pyrene in Human Urine by Gas Chromatography ... - ACS Publications

Chem. Res. Toxicol. 2000, 13, 271-280

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Determination of r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene in Human Urine by Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry Christopher D. Simpson,† Ming-Tsang Wu,‡ David C. Christiani,‡ Regina M. Santella,§ Steven G. Carmella,† and Stephen S. Hecht*,† University of Minnesota Cancer Center, 420 Delaware Street SE, Minneapolis, Minnesota 55455, Harvard School of Public Health, Boston, Massachusetts 02115, and Mailman School of Public Health of Columbia University, New York, New York 10032 Received December 13, 1999

r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BaP-tetraol) is the major hydrolysis product of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the principal ultimate carcinogen of the environmental pollutant benzo[a]pyrene (BaP). As part of a program to establish activation/detoxification profiles of urinary metabolites of BaP in humans, we developed a method for quantifying trans-anti-BaP-tetraol. Urine was collected from three groups of individuals exposed to BaP: psoriasis patients treated with a coal tar-containing ointment, steel workers, and smokers. [2H12]-trans-anti-BaP-tetraol was added to the urine as an internal standard. The urine was treated with β-glucuronidase and sulfatase, and then the BaP-tetraols were enriched by reverse-phase and phenylboronic acid solid-phase extraction. The resulting fraction was treated with sodium hydride and methylmethane sulfonate to convert BaP-tetraols to the corresponding tetramethyl ethers (BaP-TME). The mixture was purified by normal-phase HPLC and analyzed by gas chromatography/negative ion chemical ionization/mass spectrometry with selected ion monitoring. [13CH3]4-trans-antiBaP-TME was used as an external standard. Ions at m/z 376, 380, and 388 were monitored for quantitation of trans-anti-BaP-TME, [13CH3]4-trans-anti-BaP-TME, and [2H12]-trans-antiBaP-TME, respectively. The instrumental detection limit was approximately 1 fmol of transanti-BaP-TME. trans-anti-BaP-tetraol (as trans-anti-BaP-TME) was detected in 20 of 20 individuals receiving coal tar therapy (mean, 16 fmol/mL of urine), 13 of 13 exposed steel workers (mean, 4.1 fmol/mL of urine), and nine of 21 cigarette smokers (mean, 0.5 fmol/mL of urine). The means in these groups were significantly different (P < 0.0001). The urine of steel workers was also analyzed for cis-anti-BaP-tetraol and cys-syn-BaP-tetraol, but neither was found. The results of this study provide a quantitative method for determination of parts per trillion levels of trans-anti-BaP-tetraol in human urine. Ultimately, this method can be employed as part of a phenotyping approach for assessing BaP metabolites in human urine.

Introduction Polycyclic aromatic hydrocarbons (PAH)1 are products of the incomplete combustion of organic matter and are * To whom correspondence should be addressed. † University of Minnesota Cancer Center. ‡ Harvard School of Public Health. § Mailman School of Public Health of Columbia University. 1 Abbreviations: anti-BPDE, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene; BaP, benzo[a]pyrene; BaP-7,8-diol, 7,8dihydroxy-7,8-dihydrobenzo[a]pyrene; cis-anti-BaP-tetraol, r-7,t-8,9,10tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-syn-BaP-tetraol, r-7,t-8,c-9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; GC/ NICI-MS/SIM, gas chromatography/negative ion chemical ionization mass spectrometry/selected ion monitoring; MMS, methylmethane sulfonate; PAH, polycyclic aromatic hydrocarbons; SPE, solid-phase extraction; syn-BPDE, r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-anti-BaP-tetraol, r-7,t-8,9,c-10-tetrahydroxy7,8,9,10-tetrahydrobenzo[a]pyrene; [2H12]-trans-anti-BaP-tetraol, r-7,t8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydro[2H12]benzo[a]pyrene; transanti-BaP-TME, r-7,t-8,9,c-10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; [2H12]-trans-anti-BaP-TME, r-7,t-8,9,c-10-tetramethoxy[2H12]benzo[a]pyrene; [13CH3]4-trans-anti-BaP-TME, r-7,t-8,9,c-10-[13CH3]4tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-syn-BaP-tetraol, r-7,t-8,10,c-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

widely distributed in the human environment (1). Mixtures containing PAH such as coke oven emissions, coal tars, and soots are accepted causes of human cancer, particularly of the skin and lung (2, 3). PAH are also believed to play a significant role as causes of lung and oral cavity cancer in smokers (4, 5). Benzo[a]pyrene (BaP) is commonly detected in virtually all PAH-containing mixtures (1). It is a powerful locally acting carcinogen which readily induces tumors of the skin, lung, and other tissues at relatively low doses (1, 6-8). As a consequence of its ubiquitous occurrence and strong carcinogenicity, it is frequently regarded as a surrogate for other PAH. An overview of BaP metabolism is presented in Scheme 1 (1, 5, 9). In the initial step, cytochrome P450s 1A1, 1A2, 1B1, 3A4, and 2C catalyze the formation of arene oxides and phenols (10-13). The arene oxides may rearrange spontaneously to phenols or undergo hydration catalyzed by epoxide hydrolase, leading to dihydrodiols. 7,8-Dihydro-7,8-dihydroxybenzo[a]pyrene (BaP-7,8-diol) is further oxidized to anti- and syn-7,8-dihydroxy-9,10-epoxy-7,8,9,10-

10.1021/tx990202c CCC: $19.00 © 2000 American Chemical Society Published on Web 03/18/2000

