Bisdihydrodiols, rather than dihydrodiol oxides, are the principal

Heinz Frank , Mario Funk , Franz Oesch , and Karl L. Platt ... Michael George, Guy Lambert, Fabienne Meyers, Joycelyn Allison, Linda Adams, and Leon C...
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Chem. Res. Toxicol. 1994, 7, 89-97

89

Bisdihydrodiols, Rather Than Dihydrodiol Oxides, Are the Principal Microsomal Metabolites of Tumorigenic trans-3,4-Dihydroxy-3,4-dihydrodibenz[ &&]anthracene Karl L. Platt* and Michael Schollmeiert Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, 0-55131 Mainz, Germany Received May 6, 1993" Several studies on metabolism and biological activity of tumorigenic dibenz[a,hlanthracene (DBA) and its derivatives have led t o t h e conclusion t h a t the M-region dihydrodiol, trans3,4-dihydroxy-3,4-dihydro-DBA (DBA-3,4-dihydrodiol), is the precursor of the ultimate mutagenic and tumorigenic metabolite of DBA with the presumed structure of a bay-region dihydrodiol oxide. Incubations of DBA-3P-dihydrodiol (50 pM) with the microsomal hepatic fraction of Sprague-Dawley rats pretreated with Aroclor 1254 yielded more than 13 metabolites upon separation by HPLC. anti-3,4-Dihydroxy- 1,2-epoxy-1,2,3,4-tetrahydro-DBA [0.27nmol/ (nmol of P450.15 min)] could be identified for the first time by UV spectroscopy, by cochromatography with the synthetic reference compound, a n d by its nonenzymatic hydrolysis t o r-l,t-2,t-3,c-4tetrahydroxy-1,2,3,4-tetrahydro-DBA, while firm evidence for the presence of the diastereomeric syn-dihydrodiol oxide was not obtained. Major microsomal metabolites of t h e M-region dihydrodiol were however three bisdihydrodiols: trans,trans-3,4:8,9-tetrahydroxy-3,4,8,9tetrahydro-DBA [0.32nmol/(nmol of P450.15 min)], trans,trans-3,4:lO,ll-tetrahydroxy-3,4,10,ll-tetrahydro-DBA [DBA-3,4:lO,ll-bisdihydrodiol; 1.44 nmol/(nmol of P450.15 min)], and trans,trans-3,4:12,13-tetrahydroxy-3,4,12,13-tetrahydro-DBA [0.70nmol/(nmol of P450.15 min)], whose structures were verified by UV and mass spectrometry as well as cochromatography with synthetic reference compounds and by the observation t h a t they were not formed when epoxide hydrolase was inhibited (l,l,l-trichloro-2-propene oxide, 1mM). Determination of the bacterial mutagenicity in strain TAlOO of Salmonella typhimurium in the presence of ametabolic activating his+revertants/ system revealed a stronger mutagenic effect of DBA-3,4:10,11-bisdihydrodiol(52.3 nmol) as compared t o its metabolic precursor, DBA-3,4-dihydrodiol(34.5 his+revertantshmol), while the two other bisdihydrodiols contribute only little t o t h e genotoxic activity of the M-region dihydrodiol. On the basis of these observations 1,2-epoxy-3,4:10,11-tetrahydroxy-1,2,3,4,10,11-hexahydrodibenz[a,h]anthracenecan be predicted as a n ultimate mutagenic metabolite of DBA-3,kdihydrodiol playing a more important role in the genotoxicity of DBA than the simple bay-region dihydrodiol oxide. This assumption is further supported by the results of recent DNA binding studies with DBA and its derivatives.

Introduction

in experimental animals could be established (3).Although this discovery took place more than 60 years ago, the exact Polycyclic aromatic hydrocarbons (PAH)I constitute a mechanism by which DBA initiates tumorigenesis is still class of chemical compounds, which are ubiquitously not known. present in the environment as the result of incomplete The biologically inert PAH have to be enzymatically combustion of organic matter (1)and contain some of the transformed-like most other chemical carcinogens-to most powerful chemical carcinogens (2). Dibenz[a,hlelectrophilically reactive metabolites in order to covalently anthracene (DBA), a pentacyclic aromatic hydrocarbon, interact with DNA, thereby exerting their genotoxic was the first pure compound for which a carcinogenic effect activity ( 4 ) . Dihydrodiol oxides with the oxirane ring occupying part of a sterically hindered bay region have * T o whom correspondenceshould be addressed. t Present address: BU-C, P D , Ecology I, Hoechst AG, D-65926 been considered for some time ultimate genotoxic meFrankfurt,Germany. tabolites of PAH (5) although different reactive electroAbstract published in Advance ACS Abstracts,December 15,1993. philes could also be formed by mechanisms such as one1 Abbreviations: DBA,dibenz[a,hlanthracene; DBA-3,4-dihydrodiol, trans-3,4-dihydroxy-3,4-dihydrodibenz[a,hlanthracene (otherdihydrodielectron oxidation (6) and bioalkylation (7). 01s aresimilarlydesignated);DBA-3,410,1l-bisdihydrodiol, trans,trans3,410,1l-tetrahydroxy-3,4,10,1l-tetrahydrodibenz[aJ1.lanthracene (other In the case of DBA its M-region trans-3,4-dihydrodiol bisdihydrodiols are similarlydesignated);DBA-anti-3,4-dihydrodiol1,2oxide, c-3,t-4-dihydroxy-r-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,hlan- can be considered the immediate precursor of a bay-region thracene; DBA-syn-3,4-dihydrodiol1,2-oxide,t-3,~-4-dihydroxy-r-1,2- dihydrodiol oxide. Since the trans-3,4-dihydrodiol conepoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene; DBA-1,2,4/3-tetstitutes the major microsomal metabolite of DBA (8-11) rahydrotetraol,r-l,c-2,t-3,~-4-tetrahydro~l,2,3,4-tetrahydrodibe~[a,hlanthracene;DBA-l,3/2,44etrahydrotetraol, r-l,t-2,~-3,t-4-tetrahydroxy- and exhibits strong mutagenic (12, 13) and tumorigenic 1,2,3,4-tetrahydrodibenz[aJ1.lan~acene; DBA-l,4/2,3-tetrahydrotetraol, (14) activity, this metabolic pathway leading to initiation r-l,t-2,t-3,~-4tetrahy~~-l,2,3,4tetrahy~~~~[aJ1.lan~a~ne; DMF, of tumorigenesis seemed very likely. However, the search NJV-dimethylformamide;FD,fielddesorption;P A H ,polycyclicaromatic for bay-region dihydrodiol oxides or their products of hydrocarbons;T C P O , l,l,l-trichloro-2-propene oxide. 0893-228~/94/2707-0089$04.50/0 0 1994 American Chemical Society

