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Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways; by peroxidases (e.g. cytochrome P450-peroxidase) to yield radical cations; by P4501A1/1B1 monoxygenation plus epoxide hydrolase to yield diol-epoxides; and by P4501A1/1B1 monoxygenation, epoxide hydrolase plus aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung, however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6- and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation (3-hydroxyl-B[a]P, B[a]P-7,8- and 9,10-trans-dihydrodiols, and B[a]P -r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P -tetrol-1)), and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC-with in line photo-diode array and radiometric detection, and identified by LC-MS. Progress curves showed a lag-phase in the formation of 3-hydroxy-B[a]P, B[a]P-7,8-trans-dihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione over 24 h. Northern blot analysis showed that B[a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner providing an explanation for the lag-phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin, (TCDD) eliminated this lag-phase, but did not alter the levels of the individual metabolites observed, suggesting that both B[a]P and TCDD induction ultimately yield the same B[a]P-metabolic profile. The one exception was B[a]P-3,6-dione which was formed without a lag-phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH-activation yield their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.
Lung cancer is the major cause of cancer death in the US adult population and 174,470 new cases are reported annually (1). Ninety percent of all lung cancer is seen in individuals who smoke suggesting that exposure to tobacco carcinogens are cuasative. Major classes of carcinogens present in cigarette smoke, include nicotine derived nitrosamino ketones, polycyclic aromatic hydrocarbons (PAH), aldehydes, butadiene, benzene and heavy metals (2-5). PAH are also ubiquitous environmental pollutants and thus PAH exposure may be a contributing factor to never-smokers with lung cancer (6-8). Remarkably, even though human lung epithelial cells are the target cells for cellular transformation, LC/MS/MS methods have not been applied to determine the metabolic fate of B[a]P, a representative PAH in these cells.
Three major routes of B[a]P metabolism are shown in Figure 1. In the presence of a peroxide, B[a]P can undergo one-electron oxidation at C6 by cytochrome P450 (P450) peroxidase or other peroxidases (e.g. PGH-synthase or lactoperoxidase) to generate a radical-cation (9-11). In the peroxidase cycle B[a]P acts as a co-reductant of the Fe4+-protoporphyrin IX radical cation (FeV+) and forms a radical cation itself at C6 (12). The activated carbon can accept oxygen from the Fe4+=O species to form 6-hydroxy-B[a]P which autooxidizes to yield B[a]P-1,6-, 3,6- or 6,12-diones (13). These quinones undergo one electron reduction by microsomal NADPH-cytochrome P450 reductase, microsomal NADH-cytochrome b5 reductase, or mitochondrial NADH:ubiquinone oxidoreductase (14) to yield semiquinone anion radicals which in air redox-cycle back to the diones, with the generation of ROS [peroxide (O22-), superoxide anion (O2·-) and hydroxyl radical (OH·)] (15).
B[a]P can also undergo mono-oxygenation catalyzed by the microsomal NADPH-dependent P450 isoforms (1A1 and 1B1), to yield a series of arene oxides, which can either rearrange to yield 3, 7, or 9-hydroxy-B[a]P or be hydrated by microsomal epoxide hydrolase (EH) to yield the corresponding B[a]P-7,8- or 9,10-trans-dihydrodiols. The non-K-region B[a]P-7,8-dihydrodiol is further mono-oxygenated by P4501A1/1B1 to yield the reactive anti-B[a]P-7,8-diol-9,10-epoxide (anti-B[a]PDE) (16,17) which is a rodent lung carcinogen. Formation of the diol-epoxide thus requires the combined action of P450 isoforms and EH.
B[a]P -7,8-dihydrodiol is also oxidized by human aldo-keto reductases (AKR1A1, 1C1-1C4) to form a ketol which spontaneously rearranges to form a catechol (18-21). The catechol is unstable and undergoes a one-electron autooxidation in air to form an o-semiquinone anion radical followed by a second one electron autooxidation to generate a reactive Michael acceptor, B[a]P-7,8-dione and ROS (22). B[a]P-7,8-dione can be reduced enzymatically or non-enzymatically back to the catechol establishing futile redox-cycles, that result in the generation and amplification of ROS until cellular reducing equivalents are depleted (14,23). Formation of B[a]P-7,8-dione thus requires the combined action of P450 isoforms, EH and AKRs.
