anthracene Metabolites via Redox Cycles of Quinone and Uniqu

Oct 11, 2003 - Tsu, Mie 514-8507, Japan, and Department of Radiation Chemistry, Life Science Research Center,. Mie University, Tsu, Mie 514-8507, Japa...
0 downloads 0 Views 154KB Size
Download PDF
1470

Chem. Res. Toxicol. 2003, 16, 1470-1476

Oxidative DNA Damage Induced by Benz[a]anthracene Metabolites via Redox Cycles of Quinone and Unique Non-Quinone Kazuharu Seike,† Mariko Murata,† Shinji Oikawa,† Yusuke Hiraku,† Kazutaka Hirakawa,‡ and Shosuke Kawanishi*,† Department of Environmental and Molecular Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan, and Department of Radiation Chemistry, Life Science Research Center, Mie University, Tsu, Mie 514-8507, Japan Received May 27, 2003

Benz[a]anthracene (BA) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) that are ubiquitous environmental pollutants. PAH carcinogenesis is explained by DNA adduct formation by PAH diol epoxide and oxidative DNA damage by PAH o-quinone. Benz[a]anthracene-trans-3,4-dihydrodiol (BA-3,4-dihydrodiol) is a minor metabolite but shows higher mutagenicity and tumorigenicity than parent BA. We confirmed that a BA o-quinone type metabolite, benz[a]anthracene-3,4-dione (BA-3,4-dione), induced oxidative DNA damage in the presence of cytochrome P450 reductase. Interestingly, we found that BA-3,4-dihydrodiol nonenzymatically caused Cu(II)-mediated DNA damage including 8-oxo-7,8-dihydro-2′-deoxyguanosine formation and the addition of NADH enhanced DNA damage. BA-3,4-dihydrodiol induced a double-base lesion of C and G at the 5′-ACG-3′ sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. The DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H2O2 and Cu(I). Time-of-flight mass spectroscopic study suggested that BA-3,4-dihydrodiol undergoes Cu(II)mediated autoxidation leading to the formation of its hydroxylated form of BA-3,4-dihydrodiol, capable of causing oxidative DNA damage. It is noteworthy that BA-3,4-dihydrodiol can nonenzymatically induce DNA damage more efficiently than BA-3,4-dione with metabolic activation. In conclusion, oxidative DNA damage induced by BA-3,4-dihydrodiol not only via quinone-type redox cycle but also via a new type of redox cycle participates in the expression of carcinogenicity of BA and BA-3,4-dihydrodiol.

Introduction Polycyclic aromatic hydrocarbons (PAHs), ubiqitous environmental pollutants, undergo metabolic activation prior to exerting mutagenicity and carcinogenicity (13). Benz[a]anthracene (BA) is present as a major component of the total PAHs in the environment (4). Human exposure to BA occurs primarily through inhalation of polluted air, smoking of tobacco, and by ingestion of food and water contaminated by the combustion effluent (4). The International Agency for Research on Cancer (IARC) has classified BA as a group 2A carcinogen, which is probably carcinogenic to humans (4). BA is predominantly metabolized to the 5,6-, 8,9-, and 10,11-dihydrodiols (4, 5). Benz[a]anthracene-trans-3,4-dihydrodiol (BA3,4-dihydrodiol) is a minor metabolite (4, 5) but has been shown to have an extraordinary activity of carcinogenicity (6, 7). BA-3,4-dihydrodiol is highly active in causing malignant lymphomas and pulmonary adenomas in newborn mice (6). Furthermore, BA-3,4-dihydrodiol is at least 10- to 20-fold more tumorigenic than the parent BA or any other BA dihydrodiols (7). Recent epidemiological studies have suggested that PAHs can cause oxidative DNA damage in addition to * Address correspondence to the following author. Tel. and Fax: (+81) (59) 231 5011. E-mail: [email protected]. † Mie University School of Medicine. ‡ Life Science Research Center, Mie University.