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Chem. Res. Toxicol., Vol. 7,No.1, 1994

Platt and Schollmeier

DBA-tra~[3,4-~H1-3,4-dihydrodiol [4.9 MBq (132pCi)/pmoll with a chemical and radiochemical purity of 299% was obtained from DBA-3,4-quinone(22)via reduction with sodium borotritiide in a similar manner as described for the preparation of tritiumlabeled K-region trans-dihydrodiols (23). l,l,l-Trichloro-2-propene oxide (TCPO)was supplied by EGA (Steinheim, FRG). Aroclor 1254 was obtained from Bayer (Leverkusen,FRG), and trioctanoin was from Serva (Heidelberg, FRG). Biochemicals were from Boehringer (Mannheim,FRG), and solvents for HPLC were from Baker (Gross-Gerau, FRG); all other chemicalsof analytical grade were purchased fromMerck (Darmstadt, FRG). Metabolism Studies. Liver microsomes of adult male Sprague-Dawleyrats (190-240 g; Interfauna Sueddeutsche Versuchstierfarm, Tuttlingen, FRG) were prepared as previously described (24)6 days after the animals had received a single intraperitoneal injection of Aroclor 1254(500mg/kg body weight) Experimental Procedures in trioctanoin (2.5 mL/kg body weight). Protein concentrations were determined by the method of Lowry et al. (25)using bovine Caution: Since DBA is a carcinogen in laboratory animals, serum albumin for calibration. Cytochrome P450 content was its derivatives should be treated as being tumorigenic. Thus measured according to the procedure of Omura and Sat0 (26). proper care has to be taken to prevent exposure of the body, in Microsomal incubations contained a 1-2-mg protein equivalent particular, lung and skin, to these compounds. of liver microsomes,0.6 mM NADP, 8 mM glucose 6-phosphate, Chemicals. trans-1,2-Dihydroxy-1,2-dihydrodibenz[a,h]an1.2 unit of glucose-6-phosphatedehydrogenase,and 5 mM MgCl2 thracene (DBA-1,2-dihydrodiol) (I6),trans-3,4-dihydroxy-3,4in a final volume of 2 mL of 50 mM isotonic (150 mM KCl) dihydrodibenz[a,hl anthracene (DBA-3,4-dihydrodiol)(I 7),cissodium phosphate buffer (pH 7.4). In some cases the incubation 5,6-dihydroxy-5,6-dihydrodibenz[a,hlanthracene (DBA-cis-5,6dihydrodiol) (18),~-3,t-4-dihydroxy-r-l,2-epoxy-1,2,3,4-tet-mixture contained 1mM TCPO in 1OpLof acetone. This mixture was preincubated for 10min at 37 "C. the incubation was started rahydrodibenz[a,h]anthracene (DBA-anti-3,4-dihydrodiol 1,2oxide) (I9),r-l,t-2,t-3,~-4-tetrahydroxy-1,2,3,4-tetra- by the addition of 50 pM unlabeled or tritium-labeled DBA3,4-dihydrodiol [0.37 MBq (10 pCi)/pmoll in 100 pL of acetone hydrodibenz[a,h]anthracene (DBA-1,4/2,3-tetrahydrotetraol) (IO), r-l,c-2,t-3,~-4-tetrahydroxy-1,2,3,4-tetrahydrodibenz[a,h]an- and continued with shaking (80 min-1) at 37 "C. The incubation was stopped with 1.5mL of ice-coldethyl acetate followedby the thracene (DBA-1,2,4/3-tetrahydrotetraol) (IO),and r-l,t-2,c-3,taddition of 0.466 pg of DBA-cis-5,6-dihydrodiol(internal stan4-tetrahydroxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene (DBAdard) dissolved in 100pL of dimethylformamide (DMF). After 1,3/2,4-tetrahydrotetraol)(10) were obtained according to vortexing for 1 min, the organic and aqueous phases were published procedures. The preparation of t-3,c-4-dihydroxy-rseparated by centrifugation at 1OOOg. The extraction was 1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene (DBA-synrepeated twice more with 1 mL of ethyl acetate. The organic 3,4-dihydrodiol l,a-oxide) was carried out using methods (20) already applied to other PAH. trans,trans-3,4:12,13-Tetrahy- phases were combined,dried over anhydrous magnesium sulfate, and brought to dryness with a stream of nitrogen;then, the residue droxy-3,4,12,13-tetrahydrodibenz[a,hlanthracene (DBA-3,4:12,was stored at -70 "C until later HPLC separation, for which the 13-bisdihydrodiol)was prepared by a-alkylation of 5,6-dimethoxysample was dissolved in 40 pL of MezSO. 