Detection of DNA and Globin Adducts of Polynuclear Aromatic

Assieh A. Melikian*, Peng Sun, Stuart Coleman, Shantu Amin, and Stephen S. .... Rosalie K. Elespuru , Rajiv Agarwal , Aisar H. Atrakchi , C. Anita H. ...
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Chem. Res. Toxicol. 1996, 9, 508-516

Detection of DNA and Globin Adducts of Polynuclear Aromatic Hydrocarbon Diol Epoxides by Gas Chromatography-Mass Spectrometry and [3H]CH3I Postlabeling of Released Tetraols Assieh A. Melikian,* Peng Sun, Stuart Coleman, Shantu Amin, and Stephen S. Hecht American Health Foundation, Valhalla, New York 10595 Received September 25, 1995X

Gas chromatography-negative ion chemical ionization mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) was employed to detect tetramethyl ether derivatives of tetraols formed upon hydrolysis of DNA and globin adducts derived from diol epoxides of benzo[a]pyrene (BP) and other polynuclear aromatic hydrocarbons (PAH). The tetramethyl ether derivatives could also be detected by [3H]CH3I postlabeling. The methodology involves the following steps: (1) isolation of DNA or globin; (2) mild acid hydrolysis under vacuum; (3) isolation of the resulting tetraols and derivatization to the corresponding tetramethyl ethers using methyl sulfinyl carbanion and unlabeled or 3H-labeled CH3I; (4) analysis by GC-NICIMS-SIM or HPLC with radioflow detection. The optimum conditions for hydrolysis of adducts and derivatization of the resulting tetraols as well as the feasibility of this approach for detecting PAH adducts in mice and humans were explored. Using the set of four BP-tetraols that can be formed upon hydrolysis of adducts formed from r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene (anti-BPDE) or r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE) as models, the stability of the tetraols under the hydrolysis conditions was investigated. Adducts derived from anti-BPDE yield predominantly the stable r-7,t-8,9c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BP-tetraol), while adducts derived from syn-BPDE released cis-syn-BP-tetraol as a major hydrolysis product. Hydrolysis under vacuum significantly increased the recovery of tetraols. Conditions for derivatization of the BP-tetraols as well as tetraols derived from several other PAH anti-diol epoxides were investigated. Tetramethyl ethers proved to be superior derivatives that were stable, easy to prepare in high yields, and detectable with high sensitivity by GC-NICI-MS-SIM (1-50 fmol per injection). Alternatively, these derivatives could be detected by HPLC with radioflow detection if [3H]CH3I were employed for derivatization. The methodology was tested by comparing levels of DNA and globin adducts in mice treated with either unlabeled or 3Hlabeled BP. Good agreement was obtained among the GC-NICI-MS-SIM, [3H]CH3I postlabeling, and conventional radiometric methods. Moreover, analysis of human hemoglobin by GC-NICIMS-SIM resulted in detection of adducts derived from anti-BPDE and r-1,t-2-dihydroxy-t-3,4epoxy-1,2,3,4-tetrahydrochrysene. The results of this study demonstrate that GC-NICI-MSSIM of tetramethyl ethers of tetraols formed by hydrolysis of PAH diol epoxide DNA and globin adducts is a promising approach for detection and quantitation of adducts derived from a broad range of PAH.

Introduction Polynuclear aromatic hydrocarbons (PAH)1 are ubiquitous environmental carcinogens arising from incomplete combustion of organic materials and fossil fuels. Therefore, human exposure to these compounds at some levels is unavoidable. Many PAH are tumorigenic in animal bioassays (1-4). Although there is no direct evidence that an individual PAH causes cancer in humans, there is substantial evidence to suggest that complex PAH mixtures such as those in tobacco smoke, coal tar pitch, engine exhaust emissions, and shale oils cause cancer in humans (1-4). Characterization and measurement of PAH-macromolecular adducts in target tissues or in readily available surrogate cells in humans can provide qualitative and quantitative information about biologically effective doses of PAH exposure. MoreX

Abstract published in Advance ACS Abstracts, February 1, 1996.

0893-228x/96/2709-0508$12.00/0

over, quantitation of cellular macromolecular damage in humans can assist in extrapolating results of animal experiments to humans. PAH mediate their biological activities through their metabolic activation to reactive diol epoxides that bind covalently to cellular macromolecules (5-16). Methods for detecting the resulting adducts in humans such as 32P-postlabeling (17), [35S]phosphorothioate labeling (18), HPLC-linked fluorescence detection (19, 20), synchronous fluorescence spectrophotometry (21), fluorescense line narrowing (22), immunoassays (23-25), and GC-MS (2628) have been developed; however, few are quantitative or specific for individual PAH. Each of these methods has some limitation. Most of the techniques applied do not provide full characterization of unknown PAH adducts or quantitative information on individual PAH. Our long-term goal is to develop a mass spectrometric method to characterize PAH diol epoxide-derived adducts with hemoglobin and DNA in human tissues, to quantify © 1996 American Chemical Society

