, we examined oxidative DNA damage and its site specificity induced by trans-dihydrodiols of BA and B[a]P treated with DD using 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene. In addition, we investigated the formation of 8-oxo-7,8dihydro-2′-deoxyguanosine (8-oxodG), an indicator of

10.1021/tx0498814 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/02/2004

1446

Chem. Res. Toxicol., Vol. 17, No. 11, 2004

Figure 1. Autoradiogram of 32P-labeled DNA fragments incubated with BA-dihydrodiols and B[a]P-7,8-dihydrodiol. (A) The reaction mixtures (in a 1.5 mL Eppendorf microtube) containing 100 µM BA-dihydrodiols and B[a]P-7,8-dihydrodiol with 9.6 × 10-3 U/mL DD and 100 µM NAD+ were preincubated in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA at 37 °C for 1 h. (B) Another reaction mixture containing the indicated concentrations of BA-1,2-dihydrodiols with 9.6 × 10-3 U/mL DD and 100 µM NAD+ was preincubated in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA at 37 °C for 30 min. After preincubation, 32P-5′-end-labeled DNA fragments, calf thymus DNA (20 µM/base), and 20 µM CuCl2 were added to the mixtures (200 µL), followed by the incubation at 37 °C for 1 h. After piperidine treatment, DNA was electrophoresed on an 8% polyacrylamide/8 M urea gel in Tris borate/EDTA buffer. The autoradiogram was obtained by exposing X-ray film to the gel. The percentage of DNA damage was roughly estimated by using a laser densitometer.

oxidative DNA damage, in calf thymus DNA by using an electrochemical detector coupled to HPLC (HPLC-ECD). We examined the conversion of NAD+ to NADH during the reaction that DD catalyzes PAH-dihydrodiols to PAH-catechols by UV-visible spectroscopy. We detected the product in the enzymatic reaction of BA-1,2-dihydrodiol with DD in the presence of NAD+, by using time-offlight mass spectroscopy (TOF-MS). The reaction mechanism was discussed on the basis of theoretical calculations.

Materials and Methods Materials. Restriction enzymes (ApaI, EcoRI, and HindIII) and alkaline phosphatase from calf intestine were purchased

Seike et al.

Figure 2. Effects of scavengers and a metal chelator on DNA damage induced by BA-1,2-dihydrodiol in the presence of Cu(II), DD, and NAD+. The reaction mixtures containing the 32P5′-end-labeled 443 bp DNA fragments, calf thymus DNA (20 µM/ base), 20 µM CuCl2, 9.6 × 10-3 U/mL DD, 100 µM NAD+, and 50 µM BA-1,2-dihydrodiol in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 1 h, after preincubation for enzymatic reaction (37 °C, 30 min). Ethanol (5%), mannitol (0.1 M), sodium formate (0.1 M), SOD (150 units/mL), catalase (150 units/mL), bathocuproine (50 µM), or methional (0.1 M) was added before the incubation. The DNA fragment was treated as described in the legend to Figure 1. from Boehringer Mannheim GmbH (Mannheim, Germany). T4 polynucleotide kinase was obtained from New England Biolabs (Beverly, MA). [γ-32P]ATP (222 TBq/mmol) was acquired from Amersham Biosciences (Buckinghamshire, United Kingdom). Diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were procured from Dojin Chemical Co. (Kumamoto, Japan). Acrylamide, piperidine, dimethyl sulfoxide (DMSO), bisacrylamide, and β-nicotinamide adenine dinucleotide (oxidized form) (NAD+), were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CuCl2, ethanol, D-mannitol, and sodium formate were acquired from Nacalai Tesque (Kyoto, Japan). BA-1,2-dihydrodiol, BA-3,4-dihydrodiol, BA-5,6-dihydrodiol, BA-8,9-dihydrodiol, BA-10,11-dihydrodiol, and B[a]P-7,8-dihydrodiol were obtained from Midwest Research Institute (Kansas City, MO). Calf thymus DNA, calf intestine phosphatase, superoxide dismutase (SOD, 3000 units/ mg from bovine erythrocytes), and catalase (45000 units/mg from bovine liver) were obtained from Sigma Chemical Co. (St. Louis, MO). R-Cyano-4-hydroxycinnamic acid was procured from Sigma-Aldrich Japan K. K. (Tokyo, Japan). Nuclease P1 (400 units/mg) was purchased from Yamasa Shoyu Co. (Chiba, Japan). Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) was obtained from Trevigen Inc. (Gaithersburg, MD). Preparation of DD. DD, recombinant AKR1C22, belongs to a member of the AKR superfamily and was expressed in E. coli and purified to homogeneity as previously described (7). The