1-tetralone as its trimethylsilylenol ether with 1-(chloromethy1)All steps were performed under subdued light. The recovered 3,4-dimethoxynaphthalene.C1 elongation at the carbonyl group radioactivity in the organicand aqueous phase was usually >95 7% via formation of an oxirane with dimethylsulfonium methylide of that applied. and its rearrangement to an aldehyde yielded 2-[(3,4-dimethoxyl-naphthyl)methyl]-5,6-dimethoxy-l-formyl-1,2,3,4-tetrahy- Biosynthesis of trans,trans-3,4:8,9-Tetrahydroxy-3,4,8,9dronaphthalene. Cyclization with polyphosphoric acid and tetrahydrodibenz[a,h]anthracene (DBA-3,4:8,9-bisdihydehydrogenation with 2,3-dichloro-5,6-dicyano-p-benzoquinone d r ~ d i o l ) DBA-1,2-dihydrodiol(4.8 .~ mg; 77 pM) was incubated for 30 min as described above in a final volume of 200 mL using resulted in 3,4,12,13-tetramethoxydibenz[a,h]anthracene.Cleavage of the methyl ether with boron tribromide, acetylation of the a 200-mg protein equivalent of liver microsomesof Aroclor 1254biscatechol, and reduction with sodium borohydride in the treated Sprague-Dawleyrats. The incubation was stopped with 35 mL of ice-cold acetone, the precipitated protein removed by presence of oxygen (I 7)furnished DBA-3,4:12,13-bisdihydrodiol ['H-NMR (400MHz, acetone-ddMezso-d6,3:2 (v/v),D2O) 6 4.37 centrifugation (9OOOg; 10 min), and the resulting solution (dt,1H,H3;53,4=11.40Hz),4.51-4.55(m,lH,H13;512,13=9.68 extracted with ethyl acetate (6 X 70 mL). The residue obtained Hz), 4.61-4.65 (m, lH, H12; 5 1 2 ~ 3= 9.68 Hz), 4.74 (dd, lH, H4; after drying the organic phase with MgZSO, and evaporating the 5 3 , 4 = 11.40Hz),6.15 (dd, lH, H2; 51,~ = 10.08Hz, 5 2 . 3 = 2.35 Hz), solvent was subjected to preparative HPLC (seebelow) for which 7.19 (dd, lH, H1; J1,2 = 10.08 Hz, 51,3 = 2.24 Hz), 7.30-7.38 (m, it was dissolved in 110 pL of MeZSO. The yield of the 2H, H9, HlO), 7.63 (d, lH, Hll;Jlo,ll= 7.48 Hz),7.72 (d, lH, H5; bisdihydrodiol thus obtained was 417 pg, determined after calibration of the peak area at 280 nm with the eluting 5 5 , B = 8.49Hz), 7.84 (d, lH, H6;55,6= 8.49 Hz), 7.92-7.95 (m, lH, H8), 8.23 (d, lH, H14), 8.34 (s, lH, H7)l in 0.5% overall yield radioactivity of DBA-3,4:8,9-bisdihydrodiolmetabolicallyformed (21).trans,trans-3,4:l0,1l-Tetrahydroxy-3,4,1O,ll-tetrahydro- from [3H]DBA-3,4-dihydrodiol. dibenz[a,h]anthracene (DBA-3,4lO,ll-bisdihydrodiol) ['H-NMR Hydrolysis of Metabolically Formed Dihydrodiol Ep(400 MHz, acetone-de/MezSO-ds,3:2 (viv), D20) d 4.41 (d, 2H, oxides. The combined mixture of microsomal metabolites of H3, H10;53,4=510,11= 11.20Hz),4.77 (d, 2H,H4, H11; 53.4 = J i o , i i DBA-3,4-dihydrodiol(50 pM) obtained from two 2-mL incuba= 11.20 Hz), 6.20 (dt, 2H, H2, H9; J 1 , 2 = Js,9 10.10 Hz, 52,s = tions (1.02 mg of microsomal protein/mL; 40 min) performed as J 9 , l o = 2.60 Hz, 5 2 4 = Je,ll = 2.50 Hz), 7.40 (dd, 2H, H1, H8; 51,~ described above was separated into two equal parts. One part = 58,s= 10.20 Hz, J 1 , 3 = JSJO= 1.70 Hz), 7.78 (d, 2H, H5, H12; was immediately subjected to HPLC separation; the other part 55.6 = 5 1 2 ~ 3 8.80 Hz), 8.04 (d, 2H, H6, H13; J 5 , 6 = 5 1 2 ~ 3= 8.80 was mixed with dioxane (70 pL) and water (50 pL), kept in the Hz), 8.83 (s, 2H, H7, H14)l was synthesized2in a similar fashion dark for 16 h, brought to dryness with a stream of nitrogen, and as described above from 5,6-dimethoxy-l-tetralone and l-(chlosubjected to HPLC separation. The relative amount of metabromethyl)-5,6-dimethoxynaphthalenein 1.2% overall yield.

spontaneous hydrolysis,the tetraols, as metabolites of DBA or its trans-3,4-dihydrodiol led to controversial results. While Nordqvist et al. (8)obtained evidence for extensive formation of 3,4-dihydrodiol1,2-oxides, we were not able to confirm their results (10). However, on the basis of the detailed elucidation of the biotransformation of DBA, we proposed the formation of bisdihydrodiol oxides as ultimate genotoxic metabolites of this PAH (10). After a recent report giving some indications for the formation of tetra- and hexahydroxy derivatives in the metabolism of DBA and its M-region dihydrodiol (15) we now wish to present evidence that bisdihydrodiols and not dihydrodiol oxides are the major metabolites of mutagenic and tumorigenic DBA-trans-3,4-dihydrodiol.

