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Aldo-Keto Reductase- and Cytochrome P450-Dependent Formation of Benzo[a]pyrene-Derived DNA Adducts in Human Bronchoalveolar Cells Qian Ruan,† Stacy L. Gelhaus,† Trevor M. Penning,† Ronald G. Harvey,‡ and Ian A. Blair*,† Centers for Cancer Pharmacology and Excellence in EnVironmental Toxicology, UniVersity of PennsylVania School of Medicine, 854 BRB II/III, 421 Curie BouleVard, Philadelphia, PennsylVania 19104-6160, and The Ben May Institute for Cancer Research, The UniVersity of Chicago, Chicago, Illinois 60637 ReceiVed August 6, 2006
There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) induce lung cancer through metabolic activation. As part of a program to delineate the routes of PAH activation, we have examined DNA adducts that are formed in human lung cells. A stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry method was used to quantify eight anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE)-derived DNA adducts in four H358 human bronchoalveolar cell lines with different phenotypes. In P450 1A1/P450 1B1-induced H358 cells exposed to (()-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), (+)-antitrans-B[a]PDE-N2-2′-deoxyguanosine [(+)-anti-trans-B[a]PDE-N2-dGuo] was the major DNA adduct, and it formed with no lag phase. In AKR1A1-transfected H358 cells, (+)-anti-trans-B[a]PDE-N2-dGuo was also the major adduct with a 3 h lag phase before significant adduct formation was detected. In AKR1A1-transfected H358 cells with induced P450 1A1/P450 1B1, (+)-anti-trans-B[a]PDE-N2-dGuo was formed with no lag phase in amounts similar to those in the H358 cells with up-regulated P450 1A1/P450 1B1. Surprisingly, the greatest amount of (+)-anti-trans-B[a]PDE-N2-dGuo was formed in the control H358 cells. Furthermore, (+)-anti-trans-B[a]PDE-N2-dGuo formation was 2-fold higher in (-)-B[a]P-7,8-dihydrodiol-exposed H358 cells when compared with (()-B[a]P-7,8-dihydrodiol-exposed cells. The P450 1A1/1B1 inhibitor 2,4,3′,5′-tetramethoxystilbene did not attenuate DNA adduct formation in the control H358 cells, suggesting that another P450 was responsible. These data raise the intriguing possibility that P450 1A1/P450 1B1 and AKR1A1 may be protective against (+)-B[a]PDE-mediated DNA damage. Introduction Polycyclic aromatic hydrocarbons (PAHs)1 are ubiquitous environmental pollutants found in car exhaust, charbroiled food, and tobacco smoke (1-5). There is substantial evidence that PAHs are causative agents in lung (6, 7), skin (8, 9), and bladder (8, 9) cancer. PAHs require metabolic activation to their ultimate carcinogens through one or more of the three pathways shown in Scheme 1 for benzo[a]pyrene (B[a]P), a major PAH found in tobacco smoke (1). The most widely accepted pathway of activation involves cytochrome P450 (P450)-mediated formation of B[a]P-7,8oxide, which undergoes epoxide hydrolase-mediated hydrolysis to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol (B[a]P7,8-dihydrodiol) (Scheme 1) (1, 10, 11). P450 1A1 and 1B1 * To whom correspondence should be addressed. Phone: (215) 5739880. Fax: (215) 573-9889. E-mail:
[email protected]. † University of Pennsylvania School of Medicine. ‡ The University of Chicago. 1 Abbreviations: AhR, aryl hydrocarbon receptor; AKR, aldo-keto reductase; B[a]P, benzo[a]pyrene; B[a]P-7,8-dihydrodiol, 7,8-dihydroxy7,8-dihydrobenzo[a]pyrene; B[a]P-7,8-catechol, 7,8-dihydroxybenzo[a]pyrene; B[a]P-7,8-dione, 7,8-dioxobenzo[a]pyrene; B[a]P-7,8-oxide, 7,8epoxy-7,8-dihydrobenzo[a]pyrene; B[a]PDE, 7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; dGuo, 2′-deoxyguanosine; dAdo, 2′deoxyadenosine; P450, cytochrome P450; LC/MS, liquid chromatography/ mass spectrometry; MS, mass spectrometry; MS/MS, tandem MS; MRM, multiple reaction monitoring; PAH, polycyclic aromatic hydrocarbon; SAP, shrimp alkaline phosphatase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TIC, total ion current; TMS, 2,4,3′,5′-tetramethoxystilbene.
