502
Chem. Res. Toxicol. 2004, 17, 502-511
Characterization and Quantitative Analysis of DNA Adducts Formed from Lower Chlorinated PCB-Derived Quinones Shouxun Zhao,*,†,‡ Amarjit Narang,§ Xinxin Ding,†,§ and George Eadon†,§ Department of Environmental Health and Toxicology, State University of New York at Albany, Albany, New York 12201-0509, and Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, New York 12201-0509 Received November 25, 2003
Polychlorinated biphenyls are wide pollutants readily detected in environmental and human specimens. DNA adduction occurs through the corresponding quinones. Polychlorinated biphenyls are first metabolized to arene oxides, which can be further oxidized to dihydroxy metabolites by microsomal cytochrome P450s. The catechol and hydroquinone products are further oxidized by peroxidases to quinones, which are electrophilic and capable of reacting with DNA to form adducts. DNA adduction is initiated by Michael addition preferentially to guanosine followed by stabilization through enolization. Another nucleophilic attack forms a five-membered ring, which aromatizes by dehydration to form the final adduct. This report describes the characterization and quantitative study of DNA adducts formed from lower chlorinated PCB-derived quinones. Quantitative study by HPLC/ESI-MS/MS and 32P-postlabeling-HPLC gave the adduct levels in the range of 3-1200 adducts per 108 nucleotides. These results demonstrate that increasing chlorine substitution is associated with lower yields of DNA adduct. Although 32P-postlabeling is more sensitive than HPLC/ESI-MS/MS for the quantitative analysis of DNA adducts, modification levels were severely underestimated by the 32P-postlabeling assay as compared to the HPLC/ESI-MS/MS assay.
1. Introduction Polychlorinated biphenyls (PCBs) are halogenated aromatic hydrocarbons and consist of 209 theoretically possible congeners that differ in both number and position of the chlorines on the biphenyl moiety. They were first commercially manufactured in the 1930s. Because of their physical and chemical properties, such as stability and thermal characteristics, PCBs were widely used for many applications, such as heat transfer fluids in transformers and capacitors, as sealants in hydraulic fluids, as antioxidants in paints and in carbonless paper, and as organic diluters and plasticizers (1). The chemical properties that made PCBs attractive for industrial use are the same properties that cause pollution of the environment. PCBs are lipophilic and widely spread in the environment. They accumulate in wildlife and bioconcentrate in the food chain. Thus, exposure of humans to PCBs is basically due to the consumption of PCBcontaminated foodstuffs, including contaminated fish, dairy products, and animal flesh (1). PCBs display toxic effects including hepatotoxicity, neurotoxicity, immunotoxicity, carcinogenicity, and hormonal disruption (2). Evidence suggests that PCBs are hepatocarcinogens (2, 3). It was well-reported that lower chlorinated PCBs (three or fewer chlorine atoms) are first * To whom correspondence should be addressed. Tel: 212-241-7729. Fax: 212-996-9801. E-mail:
[email protected]. † State University of New York at Albany. ‡ Current address: Department of Medicine, Mount Sinai School of Medicine Box 1247, One Gustave Levy Place, New York, NY 10029. § New York State Department of Health.