DNA Damage by Benz[a]anthracene Dihydrodiols

Chem. Res. Toxicol., Vol. 17, No. 11, 2004 1447

Figure 3. Site specificity of DNA damage induced by BA-1,2-dihydrodiol treated with DD. The reaction mixtures contained 32P5′-end-labeled 443bp (ApaI 14179-EcoR* I14621) DNA fragments of the p53 tumor suppressor gene, calf thymus DNA (20 µM/base), 20 µM CuCl2, 9.6 × 10-3 U/mL DD, 100 µM NAD+, and 50 µM BA-1,2-dihydrodiol in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA (preincubation, 37 °C, 30 min; incubation, 37 °C, 1 h). After Fpg (A) or piperidine (B) treatment, the DNA fragment was treated as described in the Materials and Methods. buffer of the preparation was replaced to 10 mM sodium phosphate buffer, pH 7.8, and stored at -20 °C until used for the experiments. Preparation of 32P-5′-End-Labeled DNA Fragments. DNA fragments were obtained from the human p53 tumor suppressor gene (10). The DNA fragment of the p53 tumor suppressor gene was prepared from pUC18 plasmid. A singly 32P-5′-end-labeled 443 bp fragment (ApaI 14179-EcoRI* 14621)

and a 211 bp fragment (HindIII* 13972-ApaI 14182) were obtained according to the method described previously (11). The asterisk indicates 32P-labeling. Detection of DNA Damage Induced by BA Metabolites. Standard reaction mixtures (in a 1.5 mL Eppendorf microtube) containing the indicated concentrations of BA-dihydrodiols and B[a]P-7,8-dihydrodiol with 9.6 × 10-3 U/mL DD and 100 µM NAD+ were preincubated in 10 mM sodium phosphate buffer

1448

Chem. Res. Toxicol., Vol. 17, No. 11, 2004

Seike et al.

Figure 5. NADH formation in the reaction of NAD+, BAdihydrodiols, and DD. The reaction mixture containing 100 µM BA-dihydrodiols or B[a]P-dihydrodiol, 9.6 × 10-3 U/mL DD and 1 mM NAD+, in 1000 µL of 10 mM sodium phosphate buffer (pH 7.8) was incubated at 37 °C for 1 h. The increase of absorbance at 340 nm for NADH during 1 h was calculated as the enzymatic activity.

Figure 4. Formation of 8-oxodG in calf thymus DNA induced by BA-1,2-dihydrodiol treated with DD or not. (A) The reaction mixtures containing calf thymus DNA (100 µM/base), 20 µM CuCl2, and 100 µM NAD+ and the indicated concentrations of BA-1,2-dihydrodiol in 200 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA with or without 9.6 × 10-3 U/mL DD were incubated at 37 °C for 1 h after preincubation for DD treatment (37 °C, 1 h). (B) The reaction mixtures containing calf thymus DNA (100 µM/base), 20 µM CuCl2, 9.6 × 10-3 U/mL DD, 100 µM NAD+, 50 µM BA-1,2-dihydrodiol, and the scavenger in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 1 h after preincubation for enzymatic reaction (37 °C, 1 h). Ethanol (5%), mannitol (0.1 M), sodium formate (0.1 M), SOD (150 units/mL), catalase (150 units/mL), bathocuproine (50 µM), or methional (0.1 M) was added before the incubation. The DNA fragment was enzymatically digested into the nucleoside, and 8-oxodG formation was analyzed by using the HPLC-ECD, as described in the Materials and Methods. The results are expressed as means and SD of values obtained from three independent experiments. Symbols indicate significant differences by Student’s t-test (A, *; P < 0.01 as compared with control; B, #; P < 0.01 as compared with positive control). (pH 7.8) containing 5 µM DTPA at 37 °C for 1 h. After preincubation,32P-5′-end-labeled DNA fragments, calf thymus DNA (20 µM/base), and 20 µM CuCl2 were added to the mixtures (total 200 µL) followed by the incubation at 37 °C for 1 h. Then,