are considered to be the major P450’s involved in the activation of B[a]P (12, 13), although other isoforms including P450 1A2 are able to metabolize B[a]P (14, 15). Further activation of B[a]P-7,8-dihydrodiol occurs through P450 1A1- or 1B1mediated metabolism to form 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen (14, 15). It has also been demonstrated that B[a]PDE can arise through co-oxygenation of B[a]P-7,8-dihydrodiol during prostaglandin biosynthesis (16) or through lipid peroxyl radicalmediated epoxidation (17). B[a]PDE is detoxified by hydrolysis to the corresponding tetrols (1, 18) and by glutathione-Stransferase-mediated conversion to GSH adducts (1, 19, 20). B[a]PDE that escapes these detoxication reactions is capable of crossing the nuclear membrane and reacting with DNA to form a number of diastereomeric 2′-deoxyguanosine (dGuo) and 2′-deoxyadenosine (dAdo) adducts (Scheme 2) (1, 10, 11). A second pathway of metabolic activation involves aldoketo reductase (AKR)-mediated oxidation of B[a]P-7,8-dihydrodiol to a ketol, which rearranges to an air-sensitive catechol (7,8-dihydroxybenzo[a]pyrene, B[a]P-7,8-catechol) (19, 21, 22). AKR1A1 shows stereochemical preference for oxidation of (-)B[a]P-7,8-dihydrodiol (23), the major enantiomer formed by P450-mediated metabolism of B[a]P in rat liver microsomes (24), while AKR1C1-4 will oxidize both the (+)- and (-)stereoisomers of the dihydrodiol (25). B[a]P-7,8-catechol undergoes two sequential one-electron autoxidation reactions to form the quinone B[a]P-7,8-dione, which can potentially
10.1021/tx060180b CCC: $37.00 © 2007 American Chemical Society Published on Web 02/13/2007
B[a]P-DeriVed DNA Adducts in BronchoalVeolar Cells
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Scheme 1. Metabolic Pathways of B[a]P
Scheme 2. Stereochemistry of B[a]PDE-N2-dGuo and B[a]PDE-N6-dAdo Adducts Formed from (()-anti-B[a]PDE and (()-syn-B[a]PDE
cause covalent modifications to DNA (Scheme 1) (26-28). In the presence of cellular reducing equivalents, futile redox cycling can produce reactive oxygen species (ROS), such as hydroxyl radicals, that cause oxidative DNA damage (29, 30). A third proposed pathway of B[a]P activation involves the intermediate formation of a radical cation through the action of P450 peroxidase and other peroxidases (31-33). If the B[a]P radical cation traverses the nuclear membrane, it will react at C-8 of dGuo and N-7 of dGuo and dAdo in DNA. A one-electron oxidation of the modified DNA will result in the formation of depurinating dGuo and dAdo adducts (Scheme 1) (33). Two anti- and two syn-forms of B[a]PDE can arise from P450-mediated B[a]P metabolism through the intermediate
formation of (+)- and (-)-B[a]P-7,8-dihydrodiol (Scheme 2). All four B[a]PDE diastereomers are mutagenic in bacterial and mammalian cell assays (34, 35). However, (+)-anti-B[a]PDE is the most tumorigenic, causing pulmonary adenomas in mice (36-38). As part of a program to determine the relative importance of the P450- and AKR-mediated pathways of B[a]P metabolism (Scheme 1), we recently developed a method to specifically quantify all eight of the (()-anti-B[a]PDE-derived dGuo and dAdo adducts that are formed in naked and cellular DNA (Scheme 2) (39). This assay, which has specificity superior to that of 32P-postlabeling, was employed to quantify (()-antiB[a]PDE-mediated DNA adduct formation in bronchoalveolar H358 cells that were manipulated to express P4501A1/1B1 and/ or AKR1A1. H358 cells were exposed to (()-B[a]P-7,8-
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dihydrodiol, and the amount of each (()-anti-B[a]PDE-derived DNA adduct was quantified. The dGuo and dAdo adduct formation that occurred in the P450- and/or AKR-expressing cells was then compared with adduct formation from B[a]P, (()-B[a]P-7,8-dihydrodiol, (-)-B[a]P-7,8-dihydrodiol, and (+)B[a]P-7,8-dihydrodiol in the control H358 cells.