* H. Frank and K. L. Platt, unpublished results.

Due to its molecular symmetry DBA-3,48,9-bisdihydrodiol is structurally identical to DBA-1,2:lO,ll-bisdihydrodiol.

Metabolism of Dibenz[a,h]anthracene-3,4-diol olites was expressed as the ratio of the peak area of a given metabolite divided by the peak area of the internal standard. HPLC Analysis. Chromatographic separations were performed with a system consisting of two high-pressure pumps (Model 740; Spectra-Physics, Darmstadt, FRG), a sample injection valve (Model C6U; Valco, Schenkon, Switzerland) with a 20-pL sample loop,aUV detector (280 nm, Model 230; SpectraPhysics) connected to a recorder (ModelLS 438880; Linseis,Selb, FRG), and an integrator (Autolab System I, Spectra-Physics). In some cases UV-visible spectra of metabolites were recorded during a chromatographic run by using a diode array detector (Model 2140; Pharmacia/LKB, Freiburg, FRG) and the suitable software (Wavescan, LKB-Bromma, Sweden). For the analytical separation of the metabolites of DBA-3,4dihydrodiol LiChrospher 100 RP-18 ( 5 pm, 4 x 250 mm; Merck, Darmstadt, FRG) was used as the stationary phase. The mobile phase consisted of a mixture of methanol and 10 mM NHdHC03 (pH 7.8), with a linear increase in methanol content from 40% to 100% (v/v) over 60 min at a flow rate of 0.8 mL/min. Twenty microliters of the solution of the metabolites in MezSO was injected via a 20-pL sample loop. For the preparative separation of DBA-3,4:8,9-bisdihydrodiol, LiChrospher 100 RP-18 (5 pm, 8 X 250 mm) was used as the stationary phase. The mobile phase consisted of a mixture of 45% (v/v)methanolinwaterataflowrateof2.4mL/min.Twenty microliters of the solution of the metabolites in Me2SO was injected via a 100-pLsample loop. The peaks eluting at 19.7 and 20.5 min (diastereomers of DBA-3,4:8,9-bisdihydrodiol) were collected together, and the solvent was removed by evaporation under reduced pressure. Quantificaton of Metabolic Conversion. The metabolic formation of distinct metabolites was radiometrically determined by collecting the HPLC eluate in 20-5 fractions, adding liquid scintillator (3 mL, Lumagel; Lumac, Landgraaf, Netherlands), and measuring the radioactivity with liquid scintillation spectrometry (Tricarb 3000/4000; United Technologies, Frankfurt, FRG). Total metabolic conversion was calculated from the combinedradioactivityelutingupon HPLC before and after DBA3,4-dihydrodiol and the amount of radioactivity remaining in the aqueous phase after ethyl acetate extraction. The latter fraction contains hydrophilic and protein-bound metabolites. Spectral Methods. UV-visible absorption spectra of the metabolites in ethanol were obtained on a Shimadzu MPS-2000 spectrophotometer, and fluorescencespectra on a Spex Fluorolog 112x-1 spectrofluorometer processed with Spex DM 3000F software. Field desorption (FD) mass spectra were recorded with a Finnigan MAT 95 double-focusingspectrometer. Solutions of the compounds in acetone were loaded on the emitter, its heating current being 17 mA. Proton NMR spectra were measured on a Bruker AM 400 spectrometer at 400 MHz. Chemical shifts (in ppm) are relative to tetramethylsilane. Mutagenicity Studies. The mutagenicity experiments were performed as described by Ames et al. (27)with only minor modifications. The auxotrophic Salmonella typhimurium strain TAlOO (generously provided by Dr. B. N. Ames, Berkeley, CA) was grown overnight at 37 "C in nutrient broth (25 g of Oxoid Nutrient Broth No. 2/L of medium). For inoculation a stock culturestored at-70 OC wasused. Beforetheexperiment,bacteria were centrifuged, resuspended in medium B (1.6 g of Bacto nutrient broth and 5 g of NaCl/L of medium), and adjusted nephelometrically to a titer of (1-1.5) X lo9 bacteria (colonyforming units)/mL. Male Long-Evans rats (65-75 g; Charles River Wiga, Sulzfeld, FRG) were treated intraperitoneally with Aroclor 1254(300 mg/ kg body weight) in trioctanoin (2.5 mL/kg body weight) and sacrificed by cervical dislocation 4 days thereafter. Followingliver homogenizationin sterile ice-cold 150mM KCl (3 mL/g of liver) the homogenate was centrifuged at 9OOOg for 10 min, yielding as supernatant the postmitochondrial fraction (S-9).