the DNA fragments were treated in 10% (v/v) piperidine at 90 °C for 20 min or treated with 6 units of Fpg protein in 21 µL of reaction buffer [10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA, and 0.1 mg/mL BSA] at 37 °C for 2 h. The treated DNA was electrophoresed on an 8% polyacrylamide/8 M urea gel. The autoradiogram was obtained by exposing X-ray film to the gel (12). The extent of DNA damage was roughly estimated by using a laser densitometer (LKB 2222 UltroScan XL). The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure (13) using a DNA sequencing system (LKB 2010 Macrophor). The laser densitometer was used for the measurement of the relative amounts of oligonucleotides from the treated DNA fragments. Measurement of 8-OxodG Formation in Calf Thymus DNA. Reaction mixtures (in a 1.5 mL Eppendorf microtube) containing the indicated concentrations of BA-1,2-dihydrodiol were preincubated with 9.6 × 10-3 U/mL DD and 100 µM NAD+ in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA at 37 °C for 30 min. Following the preincubation, DNA fragments (100 µM/base) from calf thymus and 20 µM CuCl2 were added to the mixtures, incubated at 37 °C for 1 h. After ethanol precipitation, DNA was digested to nucleosides with nuclease P1 and calf intestine phosphatase and analyzed by HPLC-ECD, as described previously (14). UV-Visible Spectra Measurements. To examine the enzymatic activity, the UV-visible spectral change was measured with a UV-visible spectrometer (UV-2500PC; Shimadzu, Kyoto, Japan). The reaction mixture containing 100 µM BAdihydrodiols, 1 mM NAD+, and 9.6 × 10-3 U/mL DD in 1 mL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA was incubated at 37 °C. The difference spectra between dihydrodiols/NAD+/DD and dihydrodiols/NAD+ were measured repeatedly at 37 °C for 1 h. The appearance of a new absorption at 340 nm ( ) 6.22 × 103 M-1 cm-1) for NADH was assessed as the enzymatic activity. Theoretically, the corresponding catechols of dihydrodiols will yield at the equimolar amount of formed NADH. TOF-MS Analysis. TOF-MS analysis was performed on a Voyager B-RP (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser (337 nm, 3 ns pulse) to determine the molecular weight of the product by the reaction of BA-1,2dihydrodiol, DD, and NAD+. Reaction mixtures containing 3 mM BA-1,2-dihydrodiol, 1.45 × 10-2 U/mL DD, and 3 mM NAD+ in 132 µL of 3 mM sodium phosphate buffer (pH 7.8) were incubated at 37 °C for 1 h. Matrix solution (saturated R-cyano4-hydroxycinnamic acid: 0.1%TFA/50%acetonitrile ) 1:1) was

DNA Damage by Benz[a]anthracene Dihydrodiols

Chem. Res. Toxicol., Vol. 17, No. 11, 2004 1449

added to the sample and then air-dried on a stainless steel probe tip. Calculation. The enthalpy change (-∆H) for dehydrogenation of BA-dihydrodiols and B[a]P-7,8-dihydrodiol was estimated from semiempirical calculation at PM3 level. Structures of these molecules were optimized by calculation of equilibrium geometry at the PM3 level. Energies of the highest occupied molecular orbital (HOMO) of dehydrogenated forms of BAdihydrodiols and B[a]P-7,8-dihydrodiol were estimated from ab initio MO calculations at the Hartree-Fock 3-21G level. These calculations were performed utilizing Spartan ‘02 for Windows (Wavefunction Inc., CA).

