Chem. Res. Toxicol. 1992,5, 19-25 53-64, Taylor & Francis, New York. (3) Rietjens, I. M. C. M., Tyrakowska, B., Veeger, C., and Vervoort, J. (1990) Reaction pathways for biodehalogenation of fluorinated anilines. Eur. J. Biochem. 194, 945-954. (4) Rietjens, I. M. C. M., and Vervoort, J. (1991) Bioactivation of 4-fluorinated anilines to benzoquinoneiminea as primary reaction products. Chem.-Biol. Interact. 22, 263-281. (5) van Ommen, B., and van Bladeren, P. J. (1989) Possible reactive
intermediates in the oxidative biotransformation of hexachlorobenzene. Drug Metab. Drug Interact. 7 , 213-243. (6) Stewart, F. P., and Smith, A. G. (1986) Metabolism of the “mixed”cytochrome P-450 inducer hexachlorobenzene by rat liver microsomes. Biochem. Pharmacol. 35, 2163-2170. (7) van Ommen, B., Adang, A. E. P., Brader, L., Posthumus, M. A,, MUer, F., and van Bladeren, P. J. (1986) The microsomal metabolism of hexachlorobenzene. Origin of the covalent binding to protein. Biochem. Pharmacol. 35,3233-3238. (8) Jerina, D. M., and Daly, J. W. (1974) Arene oxides: A new aspect of drug metabolism. Science 185,573-582. (9) Daly, J. W., Jerina, D. M., and Witkop, B. (1972) Arene oxides and the NIH shift: The metabolism, toxicity and carcinogenicity of aromatic compounds. Erperientia 28, 1129-1164. (10) Rietjens, I. M. C. M., and Vervoort, J. (1989) Microsomal metabolism of fluoroanilines. Xenobiotica 19,1297-1305. (11) Hudlicky, M. (1972) Reactions of organic fluorine compounds. In Chemistry of Organic Fluorine Compounds (Hudlicky, M., Ed.) Chapter 5, pp 170-518, Ellis Horwood Ltd. Halsted Press, John Wiley & Sons, New York. (12) van de Lande, L. M. F. (1932) L’action du mgthylate de sodium
19
sur quelques d6rivgs du m6tadichlorobenzene. Rec. Trav. Chim. PUYS-BUS 51,9&113. (13) Vervoort, J., De Jager, P. A., Steenbergen, J., and Rietjens, I. M. C. M. (1990) Development of a l9F n.m.r. method for studies on the in vivo and in vitro metabolism of 2-fluoroaniline. Xenobiotica 20, 657-670. (14) Shaka, A. J., Keeler, J., and Freeman, R. (1983) Evaluation of a new broad band decoupling sequence: Waltz 16. J. Magn. Reson. 55,313-340. (15) Omura, T., and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239,2370-2318. (16) Lowry, 0. H., Rosebrough, N. L., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. (17) Rekker, R. F., and de Kort, H. M. (1979) The hydrophobic fragmental constant; an extension to a 1000 data point set. Eur. J. Med. Chem. Chim. Ther. 14,479-488. (18) Fleming, I. (1976) in Frontier Orbitakr and Organic Chemical Reactions, John Wiley & Sons, New York. (19) Guengerich, F. P. (1989) Oxidation of halogenated compounds by cytochrome P-450, peroxidases and model metalloporphyrins. J. Biol. Chem. 264,17198-17205. (20) Ortiz de Montellano, P. R. (1986) Oxygen activation and transfer. In Cytochrome P-450. Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., Ed.) pp 217-271, Plenum Press, New York. (21) Guengerich, F. P., and MacDonald, T. L. (1990) Mechanisms of cytochrome P-450 catalysis. FASEB J. 4, 2453-2459.