DNA adduct formation, which is the most widely accepted mechanism of PAHs carcinogenesis (8, 9). Exposure to air pollutant particles including PAHs induced oxidative DNA damage both in vivo (10) and in vitro (11). In addition, several reports demonstrated that a derivative of benz[a]anthracene, dimethylbenz[a]anthracene, induced 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation in mammary glands of rats (12) and 8-oxodG and 5′-hydroxymethyl-2′-deoxyuridine formation in the epidermal DNA of mice (13). Furthermore, not only DNA adducts but also oxidative DNA damage was observed in seashells exposed to waterborne benzo[a]pyrene (14). These reports suggest that exposure to ambient air pollution including BA may cause oxidative damage to DNA. Penning et al. reported that PAH o-quinone induced oxidative DNA damage with metabolic activation via a redox cycle (15). We confirmed that a metabolite of benzo[a]pyrene, a representative PAH, nonenzymatically induced oxidative DNA damage via redox cycle (16). Therefore, there remains a possibility that oxidative DNA damage also plays a role in carcinogenicity induced by BA. In this study, we examined oxidative DNA damage and its site specificity induced by BA and its metabolites, BA3,4-dihydrodiol and BA-3,4-dione, using 32P-5′-endlabeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1

10.1021/tx034103h CCC: $25.00 © 2003 American Chemical Society Published on Web 10/11/2003

DNA Damage Induced by Benz[a]anthracene Metabolites

protooncogene. Furthermore, we investigated the formation of 8-oxodG, an indicator of oxidative DNA damage, in calf thymus DNA by using an electrochemical detector coupled to high-pressure liquid chromatography (HPLCECD). A reaction product of BA-3,4-dihydrodiol was analyzed by a time-of-flight mass spectrometer (TOFMS).

Materials and Methods Materials. Restriction enzymes (ApaI, AvaI, EcoRI, BssHII, XbaI, and HindIII), alkaline phosphatase form calf intestine, and glucose-6-phosphate dehydrogenase (G-6-PDH) were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Restriction enzyme (BamHI) and T4 polynucleotide kinase were obtained from New England Biolabs (Beverly, MA). [γ-32P] ATP (222 TBq/mmol) was acquired from Amersham Biosciences (Buckinghamshire, UK). DiethylenetriamineN,N,N′,N′′,N′′-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were procured from Dojin Chemical Co. (Kumamoto, Japan). Acrylamide, piperidine, dimethyl sulfoxide (DMSO), bisacrylamide, β-nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP+), iron(III) chloride, glucose-6-phosphate monosodium salt (G-6-P), and benz[a]anthracene (BA) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cytochrome P450 reductase (P450 reductase) (10.0 mg/ mL protein from human microsomes) was purchased from Gentest Corporation (Woburn, MA). MgCl2, CuCl2, iron(III) monosodium ethylenediaminetetraacetate (Fe(III)-EDTA), iron(III) citrate-4-hydrate, ethanol, D-mannitol, and sodium formate were acquired from Nacalai Tesque (Kyoto, Japan). R-Cyano4-hydroxycinnamic acid and hemin chloride were procured from Sigma-Aldrich Japan K. K. (Tokyo, Japan). Adenosine-5′diphosphate disodium salt (ADP) was purchased from Kohjin Co. Ltd. (Tokyo, Japan). BA-3,4-dihydrodiol and BA-3,4-dione 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 (45 000 units/mg from bovine liver) were obtained from Sigma Chemical Co. (St. Louis, MO). 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 32P-5′-End-Labeled DNA Fragments. DNA fragments were obtained from the human p53 (17) and p16 (18) tumor suppressor genes and the c-Ha-ras-1 protooncogene (19). The DNA fragment of the p53 tumor suppressor gene was prepared from the 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 (20). The 5′-endlabeled 460 bp fragment (EcoRI* 9481-EcoRI* 9940) containing exon 2 of the human p16 tumor suppressor gene was obtained by pGEM-T Easy Vector (Promega Corporation). The 460 bp fragment was digested with BssHII to obtain a singly labeled 309 bp fragment (BssHII 9789-EcoRI*9481) as described previously (21). Furthermore, the DNA fragments were also prepared from plasmid pbcNI, which carries a 6.6 kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene (22, 23). A singly labeled 341 bp fragment (XbaI 1906AvaI*2246) was obtained according to the method described previously (22, 23). 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-3,4-dihydrodiol, 20 µM CuCl2, 100 µM NADH, 32P-5′-end-labeled DNA fragments, and calf thymus DNA (20 µM/base) in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 1 h. In a certain experiment, BA-3,4-dione and BA-3,4-dihydrodiol were preincubated with 1.4 × 10-3 U/mL P450 reductase, 500 µM MgCl2, 500 µM G-6-P, 250 µM NADP+, and 0.1 U/mL G-6-PDH in 10 mM sodium phosphate buffer (pH