The metabolic activating system contained 8 mM MgC12,84 mM KC1, 4 mM NADP, 5 mM glucose 6-phosphate, 50 mM

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Retention time, tR (min)

Figure 1. Reverse-phase HPLC chromatograms of the organic

ethyl acetate-extractablemetabolites obtained from incubation of DBA-trans-3,4-dihydrodiol(D; 50 pM, 40 min) with liver microsomes (1.02 mg of protein/mL) of Sprague-Dawley rata pretreated with Aroclor 1254 (A) without TCPO; (B) in the presence of TCPO (1mM). Numbers represent metabolites M-1 to M-13; IS represents internal standard (DBA-cis-5,B-dihydrodiol). For experimental conditions, see Experimental Procedures. sodium phosphate buffer (pH 7.4), and per mL an amount of S-9 equivalent to 2 nmol of cytochrome P450. The test compound dissolved in 30 p L of DMF, 500 p L of the activating system, 100 pL of the bacterial suspension, and 2 mL of warm (45 OC) top agar (0.55% agar, 0.55% NaC1, 50 pM histidine, 50 pM biotin, 25 mM sodium phosphate buffer, pH 7.4) was added sequentially to a test tube and vortexed, and the mixture was poured onto a Petri dish containing 20 mL of minimal agar (1.5 % agar,Vogel-BonnerE medium containing 2 % glucose). After incubation at 37 OC for 3 days in the dark, the colonies (his+ revertants) were counted.

Results and Discussion Identification of Microsomal Metabolites of DBA3,l-dihydrodiol. For the elucidation of its biotransformation (*)-DBA-3,4-dihydrodiol(50 pM) was incubated in the presence of an NADPH-generating system for 40 min with hepatic microsomal protein of adult male Sprague-Dawleyrats pretreated with Aroclor 1254. HPLC separation of the ethyl acetate-extractable metabolites on reverse phase with gradient elution using a mixture of methanol and aqueous NHlHCO3 (10 mM, pH 7.8) as mobile phase yielded about 16 well-separated peaks absorbing at 280 nm (Figure 1A). I n order to ascertain the purity of the peaks, they were scanned during the chromatographic run by a diode array spectrophotometer. From the fact that the peaks eluting at 15.6 and 15.9 min, at 21.3 and 22.4 min, and at 27.3 and 28.0 min exhibited the same UV spectra (data not shown), thus possessing the same chromophore, it was assumed that these double peaks probably represent optical isomers of three metabolites (M-1, -2, and -3 in Figure 1A). On the basis of the results of the spectral analysis and their elution behavior the remaining peaks were considered to represent distinct metabolites (M-4 to M-13). For normalization of retention times and for quantification of the metabolites DBA-cis-5,6-dihydrodiol was used as internal standard (28);this compound is not metabolically formed from DBA and is eluted in an area of the chromatogram where it does not interfere with microsomal metabolites of DBA (10)or of DBA-3P-dihydrodiol (data not shown). Performing the

92 Chem. Res. Toxicol., Vol. 7, No. 1, 1994

Platt and Schollmeier

Table 1. Criteria of Identity between Microsomal Metabolites of DBA-trans-3,4-dihydrodiol and Synthetic Reference Compounds identity of metabolite and reference compd as determined by change of formation in retention time UV-visible FD mass spectrum, metabolite the presence of TCPO synthetic reference compd in HPLC spectrum m/z ( M + ) 3,4:8,9-bisdihydrodiol + + 346 M-1 11 3,4:10,1l-bisdihydrodiol + + 346 M-2 11 3,4:12,13-bisdihydrodiol + + 346 M-3 11 M-4 0 1,4/2,3-tetrahydrotetraol + + 346 M-5a 0 1,3/2,4-tetrahydrotetraol + 328 M-8 t + M-10 0 1,2,4/3-tetrahydrotetraol M-12 0 syn-3,4-dihydrodioll,2-oxide + M-13 0 anti-3,4-dihydrodiol 1,2-oxide + +a -b The UV-visible spectrum of M-13was recorded during HPLC separation with a diode array detector. Isolation of M-13by HPLC resulted in conversion (hydrolysis) to M-4 (1,4/2,3-tetrahydrotetraol);this instability prevented the recording of a mass spectrum of M-13. 10.2

100-

90

-, 9 1

B E

. 3 1

3 2

80

? 3

? E

' 1

> 2

70

6 1

6 6

6 1

6 2

6.0

S P

5 1

S I

5 :

50

1 9

1 6

1 ,

4 2

6 .PPm

Figure 2. 400-MHz 'H-NMR spectrum in acetone-dslD20 (A)and FD mass spectrum (B)of biosynthetic DBA-trans,trans-3,4:8,9bisdihydrodiol. For details, see Experimental Procedures.

incubation of DBA-3,4-dihydrodiol in the presence of TCPO (1 mM) resulted in a dramatic change in the formation ofthe metabolites (Figure 1B): metabolites M-1, -2, and -3 disappeared almost completely, and the amount of metabolites M-5, -6, -7, and -8 increased considerably, while the formation of metabolites M-4, -5a, -10, -12, and -13 remained virtually unchanged. The structures of metabolites M-1, -2, -3, -4, and -13 were positively identified by a combination of chemical, biochemical,chromatographic, and spectroscopic methods while some structural indications could be obtained in the case of metabolites M-5a, -8,-10, and -12 (Table 1). From the fact that TCPO, a potent inhibitor of microsomal epoxide hydrolase (29), suppressed the formation of M-1, -2, and -3 almost completely (Figure 1B) their identity with bisdihydrodiols could be concluded. Four nonvicinal bisdihydrodiols formed from DBA-3,4-dihydrodiol can be envisaged: 3,4:5,6-, 3,4:8,9-, 3,4:10,11-, and 3,4:12,13bisdihydrodiol. In contrast to DBA-3,4:lO,ll-bisdihydrodiol and -3,4:12,13-bisdihydrodiol (see Experimental