Generation of B[a]P-radical-cations, B[a]P-diol-epoxides and B[a]P-o-quinones and ROS could each play a role in human lung carcinogenesis. For example, B[a]P-radical-cations generate depurinating adducts, including B[a]P-6-C8-Gua, B[a]P-6-N7-Gua and B[a]P-6-N7-Ade (10,11). Metabolites derived from radical cations, B[a]P-1,6- and B[a]P-3,6-dione also activate the epidermal growth factor receptor (EGFR) via the generation of hydrogen peroxide, leading to increased MCF10A cell proliferation (24) suggesting a potential role in tumor promotion.
anti-B[a]PDE is mutagenic in bacterial and mammalian cell assays (25) and causes pulmonary adenomas in mice (26). These properties result from the formation of stable bulky DNA adducts with 2′-deoxyguanosine (dGuo) (27,28). Furthermore, anti-B[a]PDE activates the c-H-ras-1 proto-oncogene to transform NIH/3T3 cells, which can be assigned to a single point mutation in the 12th codon of ras (G to T transversion) (29). Reaction of anti-B[a]PDE with the p53 tumor suppressor gene leads to adduct formation in codons 157, 158, 179, 248 and 273 (30). Mutations in these codons inactivate p53 and correspond to “hot-spots” most mutated in p53 in lung cancer patients. Thus a compelling case can be made for the role of anti-B[a]PDE in lung cancer causation.
The reactive and redox-active B[a]P-7,8-dione formed by AKRs can form stable hydrated N2-dGuo and N6-dAdo adducts as well as depurinating N7-Gua adducts (31-34). N7-Gua depurinating adducts have the potential to give rise to the G to T transversions observed in the H-ras gene. B[a]P-7,8-dione will also mutate the tumor suppressor gene p53 in vitro and in A549 human lung adenocarcinoma cells (35,36). In vitro, PAH o-quinones were only mutagenic under redox-cycling conditions. Mutagenicity was abolished with ROS scavengers, and the primary lesion detected was 8-oxo-dGuo (35,37,38). The point mutations most often seen were G to T transversions implicating 8-oxo-dGuo as the adduct responsible. Which of these three pathways of PAH activation dominate in human bronchial epithelial cells is unknown.
The individual peroxidases that form radical cations have not been assigned but it is anticipated that they will be P450 isoforms. In humans P450s most implicated in PAH activation are 1A1, 1B1 (39,40) and the AKRs most involved are (1A1, 1C1-1C4) (20,21,41). These enzymes are differentially expressed and regulated. P4501A1/1B1 are extra-hepatic enzymes that are inducible by PAHs and polyhalogenated hydrocarbons (TCDD, etc), via the aryl hydrocarbon receptor (AhR) (42-44). Of the five human AKR isoforms (1A1, 1C1-1C4) most involved in the metabolic activation of PAH trans-dihydrodiols none are directly induced by the AhR. Instead induction is achieved by electrophilic metabolites of B[a]P that signal to the antioxidant response element (ARE) (18,45). AKR1C1/2 are overexpressed in non-small cell lung carcinoma and may serve as indicators of poor prognosis (46). We showed that human lung adenocarcinoma A549 cells have high endogenous expression of AKR1C1-1C3 and convert PAH trans-dihydrodiols to PAH o-quinones (21). The competing roles of P4501A1/1B1 and AKR1A1 in the metabolic activation of B[a]P-7,8-dihydrodiol has been examined in H358 cells manipulated to express either enzyme system. We found that AKR1A1 had a dual effect: it oxidized (-)-B[a]P-7,8-dihydrodiol to B[a]P-7,8-dione, but B[a]P-7,8-dione also acted as a ligand for the AhR leading to the induction of the P4501B1 gene (47). Thus, the P450 pathway generates metabolites that induce the AKR pathway and vice versa.