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1471 7.8) containing 5 µM DTPA at 37 °C for 1 h. After preincubation, DNA fragments, calf thymus DNA (20 µM/ base), and 20 µM CuCl2 were added to the mixtures, 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 (23). 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 (24) using a DNA-sequencing system (LKB 2010 Macrophor). A laser densitometer (LKB 2222 UltroScan XL) 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. DNA fragments (100 µM/base) from calf thymus were incubated with BA-3,4-dihydrodiol and 20 µM CuCl2 in the presence or absence of 100 µM NADH 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 (25). 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 BA-3,4-dihydrodiol reaction product. Reaction mixtures containing 100 µM BA-3,4-dihydrodiol, 20 µM CuCl2, and 100 µM NADH in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) were incubated at 37 °C for 1 h. Matrix solution (saturated R-cyano-4-hydroxycinnamic acid: 0.1% TFA/50% acetonitrile ) 1:1) was added to the sample and then air-dried on a stainless steel probe tip. 32P-5′-end-labeled

Results Damage to 32P-Labeled DNA Fragments Induced by BA-3,4-Dihydrodiol and BA-3,4-Dione. Figure 1A shows an autoradiogram of DNA damage by BA metabolites in the presence and absence of P450 reductase. BA3,4-dihydrodiol induced Cu(II)-mediated DNA damage even in the absence of reductase. However, BA-3,4-dione induced DNA damage in the presence of reductase but did not in the absence of reductase. It is apparent that BA-3,4-dihydrodiol can nonenzymatically induce DNA damage more efficiently than BA-3,4-dione with metabolic activation. Furthermore, BA-3,4-dihydrodiol induced DNA damage in a dose-dependent manner, and the addition of NADH enhanced DNA damage about 2-fold (Figure 1B). Piperidine treatment increased 2-3-fold the oligonucleotides (data not shown), suggesting that BA3,4-dihydrodiol induces not only strand breakage but also base modification and/or liberation. BA-3,4-dihydrodiol alone induced no DNA damage. BA induced no DNA damage even in the presence of Cu(II) and NADH (data not shown). Oxidative DNA damage induced by BA-3,4dihydrodiol in the presence of Cu(II) was enhanced depending on the increase of buffer pH, whereas the enhancing effect of NADH on DNA damage was decreased in the increase of the pH (data not shown). When Fe(III)-EDTA, Fe(III)-ADP, Fe(III) citrate, and hemin were used instead of Cu(II), BA-3,4-dihydrodiol induced no or little DNA damage under the condition used (data not shown). Effects of Scavengers and a Metal Chelator on DNA Damage Induced by BA-3,4-Dihydrodiol and BA-3,4-Dione. Figure 2 shows the effect of scavengers and bathocuproine. Catalase and bathocuproine inhibited

1472 Chem. Res. Toxicol., Vol. 16, No. 11, 2003

Seike et al.