Materials and Methods Caution: PAHs are carcinogens and potentially tumorigenic to humans. These compounds should be handled with care (NIH Guideline for the Use of Chemical Carcinogens). Chemicals and Materials. Cell culture media and reagents were obtained from Invitrogen Co. (Carlsbad, CA). (()-B[a]PDE, (()B[a]P-7,8-dihydrodiol, (-)-B[a]P-7,8-dihydrodiol, (+)-B[a]P-7,8dihydrodiol, B[a]P, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were purchased from the NCI Chemical Carcinogen Standard Reference Repository (Midwest Research Institute, Kansas City, MO). Calf thymus DNA (ct-DNA), dAdo, dGuo, DNase, tetrahydrofuran (THF), trifluoroacetic acid (TFA), and triethylamine (TEA) were purchased from Sigma (St. Louis, MO). [15N5]dGuo and [15N5]dAdo were obtained from Spectra Stable Isotopes (Columbia, MD). 2,4,3′,5′-Tetramethoxystilbene (TMS) was purchased from Tocris Bioscience (Ellisville, MO). Nuclease P1 and shrimp alkaline phosphatase (SAP) were purchased from Roche (Germany). YMC ODS-AQ and Jupiter C18 HPLC columns were obtained from Waters (Milford, MA) and Phenomenex (Torrance, CA), respectively. A Costar spin-x nylon centrifuge tube filter was purchased from Corning Inc. (Corning, NY). HPLC grade water, methanol, acetonitrile, and ethanol were purchased from Optima, Fisher Scientific Co. (Fair Lawn, NJ). Human bronchoalveolar H358 cells were obtained from the American Type Culture Collection (ATCC No. CRL-5807). (+)-anti-trans-B[a]PDE-[15N5]-N2-dGuo and (+)anti-cis-B[a]PDE-[15N5]-N6-dAdo were synthesized as described previously (39). Construction of the Eukaryotic Expression Vector and Stable Transfection. H358 cells were transfected with AKR1A1 as described previously (19). Levels of constitutive expression of AKR1A1 were assessed spectrophotometrically by measurement of enzymatic activity. Protein expression levels were determined by Western blot analyses using polyclonal rabbit anti-human AKR1A1 antiserum (provided by Dr. John Hayes at the University of Dundee) at a 1:2000 dilution. The immunoblots were developed by incubation with the goat anti-rabbit IgG-horseradish peroxidase conjugate as a secondary antibody followed by ECL detection (Amersham Biosciences, Piscataway, NJ). Culture of Human Bronchoalveolar H358 Cells and AKR1A1Transfected Cells. Cells were maintained in an RPMI 1640 nutrient mixture with 10% heat-inactivated fetal bovine serum, 1% lglutamine, and 100 units/mL penicillin/streptomycin. Incubation of the cells was conducted at 37 °C in a humidified incubator containing 5% CO2, and the cells were passaged every 4 days at a 1:3 dilution. AKR1A1 transfectants were grown under the same conditions as the parental cell line, in the presence of 0.4 mg/mL G418-sulfate, an aminoglycoside antibiotic, to maintain selection. Transfected cells were also incubated and passaged according to the above procedure. Induction of P450 1A1/P450 1B1 by TCDD in H358 Cells and AKR1A1-Transfected Cells. P450 1A1 and P450 1B1 were induced by TCDD (confirmed by Northern and Western blot analysis) as described previously (19). In brief, when the cells were 80% confluent, H358 cells and AKR1A1-transfected cells were exposed to 10 nM TCDD (0.1% DMSO) for 6 h. Expression of P450 1A1/P450 1B1 and AKR1A1 was detected by Northern analysis and by functional assay. Northern analysis showed that P450 1B1 was the major monooxygenase induced. Functional assays, which monitored the O-deethylation of ethoxyresorufin, showed that the P450 specific activity in TCDD-exposed cells was 70 times greater than that observed in the noninduced cells. Following TCDD exposure, P450 mRNA levels remained elevated
Ruan et al. for 48 h. TCDD exposure had no effect on the expression or activity of AKR1A1 (data not shown). Formation of DNA Adducts in Human Bronchoalveolar H358 Cells and Isolation of DNA. When the cells reached 80% confluence (approximately 1 × 107 cells), the RPMI was removed and replaced with 10 mL of HBSS buffer containing 2 µM (()B[a]P-7,8-dihydrodiol, (-)-B[a]P-7,8-dihydrodiol, (+)-B[a]P-7,8dihydrodiol, or B[a]P with 400 µM β-cyclodextrin. B[a]P-7,8dihydrodiols and B[a]P were dissolved in DMSO (1 mM) so that the final concentration of DMSO in the culture medium was