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Scheme 1. Overview of BaP Metabolisma

a

For details, see refs 5 and 9-18. EH, epoxide hydrolase; DHD, dihydrodiol dehydrogenase.

tetrahydrobenzo[a]pyrene (BPDE) in a reaction catalyzed by P450s 1A1, 1A2, and 1B1, as well as other enzymes (11-13). Among the four BPDE enantiomers produced in these reactions, the (7R,8S,9S,10R)-enantiomer of antiBPDE, illustrated in Scheme 1, is generally formed to the greatest extent and shows the highest carcinogenic activity (9, 14-16). This diol epoxide reacts with DNA in vitro and in vivo, producing a major adduct in which the exocyclic amino group of deoxyguanosine undergoes trans addition to carbon 10 of BPDE (9, 14-18). Convincing evidence clearly supports the hypothesis that the formation of this and related minor DNA adducts is the major metabolic activation pathway of BaP (9, 14-19). Other pathways of BaP metabolism lead predominantly to detoxification. These include phenol formation, glutathione conjugation of arene oxides and diol epoxides involving GSTM1, GSTA1, and GSTP1, and glucuronidation of dihydrodiols. The roles of catechols produced by dihydrodiol dehydrogenase, quinones, and one-electron oxidation products in the biological activity of BaP are less clear (20-22). Extensive studies in human tissues clearly demonstrate the existence of the BaP metabolism pathways illustrated in Scheme 1 (1, 9). Large interindividual variation has been noted in the metabolic activation of BaP by human tissues (1, 9). Substantial interindividual variation is also observed in measurements of levels of BaP-DNA adducts in humans (23). These results indicate that humans differ in their abilities to activate or

detoxify BaP. Individuals who activate BaP extensively are hypothesized to be at greater risk for the carcinogenic effects of BaP than those who effectively detoxify this compound. A rapidly growing body of literature is exploring the relationship between polymorphisms in BaP metabolizing genes such as CYP1A1 and GSTM1 and cancers of various types (5, 24-26). Variants in CYP1A1 have been associated with increased lung cancer risk in Japanese populations, but not in Caucasian or AfricanAmerican populations (26). The GSTM1 null genotype, which occurs in approximately 40-50% of humans, has been associated with modest increases in lung cancer risk in smokers (24-27). Other studies indicate increased risk for lung cancer in individuals with unfavorable combinations of CYP1A1 and GSTM1 genotypes (25, 26). Considering the complexity of BaP metabolism as indicated in Scheme 1, it is unlikely that a polymorphism in a single gene can predict the outcome of BaP activation and detoxification. Rational interpretation of genotyping data with respect to carcinogen metabolism will require a more comprehensive approach involving integration of genotyping and phenotyping information. Therefore, we propose to develop an activation/detoxification metabolite profile for BaP. We hope to relate the results of such metabolite profile analyses to genotyping data, to determine how variants in BaP metabolizing genes actually relate to metabolite formation. We plan to develop methods for the analysis of several key BaP metabolites representing activation and detoxification pathways. This

BaP-Tetraols in Human Urine

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Scheme 2. Formation of BaP-Tetraols and BaP-TME from BPDE

paper reports the development of a highly sensitive and specific GC/NICI-MS/SIM method for quantitation of BaP-tetraols in human urine. BaP-tetraols are the hydrolysis products of BPDE (Scheme 2). Since most BPDE will react with H2O producing BaP-tetraols, quantitation of these metabolites will indicate how much BaP is metabolically activated by the diol epoxide pathway. The method was applied to three groups of subjects: psoriasis patients treated with a coal tar-containing ointment which has high levels of BaP (28), steel workers exposed occupationally to BaP (29), and smokers.

Experimental Procedures Caution: BaP and many of its metabolites are mutagens and/ or carcinogens. They should be handled with extreme care, using appropriate safetywear and ventilation at all times. Chemicals. r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BaP-tetraol), r-7,t-8,10,c-9-tetrahydroxy7,8,9,10-tetrahydrobenzo[a]pyrene (trans-syn-BaP-tetraol), r-7,t8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (cis-antiBaP-tetraol), r-7,t-8,c-9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (cis-syn-BaP-tetraol), and racemic r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro[1,3-3H]benzo[a]pyrene ([1,33H]-anti-BPDE, 0.55 Ci/mmol) were supplied by the National Cancer Institute Chemical Carcinogen Reference Standard Repository, Midwest Research Institute (Kansas City, MO). r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydro[2H12]benzo[a]pyrene ([2H12]-trans-anti-BaP-tetraol) was a gift from A. Melikian (American Health Foundation, Valhalla, NY). β-Glucuronidase (types B3 and H1) was purchased from Sigma Chemical Co. (St. Louis, MO). [13CH3]Methyl iodide and sodium hydride