Results Damage to 32P-Labeled DNA Fragments Induced by BA-Dihydrodiols. Figure 1A shows an autoradiogram of DNA damage by BA-dihydrodiols in the presence and absence of DD. Interestingly, BA-1,2-dihydrodiol induced Cu(II)-mediated DNA damage in the presence of DD but did not in the absence of DD. BA-1,2dihydrodiol induced DNA damage more efficiently than B[a]P-7,8-dihydrodiol with metabolic activation. Other BA-dihydrodiols induced no DNA damage even in the presence of DD. BA-3,4-dihydrodiol induced Cu(II)mediated DNA damage even in the absence of DD as reported previously (data not shown). Metabolically activated BA-1,2-dihydrodiol induced DNA damage in a dose-dependent manner (Figure 1B). Effects of Scavengers and a Metal Chelator on DNA Damage Induced by BA-1,2-Dihydrodiol Treated with DD. Figure 2 shows the effects of scavengers and bathocuproine, a Cu(I) specific chelator, on DNA damage induced by BA-1,2-dihydrodiol treated with DD. Inhibition of DNA damage by catalase and bathocuproine suggests the involvement of hydrogen peroxide (H2O2) and Cu(I). Typical free hydoxyl radical (•OH) scavengers, such as ethanol, mannitol, and sodium formate, showed no inhibitory effects on DNA damage. Methional, which scavenges not only •OH but also cryptoOH radicals, inhibited DNA damage. SOD showed a partial inhibitory effect on DNA damage. Site Specificity of DNA Damage by BA-1,2-Dihydrodiol in the Presence of DD. An autoradiogram was obtained and scanned with a laser densitometer to measure the relative intensity of DNA cleavage in the human p53 tumor suppressor gene. With Fpg treatment, the DNA cleavage occurred mainly at guanine residues (Figure 3A). BA-1,2-dihydrodiol induced piperidine labile sites preferentially at thymine, cytosine, and guanine residues (Figure 3B). BA-1,2-dihydrodiol caused Fpg sensitive and piperidine labile lesions at G in the 5′-ACG3′ sequence complementary to codon 273, a well-known hotspot of the p53 tumor suppressor gene in lung cancer (15) (Figure 3A,B). Formation of 8-OxodG by BA-1,2-Dihydrodiol in the Presence and Absence of DD. By using the HPLCECD, we measured the 8-oxodG content in calf thymus DNA treated with BA-1,2-dihydrodiol with or without DD (Figure 4A). BA-1,2-dihydrodiol significantly increased 8-oxodG formation in a dose-dependent manner in the presence of DD. In the absence of DD, BA-1,2dihydrodiol did not increase the 8-oxodG formation. Figure 4B shows the inhibitory effects of scavengers on 8-oxodG formation induced by BA-1,2-dihydrodiol treated with DD. Catalase and bathocuproine completely inhib-

Figure 6. TOF-MS spectra of BA-1,2-dihydrodiol and its reaction product by DD. Reaction mixtures containing 3 mM BA-1,2-dihydrodiol, 1.45 × 10-2 U/mL DD, and 3 mM NAD+ in 132 µL of 3 mM sodium phosphate buffer (pH 7.8) were incubated at 37 °C for 1 h. Matrix solution, saturated R-cyano4-hydroxycinnamic acid:0.1%TFA/50%acetonitrile (1:1). Mass spectra show the products of BA-1,2-dihydrodiol before (A) and after reaction (B) with DD and NAD+. The arrow indicates reaction product with m/z 260.

ited 8-oxodG formation. Methional partially inhibited 8-oxodG formation. Enzymatic Activity of DD in the Aspect of Substrate Specificity. The DD activity of AKR1C22 for several dihydrodiols was spectrophotometrically measured as the increased absorbance of NADH at 340 nm. The formation rate of NADH was plotted against the calculated -∆H for the dehydrogenation of dihydrodiols (Figure 5). The value of -∆H implies the tendency to dehydrogenation of dihydrodiols to catechol. NADH production by DD appeared to increase depending on the

1450

Chem. Res. Toxicol., Vol. 17, No. 11, 2004

Seike et al.