B[a]P metabolism has been studied in monolayer cultures of human bronchial epithelial cells (48), primary cultures of human hepatocytes (49), mammary tumor cells (50), liver microsomes and lymphocytes (51). However, these studies were performed without an appreciation for all the participating pathways, and often without the use of appropriate analytical chemistry to identify all the metabolites. We now use human bronchoalveolar H358 cells as a model to study the metabolic profile of B[a]P in human lung epithelial cells. Using LC-MS based methodology we provide evidence for the presence of all three pathways of PAH activation in these cells. While the P4501A1/1B1 and AKR enzymes are inducible, peroxidase derived B[a]P metabolites were observed without enzyme induction. The identity of the peroxidase involved remains elusive.
All PAHs are potentially hazardous and should be handled in accordance with NIH guidelines for the Use of Chemical Carcinogens.
Cell culture medium and reagents were all obtained from Invitrogen Co. (Carlsbad, CA) except fetal bovine serum from (FBS) Hyclone (Logan, Utah). [G-3H]-B[a]P (specific activity 82.0 Ci/mmol, ≥ 98% pure by HPLC) was purchased from Amersham Biosciences UK Limited (Buckinghamshire, UK). The non-radiolabeled B[a]P metabolite standards and TCDD were obtained from either the NCI Chemical Carcinogen Standard Reference Repository (Midwest Research Institute, Kansas City, Missouri) or they were synthesized according to published methods (52). The purity and identity of all B[a]P metabolites was established by LC-MS. All solvents were HPLC grade, and all other chemicals used were of the highest grade available.
Transformed human bronchoalveolar H358 cells (type II, epithelial origin) were obtained from the American Type Culture Collection (ATCC #CRL-5807) and maintained in RPMI 1640 nutrient mixture with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and were passaged every 3 days at a 1:3 dilution. Cultured cells with passage number 10 to 30 were used in the experiments to reduce variability during cell culture.
Induction experiments with TCDD (10 nM) or ethacrynic acid (EA) (70 μM), were performed in 10-mL RPMI/FBS medium for 15 h followed by [3H]-B[a]P treatment (4 μM, 2 × 106 cpm/nmol, 0.5% DMSO) in 2-mL HBSS. Before harvesting the treated cells, 0.5 nmol of B[a]P-4,5-diol was added to cell culture dishes as an internal standard to normalize for losses in the sample work-up and analysis. The total cell culture mixture was harvested by a cell scraper into a 10-mL tube and extracted twice with 3-mL water-saturated ethyl acetate. After centrifugation at 2500 × g for 10 min the organic and aqueous extracts were analyzed by scintillation counting to determine the metabolite distribution in the two phases. The organic extract was dried under vacuo. The residue was re-dissolved in 150-μL methanol and a 60-μL aliquot was subjected to chromatographic analysis. Quantitation of B[a]P metabolites generated in the cells was accomplished using a tandem Waters Alliance 2695 chromatographic system (Waters Corp., Milford, MA) with a Waters 996 photodiode array (PDA) detector and a β-RAM in-line radioactive detector (IN/US Systems Inc., Tampa, FL) as previously published (47). Chromatography was conducted on a reversed-phased (RP) column (Zorbax-ODS C18, 5 μm, 4.6 mm × 250 mm, DuPont Co., Wilmington, DE) at a flow-rate of 0.5 mL/min using the following linear gradient: MeOH/H2O (v/v) 55-70% (20 min), 70-80% (10 min), 80% (20 min), 80-95% (10 min), and 95% (20 min). Eluates from the column were introduced into the online radiometric detector following mixture of the scintillant with the HPLC effluent (IN/US system Inc.) at a flow rate 1.5 mL/min. The IN/US detector was calibrated by injecting know amounts of B[a]P determined by scintillation counting directly into the detector to determine counting efficiency (59.5%). The counting efficiency of the detector was unaffected by the composition of the mobile-phase. The detector was then programmed to correct the output signal to a cpm value. A macro was written to covert corrected cpm to dpm, which were then converted to nmoles using the specific radioactivity of the isotope.
