Chem. Res. Toxicol. 1990, 3, 111-117
111
Benzo[ a Ipyrene Diol Epoxide Adduct Formation in Mouse and Human Hemoglobin: Physicochemical Basis for Dosimetry Stephen Naylor,' Liang-Shang Gan,t Billy W. Day, Roberta Pastorelli,§ Paul L. Skipper, and Steven R. Tannenbaum* Room 56-309, Department of Chemistry, Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received September 15, 1989
T h e interactions of human hemoglobin (hHb) with the anti-diol epoxide of benzo[a]pyrene
(aBaPDE) and with the corresponding tetrols formed by hydrolysis of the epoxide were investigated with the aim of characterizing the covalent adducts formed by reaction of the epoxide with the protein. The major product (80% of the total) was determined t o be a n ester resulting from oxirane ring opening by one or several (unidentified) carboxylate group(s). Minor products were characterized as adducts formed by reaction with amino or heterocyclic nitrogen by comparison of their UV spectra with those of model compounds. There was no evidence for reaction with cysteine. Formation of ester adducts by aBaPDE with mouse hemoglobin (mHb) following administration of B a P t o mice was also investigated. It was found t h a t esters constituted t h e majority of the adducts formed by aBaPDE and a substantial fraction of the total adducts formed. T h e esters formed by m H b were significantly less stable than those formed by hHb, both in vivo and in vitro. T h e instability of m H b ester adducts is believed by be responsible for differences among previous descriptions of the in vivo binding of B a P t o mHb.
Introduction Benzo[a]pyrene (BaP),' a ubiquitous environmental contaminant, is a procarcinogen that requires metabolic activation by cytochrome P-450 and epoxide hydrolase enzymes (1-3). Certain BaP metabolites are known to bind covalently to both DNA and proteins, as does the ultimate carcinogenic metabolite, identified as (+)-7&8a-dihydroxy-9n,l0n-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (aBaPDE) (1) (for a recent review see ref 3). Interest in the binding of electrophilic metabolites of carcinogens or direct-acting carcinogens to proteins arises from the usefulness of the products in molecular dosimetry. Although protein adducts are not thought to be etiologically important, they are often a good indirect measure of DNA adduct formation ( 4 ) . Exposure assessment can also be greatly facilitated by the analysis of protein adducts. Studies of the covalent interaction of carcinogens with blood proteins have focused mainly on relatively small alkylating agents, including alkyl halides (5) and sulfonates (6), various epoxides (7, 8), and alkanediazonium ions generated from N-nitrosoamines (9). In the case of larger molecules such as the polycyclic aromatic hydrocarbons (PAH), where sites of reaction on the protein are expected to be subject to steric constraints, only a limited amount of work concerning site specificity has been reported (10). The reaction of BaP metabolites with Hb has been investigated ( I 1-15), but the results have been inconclusive and contradictory. Acid hydrolysis of the adducted protein yields isomeric 7,8,9,10-tetrahydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (BaP tetrols), as does acid hydrolysis of BaP-DNA adducts (16, 17). The reported yields of BaP
* To whom correspondence should be addressed. Present address: MRC Toxicology IJnit, Carshalton, Surrey SM5 4EF, England. Present address: Glaxo Inc., Research Triangle Park, NC 27709. *Present address: Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy. f
0893-228x/90/2703-0111$02.50/0
tetrols obtained from the acid hydrolysis of the adducted protein are highly variable. No structural characterization of the adducts has been reported. We undertook the present study to characterize the major adducts of aBaPDE with Hb and to investigate the chemical behavior of these adducts. We investigated first of all the reaction in vitro of the ultimate carcinogen aBaPDE with human hemoglobin (hHb). A preliminary report of one aspect of this work proving that the major adduct that is formed is a carboxylic ester has already appeared (18). We also investigated the binding in vivo of BaP metabolites to mouse hemoglobin (mHb) in an effort to develop a satisfactory animal model. We observed that esters formed by alkylation of carboxylate residues by aBaPDE also account for a substantial fraction of the binding of BaP to mHb and that mHb esters are considerably less stable than hHb esters. The effect that the increased lability of mHb esters has on the interpretation of previous studies of the binding of BaP to hemoglobin through its anti-diol epoxide metabolites is discussed.