Separation of (+)-sun- and (-)-anti-Benzo[ a Ipyrene Dihydrodiol Epoxide-DNA Adducts in 32P-PostlabelingAnalysis Ashok P. Reddy, Donna Pruess-Schwartz, and Lawrence J. Marnett* Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Received June 13, 1991 The (+)-enantiomer of 7,8-dihydroxy-7,&dihydrobenzo[a]pyrene(BP-7,8-diol) is a diagnostic probe for cytochrome P-450 and non-cytochrome P-450 pathways of dihydrodiol epoxidation. The principle products of epoxidation are the (+I-syn-dihydrodiol epoxide [(+)-syn-BPDE] and the (-)-anti-dihydrodiol epoxide [ (-)-anti-BPDE] . Chromatographic conditions are described that separate the major deoxynucleoside 3’,5’-bisphosphate adducts derived from these dihydrodiol epoxides on commercial poly(ethy1enimine) thin-layer plates. Inclusion of boric acid and magnesium chloride in the D4 solvent is a key feature of the separation. Reasonable separation of these bisphosphate adducts from the major deoxynucleoside 3’,5’-bisphosphate adduct derived from (+)-anti-BPDE is also observed. 3?P-Postlabeling analysis of DNA adducts produced following topical administration of benzo[a] pyrene to mouse skin suggests that cytochrome P-450 plays a major role in its metabolism to DNA binding derivatives.
Introduction Benzo[a]pyrene (BP)’ is a ubiquitous environmental pollutant and a potent animal carcinogen (1,2).Extensive experimental evidence implicates dihydrodiol epoxides (BPDE) as major contributors to the genotoxic activity of BP (3). BPDEs are formed from BP by sequential oxidation, hydration, and oxidation as indicated in Figure 1 (4-10). Four possible BPDEs exist that differ significantly in DNA binding capacity, mutagenicity, and tumor-initiating activity. The extent to which individual BPDE’s form in a particular cell is determined by the balance of *To whom correspondence should be addressed at The A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146.
enzymes that catalyze each transformation (7,8,11-13). Epoxidation of BP to BP-7,8-oxide is catalyzed by cytochromes P-450and hydration of BP-7,8-oxide to BP-7,8diol by epoxide hydrolase (5,9). Epoxidation of BP-7,8diol is catalyzed by cytochromes P-450 and by peroxidases, or it is effected by peroxyl radicals generated by lipid peroxidation, metal-catalyzed hydroperoxide decomposiAbbreviations: BP, benzo[a]pyrene;BHA,butylated hydroxyanieole;
8-NF, 8-naphthoflavone; (+)-BP-7,8-diol,7(S),B(S)-dihydroxy-7,8-dihydrobenzo[alpyrene; BPDE, 7,8-dihydroxy-9,10-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene; (+)-anti-BPDE, 7(R),8(S)-dihydroxy-9(S),lO(R)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; (-)-anti-BPDE, 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene; (+)-synBPDE, 7(S),8(R)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene; (-)-syn-BPDE, 7(R),8(S)-dihydroxy-9(R),lO(S)-epoxy7,8,9,lO-tetrahydrobenzo[aIpyrene;PEI, poly(ethy1enimine);TBA, tet-
rabutylammonium chloride; TLC, thin-layer chromatography; RAL, relative adduct labeling.
oa93-22a~/92/2705-ooi9$03.oo/o 0 1992 American Chemical Society
Reddy et al.
20 Chem. Res. Toxicol., Vol. 5, No.1, 1992
0
0
0
SP
p&y 0
0
0
@
@ 0
0
0
0
0
HO” OH
OH
7 4 a p
/
@
@ ?
@ o 0
HO”
0
OH
(-)--DE
/
\
0
0
HO”
0
(+)-.mBPDE (7R,Bs,gs,10R)
&
HO HO
,
&I
OH
f7RSSOf21W
0
\
(-)--E
.