The cells (~ 2 × 107) were treated with non-radiolabelled B[a]P (final 4 μM, 0.5% DMSO) in 2-mL HBSS for 12 h. The culture mixtures were extracted with two vols of ethyl acetate and the organic extract was dried under vacuo. The residue was re-dissolved with 50 μL methanol and an 80-μL aliquot of the pooled solution was subjected to LC-MS analysis. Mass spectrometric data were acquired using a Finnigan TSQ Quantum Ultra spectrometer (Thermo Fisher, San Jose, CA) equipped with an atmospheric pressure chemical ionization (APCI) source. The mass spectrometer was operated in the positive ion mode. On-line chromatography was performed using a Waters Alliance 2690 HPLC system (Waters Corp., Milford, MA). A RP column (Zorbax-ODS C18, 5 μm, 4.6 mm × 250 mm, DuPont Co., Wilmington, DE) was used at a flow-rate of 0.5 mL/min. Solvent A was 5 mM ammonium acetate in water containing 0.02% formic acid, and solvent B was 5 mM ammonium acetate in methanol containing 0.02% formic acid. Chromatography was conducted using the following linear gradient: 55% to 70% methanol (v/v) over 20 min, 70% to 80% methanol (v/v) for 10 min, 80% methanol (v/v) for 20 min, and 80% to 95% methanol (v/v) for 10 min. The eluant on-line was monitored by the mass spectrometer using selected reaction monitoring (SRM) and Q3 full scan modes. The mass spectrometry parameters including discharge current (30 μA), vaporization temperature (470 °C), Sheath Gas (0.525 L/min), ion sweep gas (0.3 L/min), Auxiliary gas (1.5 L/min), capillary temperature (190 °C), tube lens offset (232 V), source CID (-5 V), scan time (0.5 sec) and scan width (0.5 m/z) were automatically optimized with authentic standard compound solutions. SRM transitions for the metabolites were m/z 269 → m/z 251 for B[a]P-dihydrodiols (7,8- and 9,10-dihydrodiol), m/z 269 → m/z 251 for 3-OH-B[a]P, m/z 303 → m/z 285 for B[a]P-tetraol-1, m/z 283 → m/z 255 for B[a]P-quinones (7,8-, 1,6-, and 3,6-dione), and m/z 253 for B[a]P. The corresponding mass spectrum of each metabolite was obtained from Q3 Full Scan. Identification of the metabolite peaks was achieved by comparing chromatographic retention time and mass spectra of the metabolites with those obtained for the authentic synthetic standards.
Aliquots of the remaining aqueous phase were processed in four steps. First, the media was neutralized and extracted with ethyl-acetate. Second, the media was neutralized and treated with β-glucuronidase 50 units/mL (E. coli Type VIII, Sigma) and the released metabolites extracted with ethyl actetate. Third, the media was adjusted to pH 5.0 and extracted with ethyl acetate, and fourth the media was adjusted to pH 5.0, treated with aryl sulfatase 5 units/mL (Abalone entrails, Type VIII β-glucuronidase free Sigma) and the released metabolites extracted with ethyl acetate. No radioactivity was released by these methods. The aqueous metabolites were also analyzed by ion-pair RP-HPLC using a linear gradient of 2 - 60% acetonitrile containing 0.1% acetic acid.