Experimental Section Instrumentation. Gas chromatography-mass spectral data were obtained on a Hewlett-Packard 5987A GC-MS with a standard EI/CI source. Methane (99.999%, Med-Tech) was the moderating bath gas for NICI and reagent gas for PCI experiments, with the source pressure set at 0.4-0.6 Torr for NICI and 0.8-1 Torr for PCI. Electron energy was 170 eV, and the source temperature was 150 "C for NICI and 200 "C for PCI experiments, Abbreviations: BaP, benzo[a]pyrene; aBaPDE, 7@,8a-dihydroxy-
9a,10a-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene and 7a,8p-dihydroxy9@,10~-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene; BaP tetrol I, r-7,c10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BaP tetrol11, r-7,t-8,t-9,t-10-tetrahydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene; PAH,
polycyclic aromatic hydrocarbons; Hb, hemoglobin;hHb, human hemoglobin, mHb, mouse hemoglobin; MCPBA, m-chloroperbenzoicacid; PBS, phosphate-buffered saline; IAC, monoclonal antibody immunoaffinity column chromatography; GSH, glutathione; LSC, liquid scintillation counting.
0 1990 American Chemical Society
112 Chem. Res. Toxicol., Vol. 3, No. 2, 1990 respectively. Electron energy and source temperature were 70 eV and 240 "C, respectively, for E1 experiments. The emission current was set a t 300-400 pA. The gas chromatograph was interfaced to the MS through a direct capillary inlet maintained a t 240 "C. The injection port temperature was 250 "C. The column was either a 15 m or 30 m X 0.25 mm DB-1 fused silica capillary (J & W Scientific). Ultra-high-purity helium (Med-Tech) was the carrier gas with a flow rate of ca. 2 mL/min. Both the helium and methane lines were fitted with gas purifiers (Matheson, no. 6406). Tetrols and derivatives were pertrimethylsilylated with Trisyl Z (Pierce). Samples were injected in the splitless mode (splitless valve open for 0.5 min) and were analyzed with an initial oven temperature of 180 "C for 1 min, temperature ramp to 330 "C a t 20 "C/min, and a 4-min isothermal phase a t 330 "C. Fast atom bombardment mass spectra were taken on a Kratos MS-50 equipped with a magnet of mass range 10 000 Da a t full (8-keV) accelerating voltage. The atom beam was provided by an Ion Tech FAB gun operating with xenon a t 8 keV with a current of 30-40 MA. The magnet was scanned a t 30 s/decade. The sample matrix was 1:l thioglycerol-glycerol. HPLC analyses were performed on a Hewlett-Packard 1090 liquid chromatograph controlled by a Hewlet-Packard 300 Chem Station. UV detection was by a Hewlett-Packard 1040 diode array detector. A CISNova-Pak (Waters) reverse-phase column, or a Spheri-5 microbore C18 column (Brownlee), utilizing a gradient of 0.1% trifluoroacetic acid (TFA)/H,O (solvent A) to 0.1% TFA/MeCN (solvent B) (time: 0,85% A; 10 min, 75% A; 34 min, 72% A; 35 min, 72% A; 65 min, 70% A; 75 min, 100% B) a t a flow rate of 0.7 and 0.15 mL/min, respectively, was used in the separations of both the synthetic products and in LC analyses of the globin digests. An analytical C4 Vydac 300-A pore size column (Rainin) using a gradient of 4:1 H20-MeCN/O.l% TFA (A) to 3:7 H20-MeCN/O.l% TFA (B) (time: 0-5 min, 65% A; 35 min, 59% A; 60 min, 55% A; 65 min, 50% A) a t a flow rate of 2 mL/min was used for globin chain separation (19). The detector was operated a t a sampling interval of 640 ms over the range of 200-400 nm. Peak signals were monitored a t the wavelength approriate for the given chromophore by using a 4-nm window with a 2-nm band-pass. The base-line reference was the average of a 100-nm window centered at 550 nm. Spectra of peaks 10.5 mAU were