(60% dispersion in mineral oil) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Preparation of trans-anti-[1,3-3H]BaP-Tetraol and cisanti-[1,3-3H]BaP-Tetraol. [1,3-3H]-anti-BPDE (200 µCi) was added to 2 mL of a 75:25 H2O/acetone solution, and the mixture was hydrolyzed overnight at 37 °C. The resulting [1,3-3H]BaPtetraols were extracted into ethyl acetate, and the extract was analyzed by radioflow HPLC. Approximately 90% of the radioactivity in the extract was present as a 3:1 mixture of transanti-[1,3-3H]BaP-tetraol and cis-anti-[1,3-3H]BaP-tetraol. This mixture was used without further purification to determine recovery of BaP-tetraols at various steps in the assay described in this paper. Preparation of 7,8,9,10-Tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-TMEs). BaP-tetraols were converted to BaP-TMEs (Scheme 2) essentially as described previously (30, 31). Sodium hydride (∼2 g) was washed four times with 10 mL of hexane, and then dried overnight under a stream of N2. A solution of each BaP-tetraol isomer (∼200 µg) was placed in a 2 mL silanized glass vial with a Teflon-coated magnetic stir bar and dissolved in DMSO (400 µL). Sodium hydride (∼2 mg) was added to the vial, which was capped, and the mixture was stirred for 2 min at room temperature. Methyl iodide (∼50 µL) was added to the vial, and stirring was continued for 15 min. The reaction was quenched with 1 mL of H2O, and the mixture was extracted three times with 1 mL of benzene. The benzene extracts were combined and reduced to dryness in a Speedvac (Savant Instruments Inc., Farmingdale, NY). The crude products were purified by reverse-phase HPLC on a 25 cm × 4.6 mm, 5 µm Ultrasphere ODS column (Beckman, Fullerton, CA). The mobile phase was 75:25 CH3OH/H2O flowing at a rate of 1 mL/min. The purity of the BaP-TME standards was >98% as determined by GC/NICI-MS.

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Preparation of External Standard r-7,t-8,9,c-10-[13CH3]4Tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ([13CH3]4trans-anti-BaP-TME). This was prepared from trans-antiBaP-tetraol as described in the previous section, except that [13CH3]methyl iodide was used as the methylating agent. The concentration of [13CH3]4-trans-anti-BaP-TME was determined by HPLC/UV and by UV spectrophotometry, using a standard curve generated from trans-anti-BaP-TME, and assuming that the UV extinction coefficient was identical for both compounds. Subjects and Urine Collection. Urine samples were obtained from patients receiving Goekerman coal tar therapy for plaque-stage psoriasis (28), from workers at one of the largest steel plants in Taiwan (29), and from smokers in Minneapolis (32). For the steel workers, 13 samples were from coke oven workers and five from workers in a large administrative area, approximately 2 km from the coke oven plant (29). All subjects were sampled for the benzene-soluble fraction of total particulates at least 6 h during the workday and for three consecutive days between August 1995 and February 1996. All studies were approved by the appropriate local IRBs. Samples were stored frozen at -20 °C until required for analysis. Analysis of Urine. Urine samples were allowed to thaw by warming to room temperature. Samples were mixed thoroughly, and a representative 20 g aliquot (including suspended solids) was weighed into a 50 mL centrifuge tube. The samples were adjusted to pH ∼5 with 1 M HCl and then buffered with 5 mL of sodium acetate buffer (0.5 M, pH 5.0). A solution of [2H12]trans-anti-BaP-tetraol in CH3OH (∼400 pg) was added to each sample as an internal standard. Finally, β-glucuronidase (∼25000 units) and sulfatase (∼120 units) were added to each of the samples, which were placed on an incubated shaker bath at 37 °C for 16-20 h. Each sample was applied to a SPE system consisting of a vacuum manifold; a Sep-pak plus tC18 cartridge (Waters Corp., Milford, MA) activated with 10 mL of MeOH, and then with 10 mL of H2O; a Millex AP 25 mm prefilter (Millipore Corp., Bedford, MA); and solvent reservoir. The Sep-pak cartridge was rinsed sequentially with 15 mL of 0.15 M NH4OH, 15 mL of 0.15 M NH4OH in 30:70 CH3OH/H2O, and 10 mL of 30:70 CH3OH/H2O, and the BaP-tetraol-containing fraction eluted with 10 mL of 70:30 CH3OH/H2O into cleaned 15 mL glass centrifuge tubes. The extract was reduced to dryness in a Speedvac (Savant Instruments Inc.), the same method used in all solvent removal steps. Further sample purification was achieved with immobilized phenylboronic acid SPE cartridges (10 mL, 100 mg, Phenomonex Ltd., Torrance, CA). The cartridges were cleaned with CH3OH (1.2 mL) and H2O (1.2 mL), and then activated with disodium hydrogen phosphate buffer (1.2 mL, 0.5 M, pH 10.3) followed by potassium dihydrogen phosphate buffer (1.2 mL, 25 mM, pH 8 in 50:50 CH3OH/H2O). The sample extract was applied to the cartridge in 0.5 mL of the pH 8 buffer solution, and the cartridge was rinsed with 2.5 mL of the pH 8 buffer solution. Salicylic acid (3 mL, 25 mM in 50:50 CH3OH/H2O) was applied to the cartridge. The first 0.5 mL of the salicylic acid eluent was discarded, and the next 2.5 mL which contained the BaP-tetraols was collected into cleaned 4 mL silanized glass vials. The extract was reduced to dryness. BaP-tetraols were separated from the salicylic acid by dissolving the extract in NaOH (0.1 M, 1.5 mL), and extracting three times with ethyl acetate (1 mL). The ethyl acetate extracts were combined in cleaned 4 mL silanized glass vials, and then reduced to dryness. Permethylation of the BaP-tetraols was accomplished as follows. The extract was dissolved in DMSO (100 µL, dried over molecular sieves), and a Teflon-coated magnetic stir bar was placed in the vial. Sodium hydride was washed with hexane (4 × 10 mL) and dried under a stream of N2. Two milligrams of sodium hydride was added to the vial, which was capped and stirred for 2 min at room temperature. MMS (50 µL) was added to the vial, and stirring was continued for 20 min. The reaction was quenched with 1 mL of H2O; the mixture was extracted three times with 1 mL of benzene, and the benzene extracts