Procedures), DBA-3,4:5,6-bisdihydrodiol and -3,4:8,9bisdihydrodiol were not synthetically availabie for structural comparison with M-1, -2, and -3. However, our recent observation* that metabolite M-1 is also one of the major microsomal metabolites of DBA-1,2-dihydrodiol besides the 1,412,3- and 1,3/2,4-tetrahydrotetraols(10, 30) and therefore very likely identical to DBA-1,2:lO,ll-bisdihydrodiol provided the means for biosynthesis of DBA-3,4: 8,9-bisdihydrodiol(structurally identical to DBA- 1,2:10,llbisdihydrodiol) from synthetic DBA-1,2-dihydrodiol. The 'H-NMR spectrum of biosynthetic DBA-3,48,9-bisdihyrodiol (Figure 2A) represents the almost perfect combination of the signals of DBA-1,2-dihydrodiol(16) and -3,4dihydrodiol(l7) while its FD mass spectrum (Figure 2B) is characterized by the molecular ion at m12 = 346 and the sequential loss of two molecules of water, resulting in the fragments ml2 = 328 and 310. DBA-3,48,9-bisdihydrodiol, -3,4:10,1l-bisdihydrodiol, and -3,4:12,13-bisdihydrodiol M . Schollmeier and K . L. Platt, u n p u b l i s h e d results.

Chem. Res. Toxicol., Vol. 7,No. 1, 1994 93

Metabolism of Dibenz[aa,h]anthracene-3,4-dio1

I

200

300

COO

200

500

Figure 3. UV-visible spectra of metabolite M-1

DBA-trans,truns-3,48,9-bisdihydrodiol (- - -) Figure 1).

and of (cf. Table 1 and (-)

400

300 Wavelength, A (nm)

Wavelength, A(nm)

Figure 5. UV-visible spectra of metabolite M-3(-1 and of DBA-truns,trans-3,4:12,13-bisdihydrodiol(- -) (cf. Table 1and Figure 1). Table 2. Influence of Hydrolysis in Aqueous Dioxane on Relative Amounts of Microsomal Metabolites of DBA-trans-3,4-dihydrodiol

relative amounta relative amounta before afar before after metabolite hydrolysis hydrolysis metabolite hydrolysis hydrolysis M-1 M-2 M-3

0.85 f 0.03 0.92 f 0.09 1.12 f 0.05 1.12 f 0.08 2.24 f 0.04 2.20 f 0.05

M-4 M-8 M-13

0.27 f 0.01 1.09 f 0.08 0.60 f 0.04 0.61 f 0.07 0.43 f 0.01 0.02 f 0.01

a Ratio of peak areas at 280 nm of metabolite and internal standard (DBA-cis-5,6-dihydrodiol).Values are means f standard deviations; n = 3.

200

300 h00 Wovr1rngth.A inm)

500

M-2(-) and of DBA-trans,trans-3,410,11-bisdihydrodiol(- -) (cf. Table 1and Figure 1). Figure 4. UV-visible spectra of metabolite

coelute with M-1, -2, and -3, respectively (Table 1). The bisdihydrodiols appear as diastereomeric mixtures which are separated by HPLC (Figure 1A),forming closely eluting double peaks, each peak representing the mixture of enantiomers of one diastereomer. Due to the symmetry of DBA-3,4:lO,ll-bisdihydrodiol one portion of the double peak of M-2 contains the enantiomers 3(R),4(R),lO(R),11(R)-and 3(S),4(S),lo@),11(S)-bisdihydrodiolwhile the other portion contains just one compound, Le., the meso form with the absolute configuration 3(R),4(R),lO(S),ll(S),which is structurally identical to 3(S),4(S),lO(R),ll-

(R). The structures of metabolites M-1, -2, and -3 were furthermore confirmed by comparison of their UV-visible spectra with those of the synthetic reference compounds (Figures 3-5). Final structural proof arose from the FD mass spectra of M-1, -2, and -3, which exhibited the

molecular ion at m/z = 346 (Table 1)as the most prominent signal. The UV-visible spectra of metabolites M-4 and M-13 are virtually indistinguishable and exhibit the chromophore of 1,2,3,4-tetrahydro-DBA (datanot shown). M-4 coelutes with DBA-1,4/2,3-tetrahydrotetraol while M-13 has the same retention time as DBA-anti-3,4-dihydrodiol 1,2-oxide. When M-4 and M-13 were isolated by chromatography and subjected to FD mass spectrometry, almost identical spectra were obtained, characterized by the molecular ion m/z = 346, which is typical for a tetrahydroxytetrahydro-DBA. Since for DBA-anti-3,4dihydrodiol 1,Zoxide a molecular ion at mlz = 328 was expected, this result points to the hydrolysis of the dihydrodiol oxide to a tetrahydrotetraol during the process of isolation. Controlled hydrolysis of a mixture of metabolites of DBA-3,4-dihydrodiol immediately after the incubation leaves the amount of all metabolites unchanged except that of M-4 and M-13: while M-13 disappears almost completely, the amount of M-4 rises accordingly (Table 2). This verifies the conversion of M-13 to M-4 and is in agreement with the fact that DBA-anti-3,4dihydrodiol1,2-oxide leads to just one tetrahydrotetraol, namely, DBA-1,4/2,3-tetrahydrotetraol, upon hydrolysis (IO). Indications for the metabolic formation of the syn diastereomer of the dihydrodiol oxide M-13 were less convincing. Metabolite M-12 cochromatographs with DBA-syn-3,4-dihydrodiol1,2-oxide; however, its amount was not significantly diminished upon hydrolysis. Since

94

Chem. Res. Toxicol., Vol. 7, No. 1, 1994

Platt and Schollmeier carcinogens, e.g., benzo[alpyrene (5).

I2,69""1

,-'-\ /

--. -.

Yawlength In)

Figure 6. Fluorescence excitation and emission spectra of biosynthetic DBA-trans,trans-3,4:8,9-bisdihydrodiol (cf.Figure 2).