stored by the Chem Station for later analysis. Fluorescence spectra were taken a t room temperature on a SPEX Fluorolog double monochromator instrument as previously described (20). Chemicals. [7-14C]-(f)-aBaPDE(specific activity 57.1 or 53 mCi/mmol) and (*)-aBaPDE were purchased from the NCI Chemical Carcinogen Repository maintained by Chemsyn Science Laboratories and the Midwest Research Institute, respectively. [1,3,6-3H]BaP (specific activity 55 Ci/mmol) was obtained from New England Nuclear Research Products. H,'Q (95-98% isotopic purity) was purchased from either Aldrich Chemical Co. or MSD. Other chemicals and reagents were obtained from Sigma Chemical Co. All solvents were purchased from J. T. Baker Chemical Co. and were of the highest available purity. BaP tetrols I and I1 were prepared by hydrolysis of aBaPDE (1mg, 3.3 pmol) by stirring in 10 mL of 0.9% formic acid (pH 3) overnight a t room temperature. The aqueous solution was extracted with EtOAc (3 X 10 mL). The organic layers were combined, concentrated, dissolved in 41 H20-MeOH, and purified by HPLC to give a 101ratio of 1:II in quantitative yield: NICI-MS (per TMS derivatives), m/z (% base) 446 (M - TMSzO, 100), both tetrols. N-t-BOC-alanyl ester of BaP tetrol I was prepared from [7-14C]aBaPDEas previously described (18): +ive FAB-MS, m / z , ( % base) 987 (dimer + H, 6), 586 (M H + glycerol, 72), and 494 (M + H , 87), and 420 (M + H - Me,COH, 25), 305 [M + H - Me3COC(0)NHCH(CH3)C0,H, 1001.
Naylor et al. 2 4 (7,8,9-Trihydroxy-7,8,9,lO-tetrahydrobenzo[a Ipyren10-y1)thiolethylaminewas synthesized by adding aBaPDE (4.72 mg, 16 pmol) in 200 pL of D M F to a 4 mL of a pH 11 aqueous solution of 2-mercaptoethylamine (130 mg, 1.7 mmol) a t room temperature. T H F (150 pL) was added to dissolve the resulting precipitate, and the solution was stirred a t room temperature for 2 h. The sample was frozen a t -30 "C. Upon thawing, a precipitate persisted. The precipitate was washed with H 2 0 (4 X 750 pL), dissolved in THF, and found to be pure by C-18 HPLC (3.4 mg, 56% yield): PCI-MS (tri-TMS derivative), m/z (% base) 596 (M + H , 34), 429 [M + H - HZN(CH2)2SH - TMSOH, 251,414 [M + H - CH3 - H2N(CH,)zSH - TMSOH, 211,191 (TMS,OCH+, loo), 147 [(CH3),Si+OSi(CH3),, 201.
7,8,9-Trihydroxy-7,8,9,lO-tetrahydrobenzo[a Ipyrene-10propanoate was prepared by addition of [14C]aBaPDE (0.8 mg,
2.5 pmol) in T H F to propionic acid (7.4 mg, 250 mmol) under N2 E h N was added and the mixture was stirred a t room temperature for 2 h. The solvents were removed under vacuum, and the residue was purified by C-18 HPLC. The peak corresponding to the 9,lO-trans 10-ester amounted to 0.37 mg (40%): PCI-MS (tri-TMS derivative), m / z ( % base) 595 (M + H, loo), 522 (M H - TMS, 52), 521 (M + H - CzH&O2H, 35), 505 (M + H - TMSOH, 54). S - (7,8,9-Trihydroxy-7,8,9,1O-tetrahydrobenzo[a Ipyren10-y1)glutathione thioether was prepared by adding aBaPDE (0.5 mg, 1.67 pmol) in 200 mL of T H F to reduced glutathione (GSH, 10 mg) in 1.3 mL of 10:3 MeOH-Et3N. The resulting solution was stirred a t room temperature for 24 h, concentrated, redissolved in 4:l H20-MeOH, and purified by C-18 HPLC. Yield of the trans addition product was 20%. '*O Incorporation Studies. These were performed as previously described (18)using the N-t-BOC-alanyl ester of BaP tetrol I and hHb adducted by aBaPDE. Immunoaffinity Chromatography (21). The 8 E l l monoclonal antibody (gift from Dr. R. Santella, Columbia University) was coupled to CNBr-activated Sepharose 4B by the method of Groopman (22). The capacity of the 8Ell-Sepharose gel was 1.2 hg of [I4C]BaP tetrol I/mL. The same binding