Detection of PAH Adducts by GC-MS and [3H]CH3I

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 509

Figure 1. Structures of BP-tetraols and their corresponding tetramethyl ethers.

such adducts, and to incorporate them as biomarkers into molecular epidemiology studies. In our initial studies, described here, we have used mild acid hydrolysis of globin or DNA under vacuum to release the PAH moiety as a tetraol. This is followed by permethylation of the PAH-tetraols via tritiated or unlabeled CH3I in the 1 Abbreviations: PAH, polynuclear aromatic hydrocarbons; BP, benzo[a]pyrene; anti-BPDE, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene; syn-BPDE, r-7,t-8-dihydroxy-c-9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; anti-chrysene-DE, r-1,t-2-dihydroxyt-3,4-epoxy-1,2,3,4-tetrahydrochrysene; anti-5-MeCDE, r-1,t-2-dihydroxyt-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene; anti-6-MeCDE, r-1,t2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene; antiB[a]ADE, r-3,t-4-dihydroxy-t-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene; anti-B[c]PDE, r-3,t-4-dihydroxy-t-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; trans-anti-BP-tetraol, r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-syn-BP-tetraol, r-7,t8,10,c-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-anti-BPtetraol, r-7,t-8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-syn-BP-tetraol, r-7,t-8,c-9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-anti-chrysene-tetraol, r-1,t-2,3,c-4-tetrahydroxy1,2,3,4-tetrahydrochrysene; trans-anti-5-MeC-tetraol, r-1,t-2,3,c-4tetrahydroxy-1,2,3,4-tetrahydro-5-methylchrysene; trans-anti-6-MeCtetraol, r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydro-6-methylchrysene; trans-anti-B[a]A-tetraol, r-1,t-2,3-c-4-tetrahydroxy-1,2,3,4-tetrahydrobenz[a]anthracene; trans-anti-B[c]P-tetraol, r-1,t-2,3-c-4-tetrahydroxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; trans-anti-TME-BPtetraol, r-7,t-8,9,c-10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-syn-TME-BP-tetraol, r-7,t-8,10,c-9-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-anti-TME-BP-tetraol, r-7,t-8,9,10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-syn-TME-BP-tetraol, r-7,t-8,c-9,10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; TMEchrysene-tetraol, r-1,t-2,3,c-4-tetramethoxy-1,2,3,4-tetrahydrochrysene; GC-NICI-MS, gas chromatogaphy-negative ion chemical ionization mass spectrometry; GC-NICI-MS-SIM, gas chromatogaphy-negative ion chemical ionization mass spectrometry-selected ion monitoring.

presence of methyl sulfinyl carbanion (29, 30) and analysis of the derivatized tetraols by HPLC or gas chromatography-negative ion chemical ionization mass spectrometry-selected ion monitoring (GC-NICI-MSSIM). The set of BP-tetraol isomers shown in Figure 1 has been used as a model system for investigation of the methodology. Each BP diol epoxide forms adducts with hemoglobin and DNA that can be hydrolyzed to two isomeric tetraols (trans or cis). Methylation of each of these tetraols gives the corresponding tetramethyl ethers. Figure 1 shows the stereochemistry of the major tetraols that can be formed upon acid hydrolysis of adducts from r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) or r-7,t-8-dihydroxy-c-9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE). It also shows the structures of the corresponding tetramethyl ethers. This paper describes (a) the stability of BP-tetraols under acid hydrolysis conditions; (b) optimal hydrolysis conditions for release of BP-tetraols from DNA and globin; (c) derivatization of individual BP-tetraols and other PAH-tetraols to tetramethyl ethers; (d) GC-NICIMS analysis of the tetramethyl ethers; (e) application of the assay for quantifying globin or DNA adducts from mice treated with [3H]BP or unlabeled BP and comparison of the GC-NICI-MS-SIM results to those obtained with conventional scintillation counting methodology or by postlabeling via [3H]CH3I; and (f) detection of BP-

510 Chem. Res. Toxicol., Vol. 9, No. 2, 1996

tetraols and chrysene-tetraols in hydrolysates of human globin.