Simpson et al. were combined and reduced to dryness. The residue was purified by normal-phase HPLC on a 25 cm × 4.6 mm, 5 µm Develosil cyano column (Phenomonex Ltd.). The HPLC system consisted of a Waters Alliance HPLC module with autosampler (set to 4 °C) and column heater (set to 35 °C), a Shimadzu SPD10-AV UV/visible detector operated at 254 nm, and a fraction collector (Foxy Jr., Isco Inc., Lincoln, NE). The following solvents were used: solvent A, 99.25:0.64:0.11 hexane/ THF/CH3OH; and solvent B 70:25:5 hexane/THF/CH3OH. The sample was dissolved in 70 µL of a solution containing 1 mg/ mL benzyl alcohol (UV retention time marker) dissolved in solvent A. The HPLC gradient was as follows: solvent A, 1.2 mL/min for 20 min; solvent B, 1.2 mL/min for 15 min; recondition with solvent A at 1.5 mL/min for 20 min. The four BaPTME isomers were sufficiently well separated by HPLC that it was possible to collect separate fractions containing individual isomers. The fraction collector was programmed to collect four separate fractions into silanized 4 mL glass vials, based on defined retention time windows. After collection, the fractions were reduced to dryness. The residue was transferred to a glass microvial with three 250 µL volumes of CH3OH and concentrated to dryness again. Finally, the residue was dissolved in 10 µL of benzene containing ∼6 fmol/µL [13CH3]4-trans-antiBaP-TME (external standard) in preparation for analysis by GC/ NICI-MS/SIM. GC/NICI-MS/SIM was performed on a Finnigan TSQ 7000 instrument (FinniganMAT/Thermoquest, San Jose, CA) interfaced with a CTC A200SE autosampler (Leap Technologies, Carrboro, NC) and a HP5890 series II gas chromatograph (Hewlett-Packard Ltd., Palo Alto, CA) fitted with a 2 m × 250 µm deactivated silica retention gap and a DB-17MS (50% dimethylpolysiloxane/50% diphenylpolysiloxane) capillary column (30 m × 0.25 mm, 0.15 µm film thickness, J&W Scientific Ltd., Folsom, CA). The sample (4 µL) was injected splitless into the GC injector at 300 °C, and the carrier gas (He) flow rate was set to 2.5 mL/min. The GC oven was programmed as follows: 80 °C for 2 min, increase to 300 °C at a rate of 20 °C/ min, and maintain at 300 °C for 22 min. The transfer line to the MS was maintained at 300 °C. The MS apparatus was operated in the NICI mode, acquiring ions m/z 388, 380, and 376, corresponding to the molecular anions of [2H12]-trans-antiBaP-TME, [13CH3]4-trans-anti-BaP-TME, and trans-anti-BaPTME, respectively. The settings for the CI source were optimized with trans-anti-BaP-TME to maximize the signal at m/z 376 (peak width at base ) 1.5 amu). MS settings were as follows: source temperature of 150 °C, manifold temperature of 70 °C, electron energy of 150 eV, and electron current of 700 µA. The total scan time was 0.5 s; the scan width was 0.5 amu, and the CI (methane) gas pressure was 3300 mTorr. The data were acquired in profile mode and smoothed with the seven-point default filtering algorithm provided with Finnigan’s ICIS data acquisition and processing software. Confirmation of analyte identity was obtained using a 30 m × 0.25 mm capillary GC column with RTx-20 (80% dimethylpolysiloxane/20% diphenylpolysiloxane) stationary phase (0.1 µm film thickness) (Restek Ltd., Bellefonte, PA). The GC oven was programmed as follows: 50 °C for 2 min, increase to 250 °C at a rate of 20 °C/min, increase to 300 °C at a rate of 5 °C/ min, and maintain at 300 °C for 15 min. Statistical Analyses. Analysis of variance of log-transformed BaP-tetraol concentrations in exposed human subjects was performed by using the software package SAS, version 6.12 (SAS Institute, Cary, NC). A BaP-tetraol concentration value of 0.1 fmol/mL, equivalent to approximately one-half of the detection limit, was used for all samples in which BaP-tetraol was not detected. Spearman rank correlations were used to study the relationship between benzene-soluble fraction and BaP-tetraols in the urine of steel workers.

Results Method Development. The method for analysis of BaP-tetraols in human urine is summarized in Scheme



Scheme 3. Scheme for Analysis of BaP-Tetraols in Human Urinea

a

NP, normal-phase.

3. [2H12]-trans-anti-BaP-tetraol was used as internal standard. Urine was treated with β-glucuronidase and sulfatase to release any conjugated BaP-tetraols, as it was our goal to quantify total BaP-tetraols in urine. No information is available at present on relative levels of conjugated and free BaP-tetraols in human urine. The BaP-tetraols were enriched by SPE using reverse-phase and phenylboronic acid cartridges. The resulting fraction was reacted with NaH and MMS to convert BaP-tetraols to BaP-TME. This mixture was further purified by normal-phase HPLC prior to analysis by GC/NICI-MS/ SIM. The development of each step is discussed below. (1) Optimization of SPE of BaP-Tetraols from Human Urine. Retention of the BaP-tetraols on the C18 Sep-pak cartridges as a function of CH3OH concentration in the eluent was determined by adding trans-anti-[1,33H]BaP-tetraol and cis-anti-[1,3-3H]BaP-tetraol to human urine. Less than 2% of the added radioactivity was eluted from the Sep-pak cartridge by 30:70 CH3OH/H2O, but complete elution was achieved with 70:30 CH3OH/H2O. Subsequently, it was determined that, even after HPLC cleanup of this urine extract, there were too many interfering compounds present for analysis by GC/NICIMS/SIM. We reasoned that washing the Sep-pak cartridge with a basic solution would facilitate the selective removal of weakly acidic compounds from the extract. Therefore, the Sep-pak cartridge was washed with 0.15 M NH4OH and 0.15 M NH4OH in 30:70 CH3OH/H2O. This modification provided substantially less complex extracts than our original protocol. Recovery of [1,3-3H]BaP-tetraols from the Sep-pak cartridges was ∼80%. (2) Phenylboronic Acid Chromatography. The sample extract was further purified by phenylboronic acid SPE, which retains cis-diols with high specificity (33). Of the four isomeric BaP-tetraols, only the trans-synisomer does not possess cis-hydroxyl groups and would not form a cyclic boronate complex. However, trans-synBaP-tetraol is not expected to be present in substantial