Cellular RNA was isolated from 2 × 107 cells using Trizol reagent following either treatment with either 4 μM B[a]P for 0, 1, 2, 3, 6, 12, 24 h or treatment with 70 μM EA for 15 h in HBSS. Total RNA (30 μg) was separated by electrophoresis on 1% agarose/formaldehyde gels and transferred overnight to the Hybond-N+ nylon membrane (Amersham Biosciences UK Ltd., Little Chalfont, Bucks). The membrane was hybridized to a cDNA probe containing a fragment of the open reading frame of either AKR1C1 (GenBank accession number: NM_001353, +251 to +972 bp) or human P4501B1 (GenBank accession number: NM_000104, +385 to +1584 bp). Random priming was conducted with radiolabeled [32P]-α-dCTP, and a final specific activity greater than 109 cpm/μg of DNA fragment was obtained. Hybridization was performed in ExpressHyb hybridization solution (ClonTech, Palo Alto, CA) at 65 °C for 1 h, and the blot was subjected to a wash with 2 × SSC/0.1% SDS for 10 min at room temperature, and a wash with 0.2 × SSC/0.1% SDS for 10 min at 60 °C. The blot was then exposed to X-ray film at -80 °C overnight. To confirm equally loading of each RNA sample, 28S and 18S rRNA on agarose/formaldehyde gels were visualized by ethidium bromide under a UV transilluminator at 300 nm and photographed.
To identify B[a]P metabolites generated in H358 cells we first exposed parental naïve cells to 4 μM B[a]P. Two analytical methods were utilized RP-HPLC-with in line PDA and radiometric detection, or LC-atmospheric chemical ionization (APCI)/MS. The first method, which utilized [3H]-B[a]P provided an assessment of the complete metabolic profile and mass balance and permitted the detection of both known and unknown metabolites. The second method utilized unlabeled B[a]P and provided rigorous structural identity of the known metabolites. In the two methods the major B[a]P metabolites were assigned according to the retention time of the chromatographic peaks and/or by comparison of mass spectra to those obtained with the corresponding authentic synthetic standards. Eight significant metabolite peaks in the radio-chromatogram (Figure 2A,B) were assigned as B[a]P-tetraol-1 (M1, tr = 15.9 min), B[a]P-9,10-dihydrodiol (M2, tr = 20.7 min), B[a]P-7,8-dihydrodiol (M3, tr = 35.0 min), B[a]P-7,8-dione (M4, tr = 40.4 min), B[a]P-1,6-dione (M5, tr = 45.1 min), B[a]P-3,6-dione (M6, tr = 47.1 min), 3-OH-B[a]P (M7, tr = 59.2 min), and B[a]P (M8, tr = 78.0 min) based on their LC-UV-chromatogram (348 nm) (Figure 2A, B) and co-elution with authentic standard compounds. The metabolite, which eluted at tr = 5.9 min was not identified and was regarded as a polar phase II conjugate due to its poor retention on the column.
Relative quantities of the metabolites were determined using the specific radioactivity of the [3H]-B[a]P starting material, Figure 3A. 3-OH-B[a]P and B[a]P-dihydrodiols were the most prominent primary metabolites followed by the secondary metabolites: B[a]P-tetraol-1, B[a]P-7,8-dione and B[a]P-1,6-/3,6-diones. Over 80% of B[a]P was consumed during a 24 h incubation, Figure 3B. Radioactivity in organic extracts markedly decreased to about 40% of the total and a concomitant increase in radioactivity in the aqueous phase was noted, suggesting that B[a]P was metabolically converted to water-soluble metabolites. Attempts were made to identify the aqueous soluble metabolites by treatment with either β-glucruonidase or aryl sulfatase but these treatments failed to release radioactivity for extraction. Ion-pair RP-HPLC of the aqueous phase showed at least six minor radioactive peaks. LC/MS/MS in selected ion scan and precursor ion scan modes failed to detect transitions that could be assigned to either a glucuronide or sulfate conjugate suggesting that they are either glutathionyl or mercapturic acid conjugates. At the end of the incubation 20% of radioactivity was retained in the cells and may be present as macromolecule adducts, Figure 3C.