DBA- 1,3/2,4-tetrahydrotetraols and - 1,2,4/3-tetrahydrotetraols are the products of hydrolysis of DBA-syn-3,4dihydrodiol 1,2-oxide (10), comparison of their elution behavior with that of the metabolites of DBA-3,4-dihydrodiol revealed that they coelute with M-5a and M-10. Positive identification of metabolites M-5a, -10, and -12 by UV and mass spectrometry was precluded by their insufficient formation. The amounts of metabolites M-4, -5a, and -10 were not influenced by the presence of TCPO. Consequently, microsomal epoxide hydrolase is not involved in their formation from DBA-3,4-dihydrodiol via the corresponding dihydrodiol oxides. This is in concordance with the observation that most bay-region dihydrodiol oxides are poor substrates of this hydrolytic enzyme (ref 31 and references therein) and provides some further proof for the structure of M-4, -5a, and -10 (Table 1). Finally, the FD mass spectrum of metabolite M-8 exhibiting the molecular ion a t m/z = 328 together with its chromatographic behavior yielded an indication of its structure as a nonvicinal 3,4-dihydrodiol oxide or rather its more stable isomerization product, a DBA-3,4-dihydrodiol phenol. These results of the present study call for some comments on a recent investigation dealing with the microsomal metabolism of DBA-3,Cdihydrodiol by Lecoq et al. (15). These authors attempted the identification of the four most polar metabolites of DBA-3,4-dihydrodiol (named C, B, A, D) based solely on optical electron spectroscopic evidence. While their assignement of D to a 1,2,3,4-tetrahydrotetraol (probably identical toM-4) and of A to DBA-3,4:lO,ll-bisdihydrodiol(identical to M-2) is correct, the suggested structures of metabolites B and C as being hexahydrohexaols cannot be true. Since the excitation part of the fluorescence spectrum of C in ref 15 resembled that of the UV spectrum of DBA-3,4:8,9bisdihydrodiol (Figure 31, a fluorescence spectrum of this compound was obtained (Figure 6) which corresponded closely with the related spectrum of C (15). Hence, since metabolite C is obviously a bisdihydrodiol, metabolite B that elutes later on reverse phase (15) can hardly be identical to a hexahydrohexaol due to its higher polarity. DBA-3,4-dihydrodiol is not the first example of an M-region dihydrodiol that is metabolically transformed to bisdihydrodiols by hepatic microsomes of rats treated with inducers of cytochrome P450 1 A l and 1A2, e.g., 3-methylcholanthrene or Aroclor 1254: the related 3,4dihydrodiols of benzfalanthracene (32)and of benzo[clphenanthrene (33)exhibit the same metabolic peculiarity, uncommon to the metabolism of PAH known to be strong

Metabolic Pathways of DBA-3,4-dihydrodiol and Their Biological Significance. On the basis of our results on the structures of microsomal metabolites of DBA-3,4-dihydrodiol, its metabolic pathways can be deduced (Figure 7 ) . Cytochrome P450-dependent hepatic monooxygenase(s) of Aroclor 1254-pretreated rats epoxidize the 1,2-double bond in the M-region dihydrodiol, yielding DBA-anti-3,4-dihydrodiol 1,2-oxide, M-13, and the related tetrahydrotetraol, M-4, to a small extent. The formation of the syn diastereomer, 1, is still uncertain and only substantiated by the occurrence of its products of hydrolysis, i.e., the tetrahydrotetraols M-5a and M-10. The pronounced appearance of three of the four possible bisdihydrodiols (M-1, -2, -3) of DBA-3,4-dihydrodiol requires the formation of (nonvicinal) dihydrodiol arene oxides 2-4 (Figure 7). Their nonenzymatic aromatization should lead to dihydrodiol phenols 5-10 (Figure 7) which are very likely to be found among the metabolites M-5, -6, -7, -8,-9, and -11(Figure 1). In the case of metabolite M-8 this assumption was confirmed by mass spectroscopic evidence (Table 1). When microsomal epoxide hydrolase is inhibited, enzymatic hydrolysis of dihydrodiol arene oxides is suppressed and therefore nonenzymatic aromatization to dihydrodiol phenols should be favored. This can indeed be observed in the presence of TCPO (Figure lB), where the amount of metabolites M-5, -6, -7, -8, -9, and -11 increases considerably. Finally, bisdihydrodiols M-1, -2, and -3 could again serve as substrates for cytochrome P450-dependent monooxygenase(s),resulting in the formation of bisdihydrodiol epoxides (cf. 11 and 12 in Figure 7). In order to account for the importance of the different metabolic pathways of DBA-3,4-dihydrodiol,its metabolic conversion and the quantitative distribution of its identified metabolites were determined by radiochromatography. The total metabolic conversion of DBA-3,4dihydrodiol amounts to 8.84 nmol/(nmol of cytochrome P450.15 min) (Table 3), thus being in the same range as that of DBA itself (IO),while the dihydrodiols at the bayand K-region of DBA are much better substrates of the Aroclor 1254-inducible monoo~ygenases.~ Almost one-third of the metabolites of DBA-3,Cdihydrodiol consist of bisdihydrodiols, the 3,4:10,11-bisdihydrodiol representing the main metabolite of the M-region dihydrodiol with 16.3 % of total metabolic conversion. The anti-3,4-dihydrodiol1,2-oxide, in contrast, accounts only for 3.1% of total metabolic conversion. This amount, however, may not reflect the actual amount of DBA-anti3,4-dihydrodiol 1,2-oxide that is formed, since the dihydrodiol oxide could covalently bind to microsomal protein or to phosphate in the incubation buffer or be metabolized by monooxygenase attack followed by hydrolysis to very hydrophilic hexahydroxy derivatives of DBA, which could remain in the aqueous phase upon extraction and thus escape detection. The high amount of unknown watersoluble and protein-bound metabolites (Table 3) could be indicative of this asumption. While protein and phosphate binding has already been excluded ( l o ) ,further oxidative metabolism of DBA-anti-3,4-dihydrodiol1,2-oxide will be subject of future investigations. The biological significance of these results has to be based not only on quantitative but also on qualitative aspects.