amounts in biological samples (9, 14-16). In our procedure, CH3OH was present in the conditioning and eluting solvents to eliminate the possibility of hydrophobic secondary interactions with the phenylboronic acid cartridge. Salicylic acid eluted the BaP-tetraols from the cartridges by reducing pH and by competing for the boronate ligands (33). Recovery of trans-anti-[1,3-3H]BaPtetraol and cis-anti-[1,3-3H]BaP-tetraol from the cartridges was ∼85%. (3) Derivatization of BaP-Tetraols to BaP-TMEs. Previously, we reported a procedure for conversion of BaP-tetraols to BaP-TMEs with methylsulfinyl carbanion and methyl iodide (30, 31). We found that a modification of this procedure, based on that of Ciacanu and Kerek (34), provided a higher and more reproducible yield of BaP-TME. MMS was substituted for methyl iodide because halogens, which have a high electron-capture efficiency, could contribute to the background in the GC/ NICI-MS/SIM analysis. The overall yield was ∼60%. (4) Purification of BaP-TMEs. Normal-phase HPLC was used to purify the BaP-TMEs after the derivatization reaction. The efficacy of both normal- and reverse-phase HPLC was investigated. The normal-phase HPLC method provided less complex extracts for GC/NICI-MS/SIM. The retention times of BaP-TMEs are listed in Table 1. The four isomers were sufficiently well resolved that they could be collected as separate fractions for subsequent GC/NICI-MS/SIM analysis. This is potentially important because trans-anti-BaP-TME is not adequately separated from cis-syn-BaP-TME by GC (see Table 1). (5) GC/NICI-MS/SIM Detection of BaP-TMEs. The NICI-MS spectrum of trans-anti-BaP-TME had prominent peaks at m/z 376 (relative intensity of 75, M-), 312 (85, M- - 2CH3OH), 282 [68, M- - (2CH3OH + CH2O)], and 252 [100, M- - (2CH3OH + 2CH2O)]. The relative intensities of these four ions were strongly influenced by conditions within the MS ion source. We used m/z 376 for SIM, and similarly monitored m/z 380 and 388 for

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Table 1. Summary of Retention Time Data (minutes:seconds) for BaP-TME Isomersa trans-anti-BaP-TME trans-syn-BaP-TME cis-anti-BaP-TME cis-syn- BaP-TME [2H12]-trans-anti-BaP-TME GC normal-phase HPLC a

17:39 10:29

16:49 6:08

18:21 16:28

17:28 9:08

17:33 10:24

Details of GC and normal-phase HPLC conditions are described in Experimental Procedures.

Figure 1. GC/NICI-MS/SIM of a trans-anti-BaP-TME standard mix containing 0.74 fmol/µL trans-anti-BaP-TME, 6.0 fmol/µL [13CH3]4-trans-anti-BaP-TME, and 5.9 fmol/µL [2H12]-trans-antiBaP-TME. Four microliters was injected. Table 2. Levels of trans-anti-BaP-Tetraol Added and Found in Human Urine Samplesa amount of trans-anti-BaP-tetraol (fmol/mL) added

found

1 1 1 1 5 5 5 5 20 20 20 20

0.9 0.6 0.2 0.6 4.7 6.7 5.0 5.9 21 16 22 22

mean ( SD

0.6 ( 0.3

5.6 ( 0.9

20 ( 3

a

Aliquots (20 mL) of urine from a subject with no known exposure to elevated levels of BaP were enriched with 1, 5, or 20 fmol/mL trans-anti-BaP-tetraol and analyzed by the method illustrated in Scheme 3.

the external standard [13CH3]4-trans-anti-BaP-TME and the internal standard [2H12]-trans-anti-BaP-TME, respectively. Our low calibration standard of 3 fmol of transanti-BaP-TME was typically detected with a signal:noise ratio of >10. This translates into an instrumental limit of detection of approximately 1 fmol of trans-anti-BaPTME with a signal:noise ratio of 3. Typical ion chromatograms for the standard mixture are shown in Figure 1. Application of the Method to Human Urine Samples Spiked with trans-anti-BaP-Tetraol. The accuracy and precision of this method were established by analyzing human urine to which trans-anti-BaPtetraol had been added. Urine was collected from a volunteer with no known exposure to elevated levels of BaP, and aliquots (20 mL) were enriched with trans-antiBaP-tetraol at three different concentrations. Two 20 mL aliquots of urine to which nothing was added, as well as three samples, each consisting of 20 mL of H2O only, were also analyzed. The results of these experiments are summarized in Table 2. trans-anti-BaP-tetraol (as trans-anti-BaP-TME) was not detected in the three H2O samples, or in the two urine aliquots to which nothing was added. trans-anti-BaPTME was detected in all urine samples enriched with this