0
0
(B,SR,Of2 l a )
Figure 1. Formation of (+)-syn-and (-)-anti-BPDE as a result of BP metabolism.
tion, xenobiotic oxidation, etc. (14-17). The extent to which non-cytochrome P-450 processes play a role in BP-7,8-diol epoxidation is a matter of considerable interest. Several laboratories have utilized the stereochemistry of epoxidation of the (+)-enantiomer of BP-7,Sdiol to estimate the contribution of non-cytochrome P-450 pathways to epoxidation (18-22). Non-cytochrome P-450 pathways oxidize (+)-BP-7,8-diol predominantly to (-)-anti-BPDE whereas cytochromes P-450 oxidize (+)BP-7,8-diol exclusively to (+)-syn-BPDE (15,23-26).2 BPDE hydrolysis products or BPDE macromolecular adducts can be analyzed to quantitate BPDE formation. Our laboratory has utilized HPLC of BPDE-deoxynucleoside adducts to estimate the contribution of noncytochrome P-450 pathways to BP-7,8-diol oxidation in mouse skin (22). Commercially available [3H]BP-7,8-diol was used for these experiments, but the low level of DNA adduction necessitated the use of 10-15 animals per datum point and large amounts of radiolabel (25 pCi per animal). This limited the variety of experiments that could be performed and precluded extensive studies of the effects of inhibitors, inducers, etc. Therefore, we have explored the possibility of using 32P-postlabelingtechniques to analyze the diastereomeric BPDE-DNA adducts. The high sensitivity of postlabeling technology and the use of unlabeled BP-7,8-diol would greatly enhance the flexibility of experimental design if methods could be developed to cleanly separate the diastereomeric (+)-syn- and (-)anti-BPDE-deoxynucleoside bisphosphate adducts. In this paper, we describe the development of suitable chromatographic systems that can be used for analysis of BPDE-DNA adducts produced in vivo in a variety of tissues. In both cytochrome P-450and non-cytochrome P-450oxidations, other products are generated, but they are not important to the present analysis.
Materlals and Methods Chemicals. Unlabeled (+)-BP-7,8-diol, (+)-anti-BPDE, (-)-anti-BPDE, (it)-syn-BPDE,and 7(R),8(S),9(S)-trihydroxy10(R)-(3’-phospho-~-deoxyguanosy1)-7,8,9,10-tetrahydrobenzo[alpyrene were obtained from the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention (Bethesda, MD). 7(S),8(R),9(R)-Trihydroxy-lO(S)-
(3’-phospho-IP-deoxyguanosyl)-7,8,9,lO-tetrahydrobem[a]pyrene and 7(S),8(R),9(5’)-trihydroxy-10(R)-(3’-phospho-P-deoxyadenosyl)-7,8,9,10-tetrahydrobenzo[a]py~ene were generously provided by Karen Canella and Anthony Dipple. BP and 8-NF were purchased from Aldrich Chemical Co. (Milwaukee, WI). Micrococcal endonuclease (100 unit.a/mg) and spleen phosphodiesterase (20 unit.a/mg) were from Boehringer Mannheim (Indianapolis, IN). Deoxyribonucleotides were from Pharmacia (Piscataway, NJ). Micrococcal endonuclease (grade VI, 100 units/mg), potato apyrase, dithiothreitol, spermidine, bovine serum albumin, NADPH, ribonuclease A (bovine pancreas, type 1-A), and proteinase K (Tn’tirachiumalbum, type XI) were from Sigma Chemical Co. (St. Louis, MO). Calf thymus DNA was obtained from Sigma and purified by proteinase K and ribonuclease A digestion (27).Polynucleotide kinase was from U.S. Biochemicals (Cleveland, OH). Poly(ethy1enimine) (PEI)-celluloseTLC plates were from Machery and Nagel, FRG, purchased through Brinkmann Instruments (Westbury, NY). [y3*P]ATPwas prepared using Promega Biotech Gamma Prep-A kit (Madison, WI). Carrier-free [32p]phosphate (3000Ci/mmol) was purchased from ICN Radiochemicals (Irvine, CA). Kodak X-Omat AR film was from Fotodyne (New Berlin, WI). Animals. Female CrkCD-1 (IRC) BR mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Animals aged 6-10 weeks were shaved with surgical clippers 48 h prior to treatment (in vivo studies), and only those in the resting phase of the hair growth cycle were used. Male Sprague-Dawley rata were from Charles River Laboratories, Inc. Animals (150-175 g) were injected ip for 3 consecutive days with corn oil (uninduced) or j3-NF (100 mg/kg body weight) suspended in corn oil and sacrificed on the fourth day. Preparation of BPDE-Modified DNA. (A) (-)-antiBPDE-DNA. Calf thymus DNA [0.5 mg in 1.0 mL of 0.05 M
Diastereomeric Dihydrodiol Epoxide-DNA Adducts Tris-HC1 (pH 7.5), 5 mM MgCl,, 150 mM KCl] was incubated with 150 pg of (-)-anti-BPDE (added in 50 pL of acetone) at 37 OC for 19 h. The solution was extracted eight times with equal volumes of ethyl acetate and precipitated four times with 0.1 volume of 2 M NaCl and 2 volumes of cold ethanol. The precipitated DNA was dissolved in 0.5 mL of 0.01 M Tris-HC1, pH 7.5. (B) (+)-anti-BPDE-DNA. Calf thymus DNA [0.5mg in 1.0 mL of 0.05 M Tris-HC1 (pH 7.5),5 mM MgCl,, 150 mM KCl] was incubated with 250 pg of (+)-anti-BPDE in 20 pL of acetone at 37 "C for 6 h. The solution was extracted eight times with equal volumea of ethyl acetate; DNA was precipitated and then dissolved in 0.5 mL of 0.01 M Tris-HC1, pH 7.5. (C) (+)-syn-BPDE-DNA. Calf thymus DNA [3 mg in 0.4 mL of 0.01 M Tris-HC1 (pH 7.5)] was reacted with 1 mg of (f)-syn-BPDE [added in 0.6 mL of tetrahydrofuran/triethylamine (19/1)]. The solution was extracted eight times with equal volumes of ethyl acetate, and DNA was precipitated and then dissolved in 0.5 mL of 0.01 M Tris-HC1, pH 7.5. (D) (+)-syn-BPDE-DNA. (+)-syn-BPDE is not commercially available. Therefore, it was biosynthesized by reaction of (+)-BP-7,8-diol with rat liver microsomes in the presence of NADPH. Microsomes were prepared from 8-NF-induced male Sprague-Dawley rats by homogenization of liver and differential centrifugation (28). (+)-BP-7,&diol(ll5 pg) was added in 50 pL of acetone to a solution of 1 mM NADPH, 5 mM MgCl,, 105 mM KC1,0.05 M Tris-HC1 (pH 7.5),1 mg of liver microsomal protein, and 0.5 mg of calf thymus DNA in 1 mL total volume. After 4 h, the solution was extracted eight times with equal volumes of ethyl acetate containing 100 pM BHA. The ethyl acetate layers were back-extracted with water and then pooled and evaporated to dryness. The residue was dissolved in 50 pL of methanol and analyzed by HPLC for tetraols and unreacted BP-7,8-diol. The DNA was precipitated four times with 0.1 volume of 2 M NaCl and two volumes of cold ethanol. The final DNA pellet was dissolved in 0.5 mL of 0.01 M Tris-HC1, pH 7.5. In Vitro Mixture of (+)-syn- and (-)-anti-BPDE-DNA Adduct Standards. Ten micrograms of (+)-syn-BPDE-DNA and 5 pg of (-)-anti-BPDE-DNA were precipitated separately from 0.01 M Tris-HC1 (pH 7.5) and dissolved in 10 mM sodium succinate (pH 6.0) containing 5 mM CaCl, to give 0.5 pg of DNA/pL. A mixture of (+)-syn- and (-)-anti-BPDE-DNA standards were then prepared by keeping the concentration of (+)-sp-BPDE-DNA constant at 1 pg in all the mixtures and varying the (-)-anti-BPDE-DNA standard concentration as follows: 1, 0.7, 0.5, 0.2, 0.1, 0.05, and 0.03 pge3 In Vivo Modification of DNA. Groups of 2 mice were treated topically with either 100 pL of acetone (control) or 100 nmol of BP in 100 pL of acetone under diffuse light. After 4 h, the mice were