capacity was observed when the tetrol was applied to the column as a solution in a digest of 100 mg of globin provided the initial concentration of globin did not exceed 2.5 mg/mL. Bound material was eluted with either 4:l H,O-Me2C0 or 9:l MeOH-H20. Enzymatic Digestion. (A) Pronase. Globin was dissolved in water, and the pH was adjusted to 7.2-8.0 with 2 N NaOH. Sufficient lox PBS was added to achieve IX PBS, and enzyme was added (5% w/w). A second addition of enzyme (5%) was made after 6 h, after readjusting the p H to 8.0 with 2 N NaOH, and the digestion proceeded for an additional 12-16 h a t 37 "C with stirring. (B) Trypsin. The aqueous globin solution was adjusted to pH 7.8-8.2 with NH4HC03,and CaCl, was added to obtain a 10 mM solution. The enzyme:substrate ratio and incubation procedures were identical with those for Pronase digestions. Separation of BaP Tetrols from hHb. [14C]BaP tetrols I and I1 (120 nmol) were added to hHb (40 mg) in 1 mL of PBS. The following cleanup procedures were applied: (A) Ultrafiltration. The hHb solution was diluted with H 2 0 (19 mL) and filtered through a YM-10 (10-kDa cutoff) Amicon membrane in a 50-mL Amicon stirring cell under N, pressure. The protein and heme were retained on the membrane, and the freed BaP tetrols were in the filtrate. Radioactivity in both the retentate, after bleaching with ethanolic MCPBA, and the filtrate was determined by LSC. (B) Sephadex G-25 Chromatography. The I-mL hHb solution was eluted with H 2 0 through a 3 X 30 cm (70-mL void volume) column. The red hHb band and the fringe fractions were collected, treated with MCPBA, and counted by LSC. (C) Activated Charcoal. The 1-mL hHb solution was shaken N-(2-Aminoethyl)-N-(7,8,9-trihydroxy-7,8,9,lO-tetra- a t room temperature for 18 h with 30 mg of activated charcoal. The suspension was filtered through Whatman paper, and the hydrobenzo[a Ipyren-10-y1)aminewas prepared in quantitative filtrate was analyzed by LSC. yield by adding solid aBaPDE (0.1 mg, 330 nmol) to 2 mL of (D) Dialysis. The hHb solution was diluted with H 2 0 (19 mL) stirred ethylenediamine under N2 After 1h at room temperature and dialyzed in tubing against 2 L of H 2 0 (pH 5.8) a t 4 "C for the excess ethylenediamine was removed under vacuum and the 48 h. The H 2 0 was changed every 12-14 h. Aliquots of the residue was purified by HPLC: PCI-MS (tri-TMS derivative), dialysate were analyzed by LSC. m / z (70base) 621 (M + 41, 5), 609 (M + 29, IO), 581 (M + H , (E) Urea plus Dialysis. The hHb solution was diluted with loo), 565 (M + H - NH,, 83), 522 [M + H - NH(CHJ2NH2, IO], 9 M urea (9 mL) and left to stand a t room temperature for 48 191 (TMS02CH+,46).
+
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Chem. Res. Toxicol., Vol. 3, No. 2, 1990 113
Benzo[a]pyrene Diol Epoxide Adduct Formation Table I. Product Ratios and l 8 0 Incorporation into Hydrolysis Products of N-t-BOC-alanine-aBaPDE Ester and hHb-aBaPDE Ester Adducts as a Function of uH PH
acidic neutral 6.0
7.2-8.3
basic
9.8-10.8
N-t-BOC-alanine-aBaPDEEster ratio of BaP tetrols 1:II 48:l 3:l 601 % lSO label incorporatedointo tetrols 0 100 0 hHb-aBaPDE Ester(s) ratio of BaP tetrols 1:II 221 2:l 25:l % lSO label incorporated into tetrols ndb 60 0 Percent label incorporation determined by analysis of the molecular ion cluster of the capillary GC-PCI(CH4)-MSof the tetratrimethylsilyl derivatives of the tetrols. * Not determined. h, followed by dialysis as above for 48 h. The dialysate was analyzed by LSC. (F) HCl/Me2C0 Precipitation. The hHb solution was cooled to 4 O C and added dropwise to 300 mL of 0.015% HCl/Me2C0 maintained at