compound. The experimental determinations of transanti-BaP-tetraol were in good agreement with the expected values for urine samples to which 5 or 20 fmol had been added per milliliter. The data for urine samples to which 1 fmol of trans-anti-BaP-tetraol had been added per milliliter, however, were consistently lower than expected. In these samples, the level of trans-anti-BaPtetraol was near the detection limit of the method (0.2-2 fmol/mL of urine), and there were large peaks eluting in the region of trans-anti-BaP-TME, although they did not coelute with the analyte. Consequently, the method can be considered only semiquantitative at levels of transanti-BaP-tetraol below about 3 fmol/mL of urine. Determination of cis-anti-BaP-Tetraol, cis-synBaP-Tetraol, and trans-syn-BaP-Tetraol in Human Urines to Which BaP-Tetraols Had Been Added. The four urine aliquots to which 20 fmol of trans-antiBaP-tetraol had been added per milliliter were also analyzed for cis-anti-BaP-tetraol, trans-syn-BaP-tetraol, and cis-syn-BaP-tetraol. This was achieved by collecting a separate region from the normal-phase HPLC for each of the isomers. Each of the fractions was then analyzed by GC/NICI-MS/SIM. cis-anti-BaP-TME, trans-syn-BaPTME, and cis-syn-BaP-TME were not detected in any of the HPLC fractions from these urine samples. Therefore, it can be concluded that there is no significant conversion of trans-anti-BaP-tetraol to the other BaP-tetraol isomers using this methodology. Additionally, four urine aliquots were treated with a mixture containing cis-anti-BaPtetraol, trans-syn-BaP-tetraol, and cis-syn-BaP-tetraol (20 fmol/mL each). Four separate HPLC fractions corresponding to the retention windows of the individual BaPTME isomers were collected and analyzed by GC/NICIMS/SIM. trans-anti-BaP-TME was not detected in any of these samples, indicating that there was no significant conversion of the three isomers to trans-anti-BaP-tetraol. trans-syn-BaP-TME was not detected in these urine samples. This is not unexpected as it would not be retained on the phenylboronic acid cartridges. cis-antiBaP-TME and cis-syn-BaP-TME were readily detected in the expected HPLC fractions from these samples, although the levels were not quantified. The detection of cis-anti-BaP-TME and cis-syn-BaP-TME indicates that both of these compounds, if present in human urine, would be detected with the current methodology. Application of the Method to Urine Samples from Human Subjects Who Have Been Exposed to BaP. Urine samples from three groups of human subjects who have been exposed to BaP were analyzed for trans-antiBaP-tetraol. Representative selected ion chromatograms from urine extracts from a subject in each group are shown in Figure 2. To provide confirmation that the analyte peak in the urine samples was indeed trans-antiBaP-TME, selected positive samples were reanalyzed on a second GC column (RTx-20 stationary phase). The retention time of the analyte peak in the urine samples (20.7 min) was identical to that of standard trans-antiBaP-TME. However, the separation of trans-anti-BaPTME from other compounds in the urine extract was not


Chem. Res. Toxicol., Vol. 13, No. 4, 2000 277 Table 3. Ratios of Major trans-anti-BaP-TME Fragment Ions in Standards and in Human Urine Samplesa sample

m/z 282.3/ m/z 376.3

m/z 312.3/ m/z 376.3

trans-anti-BaP-TME CT 12 CT 12 + trans-anti-BaP-TME CT 9 CT 9 + trans-anti-BaP-TME

1.0 0.9 0.9 0.8 0.8

0.7 0.8 0.7 0.7 0.7

a Fragment ion ratios were monitored in standard trans-antiBaP-TME; in two urine samples from psoriasis patients, CT 12 and CT 9; and in these same samples to which trans-anti-BaPTME had been added.

administrative officers in the steel plant (mean of 0.3 fmol/mL, range of not detected to 0.8 fmol/mL), and 9 of 21 cigarette smokers (mean of 0.5 fmol/mL of urine, range of not detected to 1.5 fmol/mL). Average recoveries of internal standard were 30 ( 13% (range of 2-57%). Urine samples from the coke oven workers were analyzed for cis-anti-BaP-tetraol (N ) 10) and cis-syn-BaP-tetraol (N ) 19), but neither compound was detected. Analysis of variance was used to determine whether trans-anti-BaP-tetraol concentrations in human urine differed among psoriasis patients, coke oven workers, administrative workers, and smokers. An initial F-test, performed on log-transformed trans-anti-BaP-tetraol concentrations, was significant [F(3,55) ) 76.7; P < 0.001]. Subsequent pairwise comparisons (t-tests) indicated that all group means with the exception of administrative officers versus smokers were significantly different from each other (P < 0.001 for each comparison).

Discussion

Figure 2. GC/NICI-MS/SIM of trans-anti-BaP-TME (m/z 376), [13CH3]4-trans-anti-BaP-TME (m/z 380, external standard), and [2H12]-trans-anti-BaP-TME (m/z 388, internal standard) in (A) urine extract from a psoriasis patient receiving coal tar therapy, (B) urine extract from a coke oven worker, and (C) urine extract from a current smoker. Peaks at m/z 376 other than trans-antiBaP-TME do not result from other BaP tetraol isomers, since they have different HPLC retention times and were not collected in this analysis.