Replicate experiments were then performed with unlabeled B[a]P and the M1 to M8 metabolites were identified using LC-MS by comparison with SRM chromatograms (Figure 4A and 4B) and mass spectra (see, Figure 4C and Supplemental Material, Figure S-1) obtained form authentic standards. B[a]P-tetraol-1 and B[a]P-dihydrodiols (B[a]P-7,8-dihydrodiol and B[a]P-9,10-dihydrodiol) formed [M+H-H2O]+ parent ions and product ions [M+H-2H2O]+ which showed an additional loss of a H2O molecule. 3-OH-B[a]P generated the [M+H]+ parent ion which lost a molecule of water when subjected to collision induced dissociation (CID). B[a]P-diones (B[a]P-7,8-, 1,6-, and 3,6-dione) formed [M+H]+ parent ions and [M+H-CO]+ product ions when subjected to CID. B[a]P generated [M+H]+ that did not fragment. Therefore, eight metabolites were detected under the following SRM transitions: m/z 303 [M+H-H2O]+ → m/z 285 [M+H-2H2O]+ for B[a]P-tetraol-1; m/z 269 [M+H-H2O]+ → m/z 251 [M+H-2H2O]+ for B[a]P-dihydrodiols (B[a]P-7,8- and 9,10-dihydrodiol); m/z 283 [M+H]+ → m/z 255 [M+H-CO]+ for B[a]P-quinones (B[a]P-7,8-, 1,6-, and 3,6-dione); m/z 269 [M+H]+ → m/z 251 [M+H-H2O]+ for 3-OH-B[a]P; and m/z 253 [M+H]+ for B[a]P.
To verify that H358 cells have inducible P4501B1 and AKR1C1, Northern blotting analysis was performed to detect induction of P4501B1 and/or AKR1C1 by TCDD (an AhR agonist), by B[a]P (a bifunctional inducer) and by EA (a monofunctional inducer).
The results showed that P4501A1/1B1, AKR1A1 and AKR1C1 were not constitutively expressed in parental cells, however, P4501B1 and AKR1C1 were significantly upregulated by TCDD (10 nM, 15 h) and EA (70 μM, 15 h), respectively, (see Supplemental Material, Figure S-2). B[a]P induced both P4501B1 and AKR1C1 expression in a time-dependent manner, suggesting that chronic exposure to B[a]P stimulates its own metabolism through both the diol-epoxide and o-quinone pathways, Figure 5. A lag-phase was observed between the induction of P4501B1 and the induction of AKR1C1 by B[a]P and is consistent with the need to metabolize B[a]P to an electrophilic metabolite that will then activate the Keap-1/Nrf2 pathway to stimulate the ARE in the AKR1C gene promoter (18). The AKR1C cDNA probe utilized cannot distinguish between AKR1C1-AKR1C3 since they share greater than 86% sequence identity. However, the AKR1C isoform most induced by an ARE in HepG2 cells is AKR1C1 (18).
Ideally we would prefer to measure B[a]P-metabolism following chronic exposure to this PAH to induce the metabolic pathways. However, we were concerned that residual B[a]P would compromise the metabolic profiles and LC-MS analysis. Therefore we elected to measure B[a]P-metabolism in the cells following prior exposure to TCDD, Figure 6.
In un-induced cells, B[a]P-metabolism was characterized by the formation of 3-OH-B[a]P, B[a]P-7,8-dihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione, that reached a maximum after 12 h. The appearance of each of these metabolites was accompanied by a significant lag-phase consistent with enzyme induction. The one exception was B[a]P-3,6-dione, whose formation was immediate. Importantly, the formation of B[a]P-7,8-dihydrodiol preceded the formation of B[a]P-tetraol-1 and B[a]P-7,8-dione providing evidence for a precursor-product relationship. After the 12 h time point there was a significant decline in B[a]P-7,8-dihydrodiol, whereas the levels of the B[a]P-tetraol-1 and B[a]P-7,8-dione peaks remained unaltered. This suggests that B[a]P-7,8-diol was likely conjugated by phase II enzymes. Induction by TCDD led to the elimination of the lag-phases observed for the formation of B[a]P-7,8-dihydrodiol, 3-OH-B[a]P, B[a]P-tetraol-1 and B[a]P-7,8-dione. The exception was B[a]P-3,6-dione since no lag-phase was seen in its formation to begin with. Time courses in the TCDD treated cells showed that the formation of B[a]P-7,8-dihydrodiol still preceded the formation of B[a]P-tetraol-1 and B[a]P-7,8-dione demonstrating that the precursor-product relationship was retained. Interestingly, induction with TCDD did not result in elevated levels of B[a]P-tetraol-1, B[a]P-7,8-dione or 3-OH-B[a]P; the appearance of the metabolites just occurred at a faster rate.