sacrificed by cervical dislocation and the treated skin was excised. The epidermis was removed by heat treatment (29). DNA was isolated from the epidermal material using a method adapted from Diamond et al. (30) as described by Pelling and Slaga (31). DNA was quantified spectrophotometrically a t 260 nm and stored in 500 pL of 0.01 M Tris-HC1, pH 7.5. Before postlabeling, the DNA was precipitated and dissolved in 10 mM sodium succinate (pH 6.0) containing 5 mM CaC12. Synthesis of [-$'%']ATP. [y3?P]ATPwas synthesized using the Promega Gamma Prep kit (Madison, WI) as per the manufacturer's instructions. [*]Pi (2 mCi) in W p L aliquots was added to the reaction mixture and incubated at 25 "C for 50 min. The extent of reaction was monitored by spotting 8 pCi of the reaction mixture from 0 min and from 50 min on a 5 X 10 cm PEI plate which was developed to the top with 1.5 M LiC1. The PEI plate was then exposed to X-ray film and the film processed. 3'%'-Postlabeling Analysis of Adducts. (A) Digestion of DNA. (1) In Vitro Samples. Control or adducted DNA was hydrolyzed to deoxynucleoside 3'-monophosphates by incubating 2 pg of DNA (4 pL of 0.5 pg of DNA/bL) with 1r L each of 2 pg/pL micrococcal endonuclease and spleen phosphodiesterase in 24 pL of 10 mM sodium succinate (pH 6.0) containing 5 mM CaCl, at 38 "C for 4 h. The amounts of adducted DNA samples stated in the text refer to the amounts of DNA isolated following modification in vitro or in vivo. They do not represent the amount of pure adduct.
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 21
(2) In Vivo Samples. DNA samples were hydrolyzed by incubating 20 pg of DNA (40 pL of 0.5 pg/pL) with 10 pL each of 2 M / ~ Lm i c r d endonuclease and spleen phosphodiesterase in 5 p L of 10 mM sodium succinate (pH 6.01-5 mM CaClz at 38 "C for 4 h. The digest was then diluted to 80 pL with water. (B) Isolation of Adducts. To enrich the adducts prior to chromatography, the DNA digest (30 pL) was mixed with 4 pL each of 100 mM ammonium formate (pH 3.5),10 mM TBA, and 26 pL of distilled water. The mixture was extracted twice with 1volume of water-saturated 1-butanolin 1.5mL Eppendorf tubes by mixing for 40 s on a vortex mixer. Phases were separated by centrifugation on a table top microcentrifuge. The combined organic phases were back-extracted twice with 110 pL of distilled water to remove trace normal nucleotides. The butanol extract was then neutralized by adding 1 pL of 200 mM Tris-HC1 (pH 9.5) and evaporated in a Speedvac concentrator. When amounts of DNA greater than 2 pg were analyzed, the following changes were made: (a) the volume of the aqueous phase was increased by 10 pL for each additional 2 pg of DNA while keeping the concentrationsof ammonium formate and TBA constant; and (b) a proportionately higher volume of 200 mM Tris-HC1 (pH 9.5) was added to the butanol extract. (C) 3'%'-Labeling of Isolated Adducts. The adduct residue from 2 of DNA was dissolved in 10 pL of water. To this solution was added a 19-pL aliquot (150 pCi) of a radioactive mix containing 84 pL of lox buffer mix [300 mM Tris-HC1 (pH 9.5), 100 mM MgCl,, 100 mM dithiothreitol, 10 mM spermidine], 84 pL of carrier-free [-p3?P]ATP (1.7 mCi), 5 pL of polynucleotide kinase (30 units/pL), and 40 pL of distilled water. The solution was mixed by drawing it in and out of a pipet and then incubated a t 38 "C for 40 min. The labeling conditions were the same for different amounts of DNA except the amount of [ T - ~ ~ P ] A T was P increased 100-150 pCi (