as good on the RTx-20 column as on the DB-17MS column. To further confirm the identity of the analyte peak in the urine samples, fragment ions characteristic of trans-anti-BaP-TME (m/z 312 and 282) were monitored in two urine extracts from psoriasis patients. Fragment ion ratios were calculated for the trans-anti-BaP-TME standard, the analyte peak in the two urine extracts, and the analyte peak in the two urine extracts after the transanti-BaP-TME standard had been added to the extract. Data from these experiments are summarized in Table 3. It can be seen that the two fragment ion ratios were essentially identical in urine samples, independent of whether trans-anti-BaP-tetraol had been added, and were in good agreement with the fragment ion ratios for authentic trans-anti-BaP-TME. The results of the analyses of human urine are summarized in Table 4. trans-anti-BaP-tetraol (as trans-antiBaP-TME) was detected in 20 of 20 individuals receiving coal tar therapy (mean of 16 fmol/mL of urine, range of 0.9-56 fmol/mL), 13 of 13 coke oven workers (mean of 4.1 fmol/mL of urine, range of 0.7-17 fmol/mL), 2 of 5

Our results demonstrate that trans-anti-BaP-tetraol can be detected and quantified in human urine by GC/ NICI-MS/SIM. There are some important features of our method which ultimately permit detection and quantitation of this analyte at parts per trillion levels (e.g., approximately 1 pg/mL of urine). Sample purification using SPE with both reverse-phase and phenylboronic acid cartridges, followed by normal-phase HPLC purification of BaP-TMEs, allowed removal of significant amounts of interfering substances and provided selectivity for analysis of the individual BaP-TMEs. Reaction of the BaP-tetraol fraction with sodium hydride and MMS resulted in the formation of stable permethylated derivatives with sufficient volatility for GC analysis, as previously described (30, 31, 34). The internal standard [2H12]trans-anti-BaP-tetraol and the external standard [13CH3]4trans-anti-BaP-TME allowed quantitation of analyte, monitoring of GC/NICI-MS/SIM performance, and determination of recoveries. Finally, the sensitivity of GC/ NICI-MS/SIM, with a detection limit of approximately 1 fmol of BaP-TME, was essential for the success of this method. Although the method involves several steps, some of these can be automated and conveniently performed with multiple samples. Weston et al. (35) have previously described a method for the analysis of BaP-tetraols in human urine. They used immunoaffinity chromatography for sample purification and synchronous fluorescence spectroscopy for quantitation. Detection limits of 2.5-10 fmol/mL of BaPtetraol have been reported, starting with a 10 mL urine sample (35, 36). Weston et al. (35) reported BaP-tetraol levels of 240-3120 fmol/mL in the urine of four individu-

278


Simpson et al.

Table 4. trans-anti-BaP-Tetraol Concentrations in Human Urine Psoriasis Patients Treated with Coal Tar Ointment trans-anti-BaP-tetraol sample

fmol/mL of urine

fmol/µmol of creatinine

CT 1 CT 2 CT 3 CT 4 CT 5 CT 6 CT 7 CT 8 CT 9 CT 10 CT 11 CT 12 CT 13 CT 14 CT 15 CT 16 CT 17 CT 18 CT 19 CT 20 mean SD

7.0 5.9 11 4.4 11 16 6.6 22 49 8.0 49 56 4.3 10 8.7 9.1 5.3 4.5 24 0.9 16 16

2.3 3.1 3.0 4.4 6.9 2.7 1.7 4.5 7.7 2.2 14 19 1.2 2.9 4.6 2.3 2.7 1.7 3.9 0.7 4.6 4.5

Steel Workers

sample SW 1 SW 2 SW 4 SW 5 SW 6 SW 8 SW 9 SW 10 SW 12 SW 13 SW 14 SW 16 SW 17 mean SD

benzenesoluble fraction (µg/m3)

trans-anti-BaP-tetraol fmol/mL fmol/µmol of urine of creatinine

Coke Oven Workers 47 2.5 53 3.3 10.5 0.7 347 2.4 70 5.6 123 5.3 151 2.0 45 3.0 916 17 188 1.2 39 6.6 42 0.7 42 3.6 160 4.1 245 4.3 Administrative Officers 1.4 0.5 5.7 0.8 11 nd 20 nd 5.1 nd

SW 3 SW 7 SW 11 SW 15 SW 18

0.19 0.32 0.11 0.12 0.49 0.25 0.19 0.48 0.92 0.38 0.45 0.07 0.24 0.32 0.23 0.03 0.29 nd nd nd

Smokers trans-anti-BaP-tetraol sample

cigarettes/day

fmol/mL of urine

S1 S2 S3 S4 S5 S6 S7 S8 S9 S 10 S 11 S 12 S 13 S 14 S 15 S 16 S 17 S 18 S 19 S 20 S 21

38 25 20 18 20 25 25 30 30 20 25 20 40 13 40 40 25 20 25 30 26

0.2 0.3 nda nd 0.3 0.4 nd 0.3 1.5 nd 0.4 nd nd nd 0.7 nd nd nd nd nd 0.3

a

nd, not detected.