Figure 1. Autoradiogram of 32P-labeled DNA fragments incubated with BA-3,4-dihydrodiol and BA-3,4-dione. (A) The reaction mixtures (in a 1.5 mL Eppendorf microtube) containing 50 µM BA-3,4-dihydrodiol or BA-3,4-dione, 500 µM MgCl2, 500 µM G-6-P, 250 µM NADP+, and 0.1 U/mL G-6-PDH in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 1 h in the presence of 1.4 × 10-3 U/mL P450 reductase. After preincubation, the 32P-5′-end-labeled 443 bp DNA fragments (ApaI 14179-EcoRI*14621), calf thymus DNA (20 µM/base), and 20 µM CuCl2 were added to the mixtures, followed by the incubation at 37 °C for 1 h. (B) The reaction mixtures containing the 32P-5′-end-labeled 443 bp DNA fragments (ApaI 14179-EcoRI*14621), calf thymus DNA (20 µM/base), 20 µM CuCl2, and the indicated concentrations of BA-3,4-dihydrodiol in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA with or without 100 µM NADH were incubated 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.

Figure 2. Effects of scavengers and a metal chelator on DNA damage induced by BA-3,4-dihydrodiol in the presence of Cu(II) and NADH. The reaction mixtures containing the 32P-5′end-labeled 443 bp DNA fragments, calf thymus DNA (20 µM/ base), 20 µM CuCl2, 100 µM NADH, and 50 µM BA-3,4dihydrodiol in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 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 treated as described in the legend to Figure 1.

DNA damage induced by BA-3,4-dihydrodiol, suggesting the involvement of hydrogen peroxide (H2O2) and Cu(I).

Typical free hydroxyl 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 crypto-OH radicals, inhibited DNA damage. SOD showed a partial inhibitory effect on DNA damage. Similar scavenger effects were observed with BA-3,4-dione (data not shown). Site Specificity of DNA Damage by BA-3,4-Dihydrodiol in the Presence of Cu(II) and NADH. 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. BA3,4-dihydrodiol induced piperidine-labile sites preferentially at the thymine, cytosine, and guanine residues (Figure 3). With Fpg treatment, the DNA cleavage occurred mainly at the guanine and cytosine residues. In addition, two tandem bases of the 5′-TG-3′ and 5′-CT3′ sites were often damaged when taking together the Fpg and piperidine treatment (Figure 3A,B). BA-3,4dihydrodiol caused piperidine-labile and Fpg-sensitive lesions at CG in the 5′-ACG-3′ sequence, a well-known hotspot of the p53 gene (26) (Figure 3C,D). Formation of 8-oxodG by BA-3,4-Dihydrodiol in the Presence of Cu(II) and NADH. By using HPLCECD, we measured the 8-oxodG content in calf thymus DNA treated with BA-3,4-dihydrodiol. BA-3,4-dihydrodiol significantly increased 8-oxodG formation in a dosedependent manner in the presence of Cu(II) (Figure 4). When NADH was added, the 8-oxodG formation increased. BA-3,4-dione induced Cu(II)-mediated 8-oxodG formation in the presence of P450 reductase (data not shown). Determination of the Reaction Products in the Cu(II)-Mediated Autoxidation of BA-3,4-Dihydrodiol. Figure 5 shows mass spectra of the reaction product of BA-3,4-dihydrodiol before and after reaction. Before the reaction (Figure 5A), the mass spectrum with the molecular ion at m/z 262 (M+) assigned to BA-3,4-

DNA Damage Induced by Benz[a]anthracene Metabolites

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1473

Figure 3. Site specificity of DNA damage induced by BA-3,4-dihydrodiol in the presence of Cu(II) and NADH. The reaction mixtures contained the 32P-5′-end-labeled 211 bp fragment (HindIII* 13972-ApaI 14182) (A and B) and 443 bp (ApaI 14179-EcoRI* I14621) (C and D) DNA fragments of the p53 tumor suppressor gene, calf thymus DNA (20 µM/base), 20 µM CuCl2, 100 µM NADH and 20 µM BA-3,4-dihydrodiol in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After Fpg (A and C) and piperidine (B and D) treatment, the DNA fragment was treated as described in the Materials and Methods.