Figure 7. Proposed mechanism of Cu(II)-mediated oxidative DNA damage induced by BA-1,2-dihydrodiol metabolite.

-∆H for dehydrogenation to catechol. The correlation coefficiency between NADH production and -∆H was 0.8063 (P ) 0.0992). These results have shown that BA1,2-dihydrodiol can be most easily converted into the corresponding catechol form, BA-1,2-diol. Detection of the Product in the Reaction of BA1,2-Dihydrodiol, DD, and NAD+. Figure 6 shows mass spectra of the reaction product of BA-1,2-dihydrodiol before and after enzymatic reaction. Figure 6A shows the mass spectrum with a molecular ion at m/z 262 (M+) assigned to BA-1,2-dihydrodiol before the reaction. The peak with a molecular ion at m/z 260 (M+) appeared after the reaction (Figure 6B, arrow). This peak is attributed to the catechol type metabolite, BA-1,2-diol, generated by the treatment of DD. Calculated HOMO Energy of BA-Diols and B[a]P-7,8-Diol. Calculated HOMO energies of BA-diols and B[a]P-7,8-diol were as follows: 6.94 (BA-1,2-diol), 7.05 (BA-5,6-diol), 7.13 (BA-8,9-diol), 6.91 (BA-10,11-diol), and 6.69 (B[a]P-7,8-diol) eV. These calculated values suggest that the tendencies of autoxidation of these BAdiols and B[a]P-7,8-diol are in the following order: B[a]P-7,8-diol > BA-10,11-diol > BA-1,2-diol > BA5,6-diol > BA-8,9-diol. The DNA damaging activity of B[a]P-7,8-diol may be explained by the tendency of autoxidation of the corresponding catechol forms in addition to the above-mentioned -∆H.

Discussion We revealed that BA-1,2-dihydrodiol and B[a]P-7,8dihydrodiol induced Cu(II)-mediated oxidative DNA damage in the presence of DD, but other BA-dihydrodiols did not under the condition used. The observation of NADH production suggested that DD catalyzed BA-1,2dihydrodiol most efficiently to its catechol among PAHdihydrodiols tested. We also showed that BA-1,2dihydrodiol induced Cu(II)-mediated 8-oxodG formation with DD treatment. BA-1,2-dihydrodiol significantly

induced a G lesion at the 5′-ACG-3′ sequence complementary to codon 273 of the p53 tumor suppressor gene. This sequence is known as a hotspot where mutations occurred in high frequency (15). An analysis of the p53 mutational database shows that G to T transversion is a signature mutation of lung cancer. Because 8-oxodG formation can lead to G to T transvertion, 8-oxodG has attracted much attention in relation to PAH-induced lung carcinogenesis. These results suggested that DD converted BA-1,2-dihydrodiol to BA-1,2-diol, which induced metal-dependent oxidative DNA damage including 8-oxodG formation via the quinone-catechol redox cycle. Our previous study (16) and Flowers et al. (17) showed that B[a]P-7,8-dione induced oxidative DNA damage via the quinone-catechol redox cycle. Penning and his colleagues (18) examined reactive oxygen species (ROS) generation and mutations in yeast by B[a]P-7,8-dione. These observations on B[a]P-dione may be explained by assuming that AKRs activate PAH trans-dihydrodiols to yield o-quinone (19). It is noteworthy to demonstrate that PAH-dihydrodiols can induce oxidative DNA damage in the presence of DD. Catalase and bathocuproine inhibited Cu(II)-mediated DNA damage by BA-1,2-dihydrodiol metabolite, indicating the involvement of H2O2 and Cu(I). These reactive species were certainly generated during the quinonecatechol redox cycle process of BA-1,2-dihydrodiol metabolite. On the basis of these results and references, a possible mechanism of oxidative DNA damage by BA1,2-dihydrodiol can be explained as follows (Figure 7); DD catalyzes NAD+-linked oxidation of BA-1,2-dihydrodiol to BA-1,2-diol, the catechol form. It is autooxidized into the corresponding semiquinone radical with concomitant generation of Cu(I) and O2•-. O2•- dismutates to H2O2, which interacts with Cu(I) to form a copperhydroperoxo complex such as Cu(I)OOH. Furthermore, NADH nonenzymatically reduces BA-1,2-dione into BA-1,2-diol through two-electron reduction-forming re-