fmol/µmol of creatinine 0.03 0.05 nd nd 0.05 0.04 nd 0.02 0.20 nd 0.10 nd nd nd 0.08 nd nd nd nd nd 0.09

als who consumed char-broiled beef for 7 days. Bowman et al. (36) reported a mean of 150 fmol/mL BaP-tetraols in urine from psoriasis patients and 13 fmol/mL in untreated controls. Bentsen-Farmen et al. (37) were unable to detect BaP-tetraol in the urine of workers exposed occupationally to BaP. In the study presented here, the samples from psoriasis patients were a subset of those analyzed by Bowman et al. We observed no correlation between our data and theirs (data not shown), and presently have no explanation for this discrepancy. The highest levels of trans-anti-BaP-tetraol were found in the urine of the psoriasis patients. They were treated with a medicinal coal tar ointment containing relatively high concentrations of PAH. The concentration of BaP was 140 µg/g in one sample of the ointment (36). However, the dose of BaP and other PAH actually administered to these patients is unknown. There was a 60-fold variation in trans-anti-BaP-tetraol levels in the urine of these patients. This could be due to differences in dose, adsorption, or metabolism. Coke oven workers are exposed to emissions comprised of PAH, including BaP. The samples used in this study came from workers in Taiwan (29). Their exposure to PAH in coke oven emissions was estimated by determining the benzenesoluble fraction of total particulates which varied approximately 1000-fold. Levels of trans-anti-BaP-tetraol varied 24-fold, and there was no correlation with the benzene-soluble fraction. Five steel workers were administrative officers with little exposure to BaP, and these individuals had low or undetectable levels of trans-antiBaP-tetraol. The lowest levels of trans-anti-BaP-tetraol were observed in the urine of smokers. The subjects in this study smoked an average of 27 cigarettes per day. BaP concentrations in the mainstream cigarette smoke range from 20 to 40 ng/cigarette (38). Therefore, exposure to BaP in this group could be estimated as approximately 800 ng/day from smoking. This is likely to be considerably lower than in the psoriasis patients or coke oven workers, and the trans-anti-BaP-tetraol levels in urine were accordingly lower. Urinary 1-hydroxypyrene levels are reported to be higher in patients treated with coal tar than in coke oven workers, with the lowest levels being found in smokers (39). These data are consistent with the trans-anti-BaP tetraol levels measured here. While levels of trans-anti-BaP-tetraol in urine may provide an index of BaP uptake, we are not proposing to use this method for that purpose. 1-Hydroxypyrene is already established as a useful biomarker of PAH uptake, and other metabolites of BaP may be more readily quantified than BaP-tetraols (39-46). Our goal is to determine BaP metabolite ratios as an index of an individual’s ability to metabolically activate or detoxify this carcinogen. For example, the ratio of BPDE glutathione conjugates, measured as urinary mercapturic acids, to BaP-tetraols should provide an indication of one’s ability to detoxify BPDE by glutatathione conjugation. Ultimately, we hope to incorporate other BaP metabolites such as phenols, dihydrodiols, and dihydrodiol-glucuronide conjugates into a phenotyping formula. Our approach is analogous to the use of caffeine metabolite ratios for phenotyping P450 1A2 (47). We propose to relate the results of our urinary metabolite profile analysis to levels of BaP-DNA adducts in lung and to genotyping information from the same individuals. This should clarify the relationship between specific


genotypes and BaP metabolic activation in humans. Quantitation of BaP metabolites in urine is more practical than collection and analysis of feces, although it is well established that feces is the major route of BaP metabolite excretion in laboratory animals (48-51). BaP-DNA adducts provide another potentially useful measure of BaP metabolic activation (23). However, DNA adduct measurements have certain limitations, including difficulty in obtaining samples other than lymphocyte DNA, inadequate sensitivity for the determination of specific characterized adducts, and repair of adducts. Nevertheless, HPLC fluorescence appears to be a promising method for quantitation of BPDE-DNA adducts in humans, and other methods have been investigated (23, 52, 53). Another feasible approach for monitoring human metabolic activation of BaP is quantitation of hemoglobin or albumin adducts (30, 31, 54-56). Several studies, including our own, have demonstrated that BPDE adducts to protein can be quantified in humans, but this approach also has certain limitations, including requirements for high sensitivity and the necessity of establishing the relationship of levels of protein adducts to DNA adducts in the same individuals. Our method is capable of detecting cis-syn- and cisanti-BaP-tetraol, but we found only trans-anti-BaPtetraol in the urine of coke oven workers. In vitro studies with rodent and human subcellular fractions demonstrate that the (7R,8S,9S,10R)-enantiomer of anti-BPDE is generally the major product of epoxidation of (7R,8R)BaP-7,8-diol (9, 15, 16). Analysis of DNA from BaPtreated animals and from humans exposed to BaP indicates that the major adduct is produced by reaction with the (7R,8S,9S,10R)-enantiomer of BPDE (9, 16). Hydrolysis of anti-BPDE produces predominantly transanti-BaP-tetraol (57). Taken together, these results indicate that trans-anti-BaP-tetraol should be the major isomer detected in urine, and our results are consistent with this conclusion. One study of BaP metabolism in germ-free rats provides some evidence for higher levels of trans-anti-BaP-tetraol than cis-anti-BaP-tetraol in urine, but little information is available on the conversion of BaP to BaP-tetraols in vivo (58). In summary, we have developed a sensitive and specific method for quantitation of trans-anti-BaP-tetraol in human urine. Average levels of this metabolite were 16 fmol/mL of urine in psoriasis patients who used a BaPcontaining therapeutic ointment, 4.1 fmol/mL in coke oven workers exposed occupationally to BaP, and 0.5 fmol/mL in smokers. This analysis, in combination with methods for other urinary BaP metabolites, will ultimately provide an index of BaP metabolism in humans. We hope to use these data to develop carcinogen metabolite phenotyping methods that eventually can be related to cancer susceptibility in individuals exposed to BaP.

Acknowledgment. We thank Gunnar Boysen for synthesis of the BaP-TME and [13CH3]4-trans-anti-BaPTME standards and Robin Bliss and Chap Le for statistical analyses. The Biostatistics Core of the University of Minnesota Cancer Center is supported by NCI Grant CA77598. This study was supported by NCI Grant CA44377. S.S.H. is an American Cancer Society Research Professor.

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