PAH are a major class of chemical carcinogen found in tobacco smoke and fine particulate matter; and thus human lung epithelial cells are a major site of inhalation exposure. However, little information exists concerning the metabolic activation and fate of PAH in human lung cells. In this study, we used parental H358 cells (transformed bronchoalveolar cells of epithelial orgin) to study B[a]P-metabolism by RP-HPLC with in line photo-diode array and radiometric detection and by LC/APCI/MS. Three pathways of metabolic activation have been proposed to exist, the peroxidase pathway (which forms radical cations), the P4501A1/1B1 plus EH pathway (which forms diol-epoxides), and the P501A1/1B1, EH plus AKR pathway (which forms o-quinones). Each pathway can give rise to distinctive metabolites, e.g. B[a]P-1,6-dione and B[a]P-3,6-dione (peroxidase), B[a]P-tetraols (P4501A1/1B1 plus EH) and B[a]P-7,8-dione (P4501A1/1B1, EH plus AKR). Evidence for the formation of all three metabolites were obtained, suggesting that each metabolic pathway exists in human lung cells and may contribute to lung carcinogenesis. The formation of B[a]P-1,6-dione and B[a]P-7,8-dione suggest that the lung can produce two different redox-active metabolites capable of producing ROS, and ROS-derived oxidative DNA damage (that can potentially result in mutations) upon PAH exposure.
Induction with TCDD and its effects on metabolic profiles was revealing. In the absence and presence of TCDD a precursor-product relationship existed between B[a]P-7,8-dihydrodiol and the appearance of B[a]P-tetraol-1 and B[a]P-7,8-dione suggesting that the diol was the precursor of both these metabolites. Importantly, the levels of B[a]P-tetraol-1 and B[a]P-7,8-dione formed were unaltered by TCDD induction, instead their formation just occurred earlier. This suggests that TCDD (P450 inducer) and B[a]P (a P450 and AKR1C1 inducer) ultimately yield the same B[a]P-metabolic profile. B[a]P is a bifunctional inducer that works through the XRE and ARE, whereas TCDD works only through the XRE (18). However, exposure of TCDD induced cells to B[a]P will likely produce the requisite electrophilic metabolite responsible for AKR induction, and hence the metabolic profiles are ultimately similar.
A surprise in the induction experiments were the data obtained for the radical-cation metabolite B[a]P-3,6-dione. This metabolite formed immediately in the absence or presence of TCDD and suggests that the B[a]P-3,6-dione is not derived from 3-OH-B[a]P since the formation of this latter metabolite required enzyme induction. These data also suggest that the peroxidase required for B[a]P-3,6-dione formation is neither P4501A1 nor P4501B1 since these P450 isoforms required induction. Thus the peroxidase responsible for the formation of B[a]P-3,6-dione remains unidentified. Previously, incubation of B[a]P with arachidonic acid and ram seminal vesicles resulted in the formation B[a]P-1,,6-, 3,6-, and 6,12-diones on the same time course as prostaglandin (PG)H2 production suggesting that B[a]P acts as a co-reductant in the peroxidase cycle of PGH synthase (53). Other peroxidases implicated in the formation of these diones are lactoperoxidase (an airway peroxidase) (54).