Melikian et al. Scheme 1. Experimental Design of in Vivo Studies with DNA and Globin of Mice

Experimental Section Caution. PAH and many of their 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-BP-tetraol), r-7,t-8,10,c-9,-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-syn-BPtetraol), r-7,t-8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (cis-anti-BP tetraol), r-7,t-8,c-9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (cis-syn-BP-tetraol), and (()-antiBPDE (0.53 Ci/mmol) were supplied by the National Cancer Institute’s Chemical Carcinogen Reference Standard Repository, Midwest Research Institute (Kansas City, MO). r-1,t-2-Dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydrochrysene (anti-chryseneDE); r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (anti-5-MeCDE); r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4tetrahydro-6-methylchrysene (anti-6-MeCDE); r-1,t-2-dihydroxyt-3,4-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene (anti-B[a]ADE); and r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (anti-B[c]PDE) were synthesized as described previously (31, 32). [3H]BP, specific activity 25 Ci/mmol (diluted with unlabeled BP to give a specific activity of 67 mCi/mmol), and [3H]CH3I, 80 Ci/mmol (in toluene solution), were purchased from Amersham Corp. (Arlington Heights, IL). CH3I and NaH (60% dispersion in mineral oil) were obtained from Aldrich Chemical Co. (Milwaukee, WI). HCl (6 N), DMSO, and vacuum reaction tubes were bought from Pierce Chemical Co. (Rockford, IL). Preparation of (()-anti-BPDE-DNA Adducts. A solution of about 50 µg of (()-BPDE in 0.1 mL of acetone was added to purified calf thymus DNA (1 mg/mL in 0.01 M Tris buffer, pH 7.4) and incubated at 37 °C for 6 h. The unreacted BPDE that hydrolyzed to BP-tetraols was extracted five times with 2 volumes of EtOAc. DNA was precipitated by adding EtOH. Stability of BP-Tetraols under Acid Hydrolysis Conditions. Five micrograms of each isomer of BP-tetraol (purified by HPLC) in 500 µL of MeOH was transferred into vials. After removal of the MeOH, 0.5 mL of 0.1 N HCl was added to each vial and the mixtures were incubated at either 80 or 90 °C for 3 h. After cooling, each sample was adjusted to pH 5 and extracted five times with 2 volumes of EtOAc. The solvent was removed under N2, and the residue was dissolved in MeOH for HPLC analysis (system 1). Preparation of PAH-Tetraols via Hydrolysis of PAHDiol Epoxides. Each PAH-diol epoxide, anti-5-MeCDE, anti6-MeCDE, anti-B[a]ADE, and anti-B[c]PDE (200 µg), was dissolved in a minimal volume of EtOH (about 1-2 mL), added to 10 mL of 0.1 N HCl, and stirred at room temperature overnight. As an exception, anti-chrysene-DE was dissolved in 200 µL of THF and hydrolyzed in 0.9% formic acid overnight at room temperature. After the pH was adjusted to 5, the PAHtetraols were extracted five times with 2 volumes of EtOAc and purified by HPLC (system 4). The concentration of each tetraol was determined by UV. The major product in each case was the trans-anti-tetraol resulting from trans-ring opening as previously reported (11-13, 33). Animal Treatments. Seven- to eight-week-old female Crl: CD-1(1CR)BR mice were obtained from Charles River Breeding Laboratories (North Wilmington, MA). In experiment 1, [3H]BP (0.012 mmol, 0.8 mCi) in 0.2 mL of corn oil was administered by gavage to each of 28 mice. In experiment 2, each of 10 mice received the same dose of unlabeled BP in 0.2 mL of corn oil by gavage. Animals were sacrificed 24 h after treatment. Blood samples were collected in EDTA-containing vacutainers, and the livers and lungs were isolated and frozen immediately. DNA was isolated from tissues and globin from blood cells. They were analyzed as outlined in Scheme 1. Human Blood Samples. Some samples were obtained from smokers and nonsmokers through a collaborative agreement

between Dr. David Ciavarella of the Hudson Valley Blood Bank and Dr. Sharon E. Murphy of the American Health Foundation. All procedures were approved by the Institutional Review Boards of both institutions. Other samples were obtained courtesy of Dr. Nancy Haley, Metropolitan Life Insurance Co. Isolation of DNA from Tissues. DNA was isolated as described previously (34). In brief, the tissues were homogenized in 1 mM EDTA containing 1% sodium dodecyl sulfate. The homogenates were incubated with proteinase K (500 µg/mL) and extracted with phenol that contained 0.1% 8-hydroxyquinoline as an antioxidant and then with phenol/CHCl3/isoamyl alcohol and CHCl3/isoamyl alcohol. DNA was precipitated by adding EtOH. Residual unbound radioactive material was removed by repeated washing of the DNA with acetone and then with ether. Isolation of Globin from Mouse Blood. Red blood cells were isolated by centrifugation (2000 rpm, 4 °C, 10 min) and washed three times with 1 volume of physiological saline. Cells were lysed by adding 1 volume of H2O and 1 volume of 0.67 M sodium phosphate buffer (pH 6.5). Cell debris was pelleted by centrifugation (13 500 rpm, 25 min). Globin was precipitated with acetone as follows: the supernatant was added dropwise to 20 volumes of ice-cold acetone/15 mM HCl, mixed vigorously, and allowed to stand at -20 °C for 30 min. The globin precipitate was separated by centrifugation (2000 rpm, 10 min), washed three times with acetone, and then redissolved in a small volume of H2O. The precipitation was repeated twice to remove metabolites of [3H]BP and heme (21). At each step the total radioactivity released in acetone was measured. After three consecutive precipitations of globin, radioactive material was no longer detected in the acetone wash. Isolation of Globin from Human Blood. Red blood cells were isolated and lysed as described above. After centrifugation to remove cellular debris, the supernatant was dialyzed against H2O at 4°C for 48 h. Globin was precipitated by dropwise addition of the hemoglobin solution to 20 volumes of ice-cold acetone/15 mM HCl, with vigorous stirring. The precipitate was filtered and washed with ice-cold acetone. An aliquot of each globin sample was subjected to acid hydrolysis. Hydrolysis of DNA Adducts. Calf thymus DNA samples modified with (()-anti-BPDE in vitro or DNA isolated from the lungs and livers of mice treated with [3H]BP (0.66 mg/mL of H2O) were hydrolyzed in 0.1 N HCl (pre-extracted with EtOAc) for up to 4 h. Hydrolysis was carried out at 80 °C under vacuum (using vacuum reaction tubes) or under N2 or air. Aliquots were taken hourly and, after cooling, the released tetraols were extracted five times with 2 volumes of EtOAc. The radioactivity in the extracts was measured and, after removal of solvent under N2, the residue was dissolved in MeOH and analyzed by HPLC (system 2).