Figure 4. Formation of 8-oxodG in calf thymus DNA induced by BA-3,4-dihydrodiol in the presence of Cu(II) and NADH. The reaction mixtures containing calf thymus DNA (100 µM/base), 20 µM CuCl2, and the indicated concentrations of BA-3,4dihydrodiol in 400 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA with or without 100 µM NADH were incubated at 37 °C for 1 h. 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. *P < 0.05 as compared with controls.

dihydrodiol was observed. After the reaction (Figure 5B), the mass spectrum with the molecular ion at m/z 278 (M+) appeared. We speculate that this product is generated by the hydroxylation of BA-3,4-dihydrodiol such as BA-3,4-dihydro-2,3,4-triol.

Discussion The present study showed that BA-3,4-dihydrodiol and BA-3,4-dione induced oxidative DNA damage via two types of redox cycles. We confirmed that a BA o-quinone type metabolite, BA-3,4-dione, induced DNA damage in the presence of P450 reductase, as other PAH o-quinones induced oxidative DNA damage via a redox cycle (26). Notably, we found that BA-3,4-dihydrodiol nonenzy-

Figure 5. TOF-MS spectra of BA-3,4-dihydrodiol and its reaction product. The reaction mixture containing 100 µM BA3,4-dihydrodiol, 20 µM CuCl2, and 100 µM NADH in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) was incubated at 37 °C for 1 h. Mass spectra show the products of BA-3,4-dihydrodiol before (A) and after (B) reaction.

matically induced oxidative DNA damage via a nonquinone type of redox cycle. BA-3,4-dihydrodiol caused oxidative DNA damage including 8-oxodG formation in the presence of Cu(II). The addition of NADH enhanced DNA damage. Although iron did not mediate DNA damage in this system, this result cannot exclude the possibility that some kinds of iron complexes with endogenous compounds mediate oxidative DNA damage (27). Interestingly, BA-3,4-dihydrodiol significantly in-

1474 Chem. Res. Toxicol., Vol. 16, No. 11, 2003

Seike et al.

Figure 6. Proposed mechanism of Cu(II)-mediated oxidative DNA damage induced by BA-3,4-dihydrodiol.

duced the double-base lesion of G and C at the 5′-ACG3′ sequence complementary to codon 273 of the p53 gene. This sequence is known as a hotspot where mutations occur frequently (28). Furthermore, BA-3,4-dihydrodiol caused a double-base lesion at the 5′-TG-3′ and 5′-CT-3′ sequences. Relevantly, our previous studies demonstrated that DNA damage at the G residues adjacent to piperidine-labile pyrimidine sites were also induced by other chemicals such as benz[a]pyrene (16) and nitrofluorene (29). Therefore, we have speculated that the same reactive oxygen species are responsible for the site-specific DNA damage. Since cluster DNA damage is difficult to repair, we speculate that the double-base DNA damage tends toward mutation and carcinogenesis. Therefore, a BA-3,4-dihydrodiol-induced double-base lesion may give an explanation for its high carcinogenic potency. In addition, BA-3,4-dihydrodiol efficiently induced 8-oxodG formation in the presence of Cu(II) and NADH. Several studies have reported that 8-oxodG causes DNA misreplication, which can lead to mutation, particularly G f T substitutions (30, 31). 8-OxodG has attracted much attention in relation to mutagenesis and carcinogenesis. Cu(II)-mediated DNA damage by BA-3,4-dihydrodiol and BA-3,4-dione was inhibited by catalase and bathocuproine, indicating the involvement of H2O2 and Cu(I). These reactive species were possibly generated during the autoxidation process of BA-3,4-dihydrodiol. Possible mechanisms of oxidative DNA damage by BA-3,4-dihydrodiol and BA-3,4-dione have been proposed on the basis of these results (Figure 6). BA-3,4-dihydrodiol undergoes autoxidation through intermediate radicals, with a 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, which can cause