DNA Damage by Benz[a]anthracene Dihydrodiols

dox cycles, such as benzene-catechol (20). Therefore, PAH-dihydrodiols and DD may play an important role in oxidative DNA damage via BA o-quinones/catechols redox cycling. Palakal et al. reported that DD preferentially oxidized B[a]P-7,8-dihydrodiol and BA-3,4-dihydrodiol (18). We showed that BA-1,2-dihydrodiol was more efficiently oxidized to catechol form by DD than B[a]P-7,8-dihydrodiol and other BA-dihydrodiols. Reasonably, BA-1,2dihydrodiol can cause most efficiently oxidative DNA damage with enzymatic metabolic activation by DD, although it is a minor metabolite. Multiple human tissue expression array analyses showed expression of DD, suggesting that this pathway of PAH activation may be widespread in human tissue (18). DD has dual toxicological roles in the metabolism of PAHs (19, 21). The enzyme suppresses the formation of their carcinogenic dihydrodiol epoxides and DNA adducts, whereas it yields catechol and quinones, resulting in oxidative DNA damage. Reports concerning overexpression of DD in lung cancer and hepatocellular carcinoma (8, 9) may suggest the involvement of oxidative DNA damage in a carcinogenic process. Therefore, it is important to clarify the theoretically reactive mechanism for the dehydrogenation of substrate by DD and autoxidation of the corresponding catechols. The most efficient dehydrogenation in BA-1,2dihydrodiol can be explained by the highest -∆H of the reaction. The production of catechol and NADH increased almost depending on the -∆H of dehydrogenation. The degrees of dehydrogenation are involved in -∆H of substrates and partly affinity between enzyme and dihydrodiols. On the other hand, ROS generation activity of catechols (BA-diols and B[a]P-diol) depends on the oxidation potential, which is almost consistent with the HOMO energy. Among compounds used in this study, the highest DNA-damaging activity of BA-1,2-dihydrodiol can be reasonably explained by the highest -∆H, which means tendency of conversion to the catechol form. In addition, the highest HOMO energy of B[a]P-7,8-diol among the corresponding diols may contribute to relatively strong DNA-damaging activity, although its -∆H is not so high. Collectively, it is suggested that the oxidative DNA-damaging activity of BA-dihydrodiols and B[a]P-dihydrodiol may depend on -∆H of dehydrogenation and the HOMO energy. Chen et al. demonstrated that DD overexpression was assosiated with lung cancer (8). DD catalyzes PAHdihydrodiols to catechols, resulting in quinone toxicity including alkylation and oxidative stress (21). DD may play an important role in BA carcinogenesis. It is interesting to find that DD-catalyzed BA-1,2-dihydrodiol can induce oxidative DNA damage involving G damage of ACG sequence complementary to codon 273 of the p53 tumor suppressor gene, a mutational hotspot in lung cancer. Recently, we showed that BA-3,4-dihydrodiol nonenzymatically induced oxidative DNA damage via a unique nonquinone redox cycle (22). Our present and previous studies have shown a possibility that oxidative DNA damage by BA metabolites, especially BA-1,2dihydrodiol and BA-3,4-dihydrodiol, participates in carcinogenesis.