Detection of PAH Adducts by GC-MS and [3H]CH3I Scheme 2. Preparation of TME-PAH-Tetraols via CH3I

Hydrolysis of Globin Adducts. An aqueous solution of globin from mice treated with [3H]BP (5 mg/mL) was transferred into vacuum reaction tubes and hydrolyzed in 0.05-0.1 N HCl for 0-3 h at 80 or 90 °C. [3H]BP-tetraols liberated from globin were extracted and analyzed as described above. On the basis of the results of this experiment, the optimum conditions for hydrolysis of globin adducts were established as 0.1 N HCl for 3 h at 80 °C under vacuum. These conditions were used for hydrolysis of adducts in human globin samples. The pH was adjusted to 5, and the hydrolysate was extracted five times with 2 volumes of EtOAc. With each set of experiments a control sample that consisted of all reagents except globin was run. Permethylation of PAH-Tetraols. PAH-tetraol standard samples or PAH-tetraols released from biological samples were converted to tetramethyl ether derivatives according to Scheme 2 as previously described (28). Briefly, methyl sulfinyl carbanion was prepared by heating 100 mg of NaH (60% suspension in mineral oil) in 1 mL of DMSO at 90 °C under N2 for 20-30 min until generation of H2 ceased. Freshly prepared methyl sulfinyl carbanion solution (50 µL) was added to approximately 20 µg of the specified PAH-tetraol isomer dissolved in 50 µL of DMSO. The mixture was stirred for 2 min at room temperature, and CH3I (50 µL, 0.8 mmol) was added. After another 2 min, 1 mL of H2O was added and the tetramethyl ether derivatives of the PAH-tetraols were extracted with benzene. For labeling the BP-tetraols as [3H]tetramethyl ether derivatives, [3H]CH3I solution (50 µL, 0.05 mCi, 0.006 mmol) was added to the mixture of BP-tetraols (estimated to be less than 0.1 µg) in 50 µL of DMSO and 50 µL of the methyl sulfinyl carbanion solution. The [3H]CH3I derivatization was carried out in well-ventilated hoods; benzene was removed from the extracts of the [3H]tetramethyl ethers by a stream of N2. Precautions were taken to avoid release of radioactive materials into the atmosphere by passing the N2 stream through two cold traps above the freezing point of benzene and finally through H2O before it was released. Analysis of TME-BP-Tetraols from Mice. Benzene extracts containing 3H-labeled TME-BP-tetraols from experiment 2 (Scheme 1) were concentrated to dryness, redissolved in MeOH, and analyzed by HPLC (system 3). One-milliliter fractions were collected, and radioactivity was measured by scintillation counting. The benzene extracts were also analyzed by GC-NICI-MSSIM as described below. Analysis of TME-PAH-Tetraols from Human Globin. The extracts were concentrated to dryness, redissolved in MeOH, and further purified by HPLC (system 4). The fraction eluting from 25 to 45 min, which included the elution positions of four and five ring tetraols, was collected and concentrated under a stream of N2, and the residue was redissolved in benzene and analyzed by GC-NICI-MS-SIM. HPLC Analysis Conditions. Four solvent systems were employed for reversed-phase HPLC on a 5 µm Ultrasphere ODS C18 column (25 cm × 4.6 mm, Beckman, Fullerton, CA). System 1: isocratic elution with 55:45 MeOH/H2O for 30 min followed by a linear gradient to 100% MeOH in 20 min, at a flow rate of 1 mL/min. System 2: isocratic elution with 15:85 MeOH/H2O for 30 min, a linear gradient to 45:55 MeOH/H2O in 10 min, isocratic for 35 min, a linear gradient to 55:45 MeOH/H2O in 10 min,