Two of the organic soluble metabolites, B[a]P-7,8-dihydrodiol and 3-OH-B[a]P showed a dramatic decline in levels after reaching a peak suggesting that they were forming water soluble conjugates. The presence of water soluble conjugates is notable in the mass balance since up to 40% of B[a]P becomes aqueous soluble. Treatment of the aqueous phase with either β-glucuronidase or sulfatase failed to liberate radioactivity that was organic extractable suggesting the presence of other water-soluble conjugates e.g. GSH or mercapturic acid metabolites
Knowledge of the metabolic profiles described in the present study has two major utilities. First, they can identify the major routes of PAH activation in lung and second they can be used to identify the most valuable biomarkers for PAH inhalation exposure in terms of detecting urinary and plasma metabolites. With regard to PAH activation, we have shown that when parental H358 cells are exposed to B[a]P the formation of (+)-trans-anti-B[a]PDE-N2-dGuo adducts occurred without a lag-phase. Moreover, more (+)-trans-anti-B[a]PDE-N2-dGuo adducts were formed in parental cells treated with (-)-B[a]P-7,8-dihydrodiol than in cells treated with TCDD (55). Thus, while diol-epoxides are the source of these adducts, and B[a]P-tetraol-1 levels may reflect anti-B[a]PDE formation, the enzyme system responsible for diol-epoxide-DNA adduct formation may not be P4501A1/1B1. This view was supported by the inability of 2,4,3′,5′-tetramethylstilbene, a P4501A1/1B1 inhibitor, to block anti-B[a]PDE adduct formation (unpublished data Gelhaus and Blair). Other P450’s that are expressed in human lung cells include, 2A6, 2A13, 2B6, 2E1, 2C18, 2F1, 2J2, 2S1, 3A5 and 4B1 (56-60). Of these, P450 3A5 has been shown to catalyze the metabolism of (-)-B[a]P-7,8-dihydrodiol to form (+)-anti-B[a]PDE (61). The rapid formation of anti-B[a]PDE in the TCDD treated cells may instead lead to improved detoxication rather than DNA-adduct formation. We have suggested that induction of P4501A1/1B1 may provide a protective mechanism against anti-B[a]PDE-N2-dGuo adduct formation (55).
The most widespread biomarker of PAH exposure has been 1-hydroxypyrene, since pyrene is present in many PAH mixtures (62,63). However, pyrene is not carcinogenic by itself. Since B[a]P is now considered by IARC as a human carcinogen it would be superior to have a biomarker of response based on its exposure (64). The abundance of 3-OH-B[a]P in our studies suggest that this metabolite may be a superior biomarker of exposure to carcinogenic PAH than 1-hydroxypyrene. Several studies have been performed to validate this metabolite as a biomarker. The largest single problem has been one of sensitivity, since levels of 3-OH-B[a]P are 103 to 105 less than that seen for 1-hydroxypyrene (63). Attempts to solve this problem have utilized LC-APCI/MS methodology with some success (62). The remaining issue is that 3-OH-B[a]P does not reflect levels of B[a]P metabolites that would have carcinogenic properties. By contrast, the formation of metabolites that come from electrophiles that are able to form lesions with DNA, e.g. B[a]P-tetraol-1 (anti-B[a]PDE), B[a]P-3,6-dione (radical cation) and B[a]P-7,8-dione (o-quinone) suggest that levels of these metabolites might be used as superior biomarkers of PAH exposure and response. Progress in detecting and quantitating B[a]P-tetraol-1 in human urine by GC/negative-ion (NI)CI/MS has been reported (65). This method gave fmol sensitivity and could detect B[a]P exposures in psorarsis patients receiving coal-tar treatments, steel workers, an smokers. Lowest levels were observed in smokers, and in some smokers B[a]P-tetraol-1 was not detected suggesting that increased sensitivity may be desirable.
In summary, we have conducted studies on the metabolism of B[a]P in human lung cells using LC/APCI/MS methodologies. We provide evidence for the existence of three pathways of B[a]P activation which include the formation of radical cations, diol-epoxides, and o-quinones. Measurement of the respective organic soluble metabolites from each of these pathways suggest that they are relatively minor with respect to the formation of 3-hydroxy-B[a]P. The contributions of each of these pathways to B[a]P-activation based on the relative abundance of their respective DNA-adducts in lung cells remains to be addressed.
This work was supported by grants R01 CA39504 and P01 CA92537 awarded to T.M.P. and by P30 ES013508-01. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS and NIH.