metabolized to arene oxides, followed by cytochrome P450 enzyme-catalyzed hydroxylation to form monohydroxylated or bihydroxylated PCBs. When the hydroxyl groups are ortho or para to each other, oxidation to quinones may be catalyzed by peroxidases and/or prostaglandin H synthase in the cell. The resulting (semi)quinones frequently react with S- and/or N-centered nucleophiles (4-7) (Figure 1). The binding of radiolabeled PCBs to microsomal proteins, RNA, and DNA was previously attributed to arene oxide intermediates (8). Recent studies presented evidence showing that arene oxide metabolites were minor contributors to macromolecular binding. Instead, quinones or semiquinones are the ultimate reactive agents that bind to DNA (5, 9). Oakley et al. demonstrated that lower chlorinated biphenyls were metabolically activated to electrophilic quinonoid species in vitro. 32P-postlabeling showed that these PCB-derived quinones can bind to DNA with an apparent preference for guanine residues (9, 10). It was also found that PCBderived para-quinones rather than ortho-quinones are involved in the major DNA adduction (10). However, previous studies concerning DNA adduction with quinonoid PCB metabolites have not established the structures of these adducts due to the limitations of the 32Ppostlabeling technique. The detailed structural information for these DNA adducts is of significant importance in understanding DNA adduction mechanisms and PCB metabolic polymorphism. Identification and characterization of the chemical structures of these DNA adducts may also facilitate efforts to determine the effects of the degree and position of chlorination of PCBs on the DNA adduc-
10.1021/tx034245b CCC: $27.50 © 2004 American Chemical Society Published on Web 02/28/2004
DNA Adducts from PCB-Derived Quinones
Chem. Res. Toxicol., Vol. 17, No. 4, 2004 503
Figure 1. Proposed routes for the activation of lower chlorinated biphenyls via (semi)quinone intermediates. PCBs are first metabolized to arene oxides, followed by cytochrome P450-catalyzed hydroxylation to form monohydroxylated or bihydroxylated PCBs. These catechol and hydroquinone products can be further oxidized by peroxidases to quinones.
tion, which is of significant importance for understanding in vitro structure-activity relationships of PCB metabolites concerning adduction with DNA. The basic structural core of para-dihydroxy-PCB metabolites is represented by 2-phenyl-1,4-benzoquinone, a metabolite of the fungicide ortho-phenylphenol (11). Our previous study in characterizing DNA adducts formed from ortho-phenylphenol metabolite demonstrated that 2-phenyl-1,4benzoquinone can bind to nucleotides by the Michael addition mechanism (12). The reaction is initiated by nucleophilic attack of the exocyclic amine nitrogen, N2, of the deoxyguanosine on the electrophilic carbon from the quinone, followed by stabilization through enolization. Another nucleophilic attack forms a five-membered ring, followed by loss of a water molecule, thus forming an aromatic system that stabilizes the molecule. It was also found that guanosine is the preferred nucleoside for the DNA adduction because it formed adducts with 2-phenyl-1,4-benzoquinone in amounts at least 10 times of that for adenosine or cytidine (12). Formation of DNA adducts with quinones was reported to play a causative role in human carcinogenesis (13, 14). The aim of the present investigation was to determine the structures of DNA adducts formed from para-quinone-like PCB metabolites and to quantify these adducts by using HPLC/ ESI-MS/MS and 32P-postlabeling-HPLC assays.
2. Materials and Methods Caution: Phenyl-1,4-benzoquinone and chlorinated phenyl1,4-benzoquinones are highly toxic and should be handled carefully. 2.1. Chemicals. Aniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, 2,3-dichloro-aniline, 2,6-dichloroaniline, 3,4dichloroaniline, 1,4-benzoquinone, chloro-1,4-benzoquinone, sodium nitrite, anhydrous dimethyl sulfoxide (DMSO), tetrohydrofuran, KH2PO4, sodium acetate, hexane, toluene, acid alumina (80-200 mesh, Brockman Activity I), silica gel (5-25 µm), acetic acid, and formic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). Micrococcal nuclease, T4 polynucleotide
kinase, spleen phosphodiesterase, calf thymus DNA, nuclease P1, 2′-deoxynucleosides, and 2′-deoxynucleoside-3′-monophosphates were purchased from Sigma Chemical Co. (St. Louis, MO). [γ-32P]ATP with an original specific activity of ∼6000 Ci/ mmol was purchased from Perkin-Elmer Life Sciences (Boston, MA). 2.2. Synthesis of Chlorinated Phenyl-1,4-benzoquinones. 2.2.1. Synthesis of 2-(x′-Chlorophenyl)-1,4-benzoquinone. Chemical synthesis of chlorinated phenyl-1,4-benzoquinones was carried out in this study according to the method described by Bagli et al. (15). A 1.28 g amount of 4-chloroaniline (or 2-chloroaniline, 3-chloroaniline, or 2,3-dichloro-aniline, 2,6dichloroaniline, or 3,4-dichloroaniline) in 2.57 mL of concentrated HCl and 5 mL of H2O was cooled by ice water (