Acknowledgment. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

Chem. Res. Toxicol., Vol. 17, No. 11, 2004 1451

References (1) Yu, D., Berlin, J. A., Penning, T. M., and Field, J. (2002) Reactive oxygen species generated by PAH o-quinones cause change-infunction mutations in p53. Chem. Res. Toxicol. 15, 832-842. (2) IARC Working Group (1983) General remarks. IARC monographs on the evaluation of the carcinogenic risk of chemicals to human. Polynuclear Aromatic Compounds, Part 1, Vol. 32, pp 33-91, IARC, Lyon. (3) Mastrangelo, G., Fadda, E., and Marzia, V. (1996) Polycyclic aromatic hydrocarbons and cancer in man. Environ. Health Perspect. 104, 1166-1170. (4) Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 91, 1194-1210. (5) IARC Working Group (1973) Benz[a]anthracene. IARC monographs on the evolution of the carcinogenic risk of chemicals to human. Certain Polycyclic Aromatic Hydrocarbons and Heterocyclic Compounds, Vol. 3, p 45, IARC, Lyon. (6) Sahali, Y., Kidd, L. R., Skipper, P. L., and Tannenbaum, S. R. (1994) Metabolism of benz[a]anthracene by human liver microsomes. Cancer Lett. 83, 299-303. (7) Deshiyashiki, Y., Takatsuji, T., and Hara, A. (2003) Molecular cloning and characterization of dihydrodiol dehydrogenase from the mouse. In Aldo-Keto Reductases and Toxicant Metabolism (Penning, T. M., and Petrash, J. M., Eds.), Chapter 7, pp 101114, American Chemical Society, Washington, DC. (8) Chen, C. Y., Hsu, C. P., Hsu, N. Y., Shih, C. S., Lin, T. Y., and Chow, K. C. (2002) Expression of dihydrodiol dehydrogenase in the resected stage I nonsmall cell lung cancer. Oncol. Rep. 9, 515519. (9) Yang, M. D., Wu, C. C., Chiou, S. H., Chiu, C. F., Lin, T. Y., Chiang, I. P., and Chow, K. C. (2003) Reduction of dihydrodiol dehydrogenase expression in resected hepatocellular carcinoma. Oncol. Rep. 10, 271-276. (10) Chumakov, P. (1990) EMBL Data Library, accession number X54156. (11) Yamashita, N., Murata, M., Inoue, S., Hiraku, Y., Yoshinaga, T., and Kawanishi, S. (1998) Superoxide formation and DNA damage induced by a fragrant furanone in the presence of copper(II). Mutat. Res. 397, 191-201. (12) Yamamoto, K., and Kawanishi, S. (1991) Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions; the role of hydroxyl radical and hydrogen atom. J. Biol. Chem. 266, 1509-1515. (13) Maxam, A. M., and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499-560. (14) Ito, K., Inoue, S., Yamamoto, K., and Kawanishi, S. (1993) 8-Hydroxy-deoxyguanosine formation at the 5′ site of 5′-GG-3′ sequences in double-stranded DNA by UV radiation with riboflavin. J. Biol. Chem. 268, 13221-13227. (15) Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 274, 430-432. (16) Ohnishi, S., and Kawanishi, S. (2002) Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene. Biochem. Biophys. Res. Commun. 290, 778-782. (17) Flowers, L., Bleczinski, W. F., Burczynski, M. E., Harvey, R. G., and Penning, T. M. (1996) Disposition and biological activity of benzo[a]pyrene-7,8-dione. A genotoxic metabolite generated by dihydrodiol dehydrogenase. Biochemistry 35, 13664-13672. (18) Palackal, N. T., Burczynski, M. E., Harvey, R. G., and Penning, T. M. (2001) The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation. Biochemistry 40, 10901-10910. (19) Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D., Palackal, N. T., and Tsuruda, L. S. (1999) Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: Generation of reactive and redox active o-quinones. Chem. Res. Toxicol. 12, 1-18. (20) Hirakawa, K., Oikawa, S., Hiraku, Y., Hirosawa, I., and Kawanishi, S. (2002) Catechol and hydroquinone have different redox properties responsible for their differential DNA-damage ability. Chem. Res. Toxicol. 15, 76-86. (21) Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135-160. (22) Seike, K., Murata, M., Oikawa, S., Hiraku, Y., Hirakawa, K., and Kawanishi, S. (2003) Oxidative DNA damage induced by benz[a]anthracene metabolites via redox cycles of quinone and unique nonquinone. Chem. Res. Toxicol. 11, 1470-1476.

TX0498814