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 511 isocratic for 25 min, and, finally, a linear gradient to 100% MeOH, at a flow rate of 1 mL/min. System 3: isocratic elution with 100% H2O for 10 min, a linear gradient to 50:50 MeOH/H2O in 5 min, isocratic for 30 min, a linear gradient to 80:20 MeOH/H2O in 40 min, and finally a linear gradient to 100% MeOH in 20 min, at a flow rate of 1 mL/min. System 4: isocratic elution with 40:60 MeOH/H2O for 10 min followed by a linear gradient to 100% MeOH in 40 min at a flow rate of 1 mL/min. GC-NICI-MS Analysis Conditions. Permethylated PAHtetraol standards or permethylated tetraols released from DNA or globin were analyzed by GC-NICI-MS or GC-NICI-MS-SIM on a Hewlett-Packard Model 5988A system using on-column injection with a 10-µL syringe equipped with a 15-cm fused silica needle. An SE-54 (30 m × 0.25 mm i.d. × 0.25 µm film thickness) capillary GC column (Alltech Associates, Deerfield, IL) was interfaced directly with the MS source. The GC oven temperature was held at 50 °C for 2 min, followed by programming to 300 °C at 20 °C/min. The temperature was then kept at 300 °C for 20 min. MS conditions were as follows: ion source, 150 °C; emission current, 250 µA; electron energy, 110 eV. Ultrahigh-purity methane was the reagent gas. Solvent blanks were injected between runs to ensure that there was no carryover from one sample to the next.

Results and Discussion Stability of BP-Tetraol Isomers under Acid Hydrolysis Conditions. Shugart et al. have demonstrated that BPDE adducts to globin or DNA are hydrolyzed to BP-tetraols under mild acid hydrolysis conditions (35, 36). Four BP-tetraol isomers can be liberated. We have studied the stability of these tetraols under the hydrolysis conditions. The results are summarized in Table 1. trans-anti-BP-tetraol (Figure 1) was the most stable tetraol under these hydrolysis conditions; about 80% of the original material was recovered unchanged. cis-antiBP-tetraol was unstable; approximately 70% of it was converted to trans-anti-BP-tetraol. Similarly, trans-synBP-tetraol was unstable and epimerized to cis-syn-BPtetraol (59-67%) and only about 16% of the original compound remained. In contrast, cis-syn-BP-tetraol did not epimerize but decomposed to unknown products which eluted from the HPLC column after cis-syn-BPtetraol (data not shown). The instability of cis-syn-BPtetraol was reported previously (37, 38). Jansen et al. have shown a similar epimerization of BP-tetraols under acid hydrolysis conditions (39). Epimerization of BPtetraols occurred immediately after 0.1 N HCl was added; heating was not a major factor. However, when the hydrolysis temperature was increased from 80 to 90 °C, the decomposition of cis-syn-BP-tetraol into unknown products increased from 23% to 38%. cis-anti-BP-tetraol exists in a half-chair conformation with C7-H and C8-H being pseudodiequatorial and C10OH being pseudodiaxial (40). This causes an unfavorable steric interaction between C10-OH and C8-OH, which is relieved upon epimerization to trans-anti-BP-tetraol. Similar relief of 1-3 transannular interactions between hydroxyl groups can account for the epimerization of trans-syn-BP-tetraol. The results of these experiments indicate that transanti-BP-tetraol will be the major product detected upon hydrolysis of adducts formed from anti-BPDE, independent of their being trans or cis adducts. Similarly, synBPDE adducts will give mainly cis-syn-BP-tetraol. Derivatization of Racemic BP-Tetraols to Tetramethyl Ethers. The application of GC-MS for detect-

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Melikian et al.

Table 1. Stability of BP-Tetraols under Acid Hydrolysis Conditionsa,b % of each tetraol recovered after hydrolysis at 80 °C (90 °C) tetraol subjected to hydrolysis trans-anti-BP-tetraol cis-anti-BP-tetraol trans-syn-BP-tetraol cis-syn-BP-tetraol unknown products trans-anti-BP-tetraol cis-anti-BP-tetraol trans-syn-BP-tetraol cis-syn-BP-tetraol

80(72) 68(78) 4 7(11)

12(19) 25(13) 3 1

6(9) 7(9) 16(16) 14

2 67(59) 55(50)

10 (25) 23 (38)

a Hydrolysis was performed in 0.1 N HCl for 3 h at 80 or 90 °C in open air. b BP-tetraols were assigned on the basis of HPLC retention times only.