oxidative DNA damage. Oxidative DNA damage induced by BA-3,4-dihydrodiol in the presence of Cu(II) was enhanced depending on the increase of buffer pH (data not shown), suggesting that the hydroxylation is due to the reaction with OH-. TOF-MS revealed that the reaction product contained a molecular ion at m/z 278 (M+), supporting that the hydroxylated form of BA-3,4-dihydrodiol was generated. Cu(II) ion may easily interact with the hydroxyl groups at the 3,4-positions of BA-3,4dihydrodiol (32). Thus, the double bond in the benzo ring near the Cu(II)-interacted hydroxyl groups may be subject to undergo a Cu(II)-mediated hydroxylation. Ab initio molecular orbital calculation at the Hartree-Fock/ 6-31G* level showed that electron density at the 2-position of BA-3,4-dihydrodiol is 10-fold higher than that at the 1-position, suggesting that the carbon of BA-3,4dihydrodiol at the 2-position is much more likely to undergo hydroxylation. Therefore, we speculate that the final reaction product is BA-3,4-dihydro-2,3,4-triol. In the presence of an endogenous reductant NADH, the carboncentered cation radical may be reduced to BA-3,4dihydrodiol, and autoxidation occurs again, resulting in the formation of the redox cycle. When the buffer at a higher pH was used, the enhancing effect of NADH on DNA damage decreased, possibly due to the promotion of the hydroxylation of the cation radical. This result suggests that the cation radical reacts with NADH and supports the previous redox cycle. In general, PAHs are metabolized initially by the microsomal cytochrome P450 monooxygenase system to several arene oxides, which undergo hydration to dihydrodiols by epoxide hydrolase. PAH-dihydrodiols were also converted to diol epoxide by cytochrome P450 (1). Benzo ring diol epoxide, in which the epoxide forms part

DNA Damage Induced by Benz[a]anthracene Metabolites

of the bay region of the hydrocarbon molecule, has a high chemical reactivity (1). DNA adduct formation by diol epoxide metabolites is considered to be essential for PAH carcinogenesis (1, 28, 33). In another pathway, PAHdihydrodiol is oxidized by dihydrodiol dehydrogenase (aldo-keto reductase) to form PAH catechol (25, 33). The catechols undergo autoxidation to PAH o-quinones with the generation of a reactive oxygen species (ROS). In the presence of P450 reductase, PAH o-quinones can be converted back to the catechols forming the redox cycle (34). Finally, PAH o-quinones/catechols-generated ROS and subsequent oxidative DNA damage (14, 33, 35) are considered to be causal factors. In fact, we detected that BA-3,4-dione induced DNA damage in the presence of reductase, suggesting that BA o-quinones/catecholsgenerated ROS induced oxidative DNA damage. Moreover, we have first demonstrated that BA-3,4-dihydrodiol can induce oxidative DNA damage through a unique nonquinone type of redox cycle. It is noteworthy to find that BA-3,4-dihydrodiol can cause DNA damage without further metabolic activation. It is generally accepted that DNA adduct formation participates in tumor initiation (36), while oxidative DNA damage would be involved in tumor promotion (37) and progression (38). Chemical compounds can induce both DNA adduct formation and oxidative DNA damage (39). The literature described that control values of DNA adduct formation and 8-oxodG formation were 0.27 adducts/108 nucleotides and 300 8-oxodG/108 dG, respectively (40). The basal levels of these DNA lesions are quite different. Canova et al. reported that benzo[a]pyrene increased significantly the DNA adduct levels from 0.063 to 0.486 adducts/108 nucleotides and 8-oxodG levels from 14.01 to 28.51 8-oxodG/105 dG in the digestive gland of mussels (14). This study has shown a possibility that oxidative DNA damage by BA metabolites participates in carcinogenesis, although we have no data to judge whether oxidative DNA damage is more or less important than DNA adduct formation. Findings of DNA damage by BA-3,4-dihydrodiol itself via a new type of redox cycle may provide an insight into the mechanism of carcinogenesis by PAH, in addition to already known types of DNA damage.