Figure 2. HPLC elution profiles (system 3) of tetramethyl ethers of BP-tetraols prepared by derivatization with CH3I: (A) trans-anti-TME-BP-tetraol; (B) cis-anti-TME-BP-tetraol; (C) trans-syn-TME-BP-tetraol; and (D) cis-syn-TME-BP-tetraol. Table 2. HPLC and GC Retention Times of BP-Tetraols and Their Corresponding Tetramethyl Ethers retention time (min) compound

HPLCa

trans-anti-BP-tetraol cis-anti-BP-tetraol trans-syn-BP-tetraol cis-syn-BP-tetraol

38.0 42.6 39.6 51.6

trans-anti-TME-BP-tetraol cis-anti-TME-BP-tetraol trans-syn-TME-BP-tetraol cis-syn-TME-BP-tetraol

85.7 86.5 92.0 91.2

GCb

22.54 23.43 21.67 22.54

a Using HPLC elution system 3. b The retention times change as portions of the GC column are broken off to remove contamination.

ing and quantifying PAH-tetraols requires derivatization to increase volatility and, potentially, to increase sensitivity. In the course of developing the method several perfluorinated derivatives of BP-tetraols were prepared and analyzed. Under the derivatization conditions, the tetraols tended to aromatize or oxidize, yielding more than a single product. However, methylation via CH3I (Scheme 2), described by Hakomori for glycolipids, is applicable to the derivatization of PAH-tetraols and yields only a single product (28, 29). HPLC profiles of the tetramethyl ethers obtained via derivatization of the four BP-tetraols are illustrated in Figure 2. The HPLC analyses demonstrate that derivatization of each tetraol was complete without formation of appreciable amounts of side products. Each tetraol was derivatized primarily to a single less polar product (Figures 2 and Table 2), and each showed a pyrene absorption in its UV spectrum, similar to the UV spectra of the BP-tetraols (data not shown). HPLC elution times of the BP-tetraols, as well as GC and HPLC retention

Figure 3. GC-NICI-MS of trans-anti-TME-BP-tetraol.

times of their corresponding tetramethyl ethers, are shown in Table 2. Two of the tetramethyl ethers, transanti-TME-BP-tetraol and cis-syn-TME-BP-tetraol, had GC retention times of 22.54 min, yet they can be separated by HPLC (85.7 and 91.2 min, respectively; Table 2). Thus, applying HPLC before GC-NICI-MS can give some information about the stereochemistry of the liberated tetraols. The NICI-MS of trans-anti-TME-BP-tetraol is illustrated in Figure 3. The characteristic MS shows a molecular ion at m/z 376 (M)- which loses one molecule of CH3OH to produce m/z 344 (M - 32)-. The ion at m/z 344 again eliminates CH3OH, resulting in m/z 312 (M - 64)-, or loses two CH3O, producing m/z 314 (M 62)-. Subsequently, the ion at m/z 312 loses one molecule of HCHO to form m/z 282 (M - 94)- or two molecules of HCHO to form m/z 252 (M - 124)-. Similar fragmentation patterns were observed for the other TMEBP-tetraols. These data are consistent with previously reported MS data for TME-BP-tetraols (28). Each major tetraol product, formed by trans-ring opening in the hydrolysis of several other PAH anti-diol epoxides, was converted to its corresponding tetramethyl ether derivative. Their GC retention times and fragmentation patterns are summarized in Table 3. Tetramethyl ethers of PAH-tetraols were superior to the other derivatives studied because (a) the availability of [3H]CH3I at high specific activity (80 Ci/mmol) complements the GC-NICI-MS-SIM analysis; (b) they are stable with shelf lives of greater than 2 months, whereas other derivatives such as trimethylsilyl ethers are labile (27); (c) their preparation is relatively simple and rapid and proceeds in good yield (80-90% for conversion of 1 ng of corresponding tetraol); and (d) they can be detected with

Detection of PAH Adducts by GC-MS and [3H]CH3I

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 513

Table 3. GC Retention Times and NICI-MS Data for Tetramethyl Ethers of PAH-Tetraols compound

GC retention time (min)

NICI-MS m/z (rel intensity)

TME-chrysene-tetraola TME-B[a]A-tetraol TME-B[c]P-tetraol TME-5-MeC-tetraol TME-6-MeC-tetraol