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

References (1) 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. (2) Mastrangelo, G., Fadda, E., and Marzia, V. (1996) Polycyclic aromatic hydrocarbons and cancer in man. Environ. Health Perspect. 104, 1166-1170. (3) Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 91, 1194-1210. (4) IARC working group (1983) Benz[a]anthracene. IARC monographs on the evolution of the carcinogenic risk of chemicals to human; Polynuclear Aromatic Compounds, Part 1, Vol. 32, pp 135-145, IARC, Lyon. (5) 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. (6) Wislocki, P. G., Kapitulnik, J., Levin, W., Lehr, R., SchaeferRidder, M., Karle, J. M., Jerina, D. M., and Conney, A. H. (1978) Exceptional carcinogenic activity of benz[a]anthracene 3,4-dihy-

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1475

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17) (18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

drodiol in the newborn mouse and the bay region theory. Cancer Res. 38, 693-696. Wood, A. W., Levin, W., Chang, R. L., Lehr, R. E., Schaefer-Ridder, M., Karle, J. M., Jerina, D. M., and Conney, A. H. (1977) Tumorigenicity of five dihydrodiols of benz[a]anthracene on mouse skin: exceptional activity of benz[a]anthracene 3,4-dihydrodiol. Proc. Natl. Acad. Sci. U.S.A. 74, 3176-3179. Toraason, M., Hayden, C., Marlow, D., Rinehart, R., Mathias, P., Werren, D., Olsen, L. D., Neumeister, C. E., Mathews, E. S., Cheever, K. L., Marlow, K. L., DeBord, D. G., and Reid, T. M. (2001) DNA strand breaks, oxidative damage, and 1-OH pyrene in roofers with coal-tar pitch dust and/or asphalt fume exposure. Int. Arch. Occup. Environ. Health 74, 396-404. Loft, S., Poulsen, H. E., Visitisen, K., and Knudsen, L. E. (1999) Increased urinary excretion of 8-oxo-2′-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers. Mutat. Res. 441, 11-19. Sorensen, M., Autrup, H., Hertel, O., Wallin, H., Knudsen, L. E., and Loft, S. (2003) Personal exposure to PM(2.5) and biomarkers of DNA damage. Cancer Epidemiol. Biomarkers Prev. 12, 191196. Greenwell, L. L., Moreno, T., Jones, T. P., and Richards, R. J. (2002) Particle-induced oxidative damage is ameliorated by pulmonary antioxidants. Free Radical Biol. Med. 32, 898-905. Frenkel, K., Wei, L., and Wei, H. (1995) 7,12-Dimethylbenz[a]anthracene induces oxidative DNA modification in vivo. Free Radical Biol. Med. 19, 373-380. El-Bayoumy, K., Chae, Y. H., Rosa, J. G., Williams, L. K., Desai, D., Amin, S., and Fiala, E. (2000) The effects of 1-nitropyrene, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and 7,12-dimethylbenz[a]anthracene on 8-hydroxy-2′-deoxyguanosine levels in the rat mammary gland and modulation by dietary 1,4-phenylenebis(methylene) selenocyanate. Cancer Lett. 151, 7-13. Canova, S., Degan, P., Peters, L. D., Livingstone, D. R., Voltan, R., and Venier, P. (1998) Tissue dose, DNA adducts, oxidative DNA damage, and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[a]pyrene. Mutat. Res. 399, 17-30. Flowers, L., Ohnishi, S. T., and Penning, T. M. (1997) DNA strand scission by polycyclic aromatic hydrocarbon o-quinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling, and o-semiquinone anion radicals. Biochemistry 36, 8640-8648. 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. Chumakov, P. (1990) EMBL Data Library, accession number X54156. Serrano, M., Hannon, G. J., and Beach, D. A. (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366, 704-707. Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H., and Goeddel, D. V. (1983) Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302, 33-37. 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. Oikawa, S., Hirosawa, I., Hirakawa, K., and Kawanishi, S. (2001) Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol. Carcinogecnesis 22, 1239-1245. Kawanishi, S., and Yamamoto, K. (1991) Mechanism of sitespecific DNA damage induced by methylhydrazines in the presence of copper(II) or manganese(III). Biochemistry 30, 3069-3075. 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. Maxam, A. M., and Gilbert, W. (1980) Sequencing end-labeled DNA with base specific chemical cleavages. Methods Enzymol. 65, 499-560. 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. 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. Barbouti, A., Doulias, P. T., Zhu, B. Z., Frei, B., and Galaris, D. (2001) Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage. Free Radical Biol. Med. 31, 490-498.