18.6 18.3 18.8 19.4 19.5

353 (M- + 1, 28), 352 (M-, 10), 351 (M- - 1, 20), 336 (34), 298 (11), 287 (100), 257 (17) 352 (M-, 100) 352 (M-, 100), 320 (17) 367 (M- + 1, 33), 366 (M-, 23), 365 (M- - 1, 76), 350 (32), 301 (100), 271 (25), 229 (4) 367 (M- + 1, 60), 366 (M-, 26), 365 (M- - 1, 84), 350 (40), 301 (100), 304 (30), 271 (10), 257 (100), 229 (4)

a

EI-MS (70 eV), 352 (M+, 3), 254 (M+ - [CH3OCHOCH3], 30), 249 (M+ - [CH3OCHOCH3], 45); similar to that reported in ref 28. Table 4. Binding of [3H]BP Metabolites to the DNA of Mouse Lung, Liver, and Globin and Time Course of EtOAc-Extractable Metabolites Released upon Acid Hydrolysisa,b [3H]BP

tissue

total binding of metabolites to DNA or globin (pmol/mg of DNA or globin)

liver lung blood

113 14.6 3.9c

EtOAc-extractable [3H]BP metabolites released from DNA upon HCl hydrolysis (pmol/mg of DNA or globin) after hydrolysis time of 1h 2h 3h 4h 9.3 7.6 0.32

12.3 9.1 0.41

12.6 8.8 0.41

12.5 9.2 0.40

a DNA was isolated from a group of mice 24 h after oral administration of 0.012 mmol of [3H]BP/mouse (experiment 1, Scheme 1); hydrolysis was carried out with 0.1 N HCl at 80 °C under vacuum. b Mean of two measurements. c Estimated on the basis of the weight of globin.

high sensitivity by GC-NICI-MS-SIM. The GC-NICI-MSSIM detection limit for trans-anti-TME-BP-tetraol was about 1 fmol per injection, and intensities of selected ions (m/z 376, 282, 252) increased linearly at concentrations between 1 fmol and 1 pmol. The detection limit of BPtetraols by [3H]CH3I postlabeling was approximately 1 fmol (500 dpm). The GC-NICI-MS-SIM detection limits for TME-chrysene-tetraol and TME-B[a]A-tetraol were approximately 50 and 1 fmol per injection, respectively. Application of Methodology to Mice Treated with BP. As summarized in Scheme 1, two experiments were carried out to test the proposed methodology for DNA and globin analysis. In experiment 1, mice were treated with [3H]BP and the released [3H]BP-tetraols were either analyzed by HPLC or derivatized and analyzed by GCNICI-MS-SIM. In experiment 2, mice were treated with unlabeled BP and the unlabeled tetraols were derivatized with [3H]CH3I and analyzed by HPLC. a. Detection of BP-Tetraols by Radioflow HPLC or GC-MS. The total binding of [3H]BP metabolites to DNA and globin (experiment 1, Scheme 1) and the levels of released EtOAc-extractable metabolites as a function of hydrolysis time are summarized in Table 4. Maximal release of EtOAc-extractable radioactivity was achieved after 2 h. Total binding of [3H]BP metabolites to liver DNA exceeded that to lung DNA. However, only about 10% of the bound material was released as EtOAcextractable from liver DNA or globin, while hydrolysis of lung DNA released about 60% of the total radioactivity. These observations are in agreement with previous studies which indicate that about 90% of the radioactivity associated with liver DNA is from unknown products (41, 42). The HPLC profile of EtOAc-extractable materials from lung DNA is illustrated in Figure 4. [3H]BP-tetraols contributed about half of the products in the extract and the remaining were unknowns (peak X of Figure 4.). These unidentified products were also present in liver DNA and globin but to different extents (data not shown). Recovery of [3H]BP-tetraols was enhanced by about 25% when hydrolysis was performed under N2 or vacuum. Lowering the hydrolysis temperature also improved the yield of tetraols. For example, EtOAc-extractable materials released from globin incubated under vacuum at 80 or 90 °C were about the same, but the recovery of tetraols at 80 °C was about 10% greater than at 90 °C.

Figure 4. HPLC radiochromatogram of EtOAc-extractable products released upon acid hydrolysis of DNA isolated from lungs of mice treated with [3H]BP (experiment 1, Scheme 1).

Figure 5. GC-NICI-MS-SIM of tetramethyl ethers of (A) transanti-BP-tetraol standards; (B) [3H]BP-tetraol released from the lung DNA of mice treated with [3H]BP (experiment 1, Scheme 1); and (C) [3H]BP-tetraol released from mouse globin treated with [3H]BP (experiment 1, Scheme 1).

The [3H]BP-tetraols released from globin and DNA were derivatized to their corresponding tetramethyl ethers and also were analyzed by GC-NICI-MS-SIM (Figure 5B,C). The selected ions monitored were m/z 376, 282, and 252. The results shown in Figure 5 confirm the identity of the [3H]BP-tetraols released from mouse DNA and globin. The concentrations of these analytes were estimated from the intensities of total selected ions and were comparable with levels determined by the conventional radioactive tracer method (Table 5). b. Detection of BP-Tetraols by [3H]CH3I Postlabeling. The HPLC radiogram of the [3H]tetramethyl

514 Chem. Res. Toxicol., Vol. 9, No. 2, 1996

Melikian et al.