1476 Chem. Res. Toxicol., Vol. 16, No. 11, 2003 (28) 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. (29) Murata, M., Yoshiki, Y., Tada, M., and Kawanishi, S. (2002) Oxidative DNA damage by a common metabolite of carcinogenic nitrofluorene and N-acetylaminofluorene. Int. J. Cancer 102, 311-317. (30) Shibutani, S., Takeshita, M., and Grollman, A. P. (1991) Insertion of specific bases during DNA synthesis past the oxidationdamaged base 8-oxodG. Nature 349, 431-434. (31) Efrati, E., Tocco, G., Eritja, R., Wilson, S. H., and Goodman, M. F. (1999) Action-at-a-distance mutagenesis. 8-oxo-7,8-dihydro-2′deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase β. J. Biol. Chem. 274, 15920-15926. (32) Hirakawa, K., Oikawa, S., Hirosawa, Y., and Kawanishi, S. (2002) Catechol and hydroquinone have different redox properties responsible for their differential DNA-damage ability. Chem. Res. Toxicol. 15, 76-86. (33) Viezzer, C., Norppa, H., Clonfero, E., Gabbani, G., Mastrangelo, G., Hirvonen, A., and Celotti, L. (1999) Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers. Mutat. Res. 431, 259-269. (34) Klein, J., Post, K., Thomas, H., Worner, W., Setiabudi, F., Frank, H., Oesch, F., and Platt, K. L. (1990) The oxidation of the highly tumorigenic benz[a]anthracene 3,4-dihydrodiol by rat liver dihydrodiol dehydrogenase. Chem. Biol. Interact. 76, 211-226.

Seike et al. (35) 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. (36) Devanesan, P., Todorovic, R., Zhao, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (2001) Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian golden hamsters treated with 4-hydroxyestradiol: potential biomarkers for estrogeninitiated cancer. Carcinogenesis 22, 489-497. (37) Rusyn, I., Denissenko, M. F., Wong, V. A., Butterworth, B. E., Cunningham, M. L., Upton, P. B., Thurman, R. G., and Swenberg, J. A. (2000) Expression of base excision repair enzymes in rat and mouse liver is induced by peroxisome proliferators and is dependent upon carcinogenic potency. Carcinogenesis 21, 21412145. (38) Jaloszynski, P., Jaruga, P., Olinski, R., Biczysko, W., Szyfter, W., Nagy, E., Moller, L., and Szyfter, K. (2003) Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx. Free Radical Res. 37, 231240. (39) Kawanishi, S., Hiraku, Y., Murata, M., and Oikawa, S. (2002) The role of metals in site-specific DNA damage with reference to carcinogenesis. Free Radical Biol. Med. 32, 822-832. (40) Dybdahl, M., Rison, L., Moller, P., Autrup, H., Wallin, H., Vogel, U., Bornholdt, J., Daneshvar, B., Dragsted, L., Weimann, A., Poulsen, H. E., and Loft, S. (2003) DNA adduct formation and oxidative stress in colon and liver of Big Blue rats after dietary exposure to diesel particles. Carcinogenesis, in press.

TX034103H