Table 5. Levels of BP-Tetraols Released from Mouse Globin and Lung and Liver DNA pmol BP-tetraols/mg DNA or globin by radioflow by radioflow by GC-NICI-MS-SIM HPLC of HPLC of 3 a a 3 [ H]BP-tetraols of TME-BP-tetraols [ H]TME-BP-tetraolsb lung liver globin

3.4 ( 0.1c 3.7e 0.2

2.9d NAf 0.3g

2.9e 4.1e

a Experiment 1, Scheme 1. b Experiment 2, Scheme 1. c Mean ( SE, n ) 3. d Estimated amount. e Mean of two analyses. f Not analyzed. g Using d12-BP-tetraol as internal standard.

Figure 7. GC-NICI-MS-SIM (selected ions for TME-BP-tetraol) of HPLC-purified permethylated derivatives of globin hydrolysates from a nonsmoker. The inset shows the chromatogram of the same sample for the major selected ion before HPLC purification.

Figure 6. HPLC radiochromatograms of BP-tetraols after derivatization with [3H]CH3I: (A) [3H]trans-anti-TME-BP-tetraol standard; (B) [3H]TME-BP-tetraols obtained upon hydrolysis of calf thymus DNA modified with (()-anti-BPDE; and (C) [3H]TME-BP-tetraols obtained upon hydrolysis of lung DNA after treatment of mice with BP (experiment 2, Scheme 1).

ethers of BP-tetraols released from mouse lung DNA (experiment 2, Scheme 1) is shown in Figure 6C. This profile had the identical peaks 1 and 2 that were observed upon HPLC analysis of the standard (Figure 6A) or analysis of [3H]TME-tetraols released from calf thymus DNA modified with anti-BPDE (Figure 6B). Peak 1 of Figure 6C coeluted with trans-anti-TME-BP-tetraol standard (Figure 2A). This indicates that the major BPtetraol released from mouse lung DNA is derived from anti-BPDE. Quantitative results, which are summarized in Table 5, show good agreement between the conventional radioactive tracer method (experiment 1), GCNICI-MS-SIM, and postlabeling via [3H]CH3I (experiment 2). This demonstrates the feasibility of postlabeling via [3H]CH3I for animal model studies. Levels of BPtetraols quantified from experiment 1 or 2 were similar to the concentration of [3H]BPDE-DNA adducts reported previously in a similar bioassay (41, 42). Application of GC-NICI-MS-SIM Methodology to Human Hemoglobin. Results of analysis of hemoglobin from a nonsmoker are illustrated in Figures 7 and 8,

Figure 8. GC-NICI-MS-SIM (selected ions for TME-chrysenetetraol) of HPLC-purified permethylated derivatives of globin hydrolysates from a nonsmoker. The inset shows the chromatogram of the same sample for the major selected ion before HPLC purification.

which demonstrate the presence of TME-BP-tetraol and TME-chrysene-tetraol in the hydrolysates. These analytes cochromatographed with standard samples (data not shown). TME-BP-tetraol could be either trans-antior cis-syn. The insets in Figures 7 and 8 show the results of the GC-NICI-MS-SIM analysis without prepurification by HPLC. BP-tetraol was detected in all 20 samples, while chrysene-tetraol was detected in 7 of 10 smokers and 2 of 5 nonsmokers. Control samples had no detectable amounts of BP-tetraols or chrysene-tetraols. Further studies are in progress on quantitation of these adducts in human hemoglobin. Some samples showed evidence for the presence of B[a]A-tetraol, but this requires confirmation. These results are consistent with previous analyses of human hemoglobin by GC-NICI-MS-SIM of trimethylsilylated tetraols, in which tetraols derived from BP and chrysene have been detected (27, 43, 44). The tetra-

Detection of PAH Adducts by GC-MS and [3H]CH3I

methyl ether derivatives employed in our work may have advantages over trimethylsilylated tetraols because of their greater stability. Conclusion. We have defined the optimum conditions for hydrolysis of BP DNA and globin adducts to the corresponding tetraols and have established the stabilities of these tetraols under the hydrolysis conditions. We have found that derivatization of the tetraols to their tetramethyl ethers is a superior method with many advantages. These stable derivatives can be detected with great sensitivity by either [3H]CH3I postlabeling or GC-NICI-MS-SIM. The method also appears to be applicable to other PAH tetraols. We have validated the postlabeling and GC-NICI-MS-SIM methods by comparing them to conventional radiochemical techniques for determination of BP DNA and globin adducts in mice. Moreover, the GC-NICI-MS-SIM method is applicable to hydrolysates of human hemoglobin, in which we have confirmed the presence of adducts of anti-diol epoxides of BP and chrysene. It is likely that this methodology will ultimately be applicable to quantitation of a broad range of PAH diol epoxide adducts in human samples.

Acknowledgment. This study was supported by Grants CA-29580 and CA-44377 from the U.S. National Cancer Institute. We thank Elizabeth Appel for preparation of the manuscript and schemes.

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