Chem. Res. Toxicol., Vol. 7,No. 1, 1994 95

Metabolism of Dibenz[a,h]anthracene-3,4-diol

U A,..

u - 4

bisdihydrodiol oxides

0.780

Lo-

\

t

@&:

0.658

000

M-L

"#

01Q

0 9

Figure 7. Metabolic pathways of DBA-trans-3,4-dihydrodiol with liver microsomes of Sprague-Dawleyrats pretreated with Aroclor 1254. M-1, -2, -3, -4, and -13 represent identified metabolites (cf. Table 1); 1-12 represent proposed metabolites (see text); values of delocalization energy ( h E d & / O ) of benzylic carbocations obtained by heterolytic cleavage of the oxirane ring are shown for M-13,11,

and 12.

Table 3. Quantitative Distribution of Principal Metabolites upon Incubation of [WIDBA-trans-3,4-dihydrodiol with the Microsomal Liver Fraction of Sprague-Dawley Rats after Aroclor 1254 Treatment

metabolite formation" [nmol/(nmolof metabolite cytochrome P450.15 min)] 0.32 0.020b (3.6)' 3,4:8,9-bisdihydrodiol(M-1) 1.44 0.011 (16.3) 3,4:10,1l-bisdihydrodiol(M-2) 3,4:12,13-bisdihydrodiol(M-3) 0.70 0.009(7.9) 0.06 0.021 (0.7) 1,4/2,3-tetrahydrotetraol(M-4) anti-3,4-dihydrodiol l,2-oxide (M-13) 0.27 0.159 (3.1) 2.05 f 0.160 (23.2) unknown ethyl acetate-extractable metabolites (M-5 to M-12) 4.00 f 0.250 (45.2) water-soluble and protein-bound metabolitesd 8.84 f 0.353 (100) total metabolic conversion a From incubation of 50 pM DBA-3,4-dihydrodiol. Values are means f standard deviation; n = 3. Values in parentheses refer to percentage of total metabolic conversion. Calculated from radioactivity remaining in the aqueous phase after extraction with ethyl acetate.

* * * *

Bisdihydrodiol epoxides originating from M-1 hardly contribute anything to the biological activity of DBA3,4-dihydrodiolbecause of the weak bacterial mutagenicity (12,34) and tumorigenicity (14)of DRA-1,2-dihydrodiol, which is a metabolic precursor of bisdihydrodiol M-1 (see above). The putative biological activity of dihydrodiol oxide M-13 and 1 as well as of bisdihydrodiol epoxides 11 and

12 can be estimated on the basis of the "bay-region theory" (35) by comparing h E d & JP of the benzylic carbonium

ions resulting from opening of the oxirane ring; these values are 0.738 for M-13 and 1, 0.780 for 11, and 0.658 for 12. Thus it can be expected that the bisdihydrodiol oxide 11 formed from DBA-3,4:lO,ll-bisdihydrodiolis more reactive than the vicinal dihydrodiol oxide M-13 or the bisdihydrodiol oxide M-12 originating from DBA-3,4:12,13-bisdihydrodiol. Since DBA-3,4:lO,ll-bisdihydrodiol is the immediate precursor of bisdihydrodiol oxide 11 while DBA-3,4dihydrodiol needs two additional enzymatic steps to be converted to 11, DBA-3,4:lO,ll-bisdihydrodiolshould exhibit a higher genotoxic activity than DBA-3,Cdihydrodiol when activated by the same metabolizing system. This could indeed be confirmed by determining the bacterial mutagenicity in S. typhimurium TAlOO (Figure 8). The difference in mutagenic activity of DBA-3,4:10,11-bisdihydrodiol, 52.3 his+revertants/nmol, as compared to that of DBA-3,4-dihydrodiol,34.5his+revertants/nmol, reflects well the extent of binding of these two metabolites of DBA to calf thymus DNA (36).The lack of metabolic activation of DBA-3,4:12,13-bisdihydrodiol to mutagenic species is somewhat surprising since the related bay-region dihydrodiol oxides of phenanthrene and chrysene exhibiting similar h E d e l d @values 10.658 and 0.639, respectively (35)l for their benzylic carbonium ions as the bisdihydrodiol oxide 12 are strong bacterial mutagens in strain

96 Chem. Res. Toxicol., Vol. 7, No. 1, 1994

i

0

/

/

Platt and Schollmeier

w i t h Aroclor 1 2 5 1

50 Concentration ( nmol / p l a t e

100

300

I

Figure 8. Bacterial mutagenicity in S. typhimurium TAlOO of DBA-trans-3,4-dihydrodiol,DBA-trans,trans-3,410,ll-bisdihydrodiol, and DBA-trans,trans-3,4:12,13-bisdihydrodiol;each point represents the average of three experiments, with the vertical bars showing the standard deviation. TAlOO (37). It is therefore assumed that the hydroxyl group in the 13-position of M-3 (cf. Figure 7) alters the interaction of the bisdihydrodiol with the catalytic binding site of cytochrome P450 in such a way that epoxidation of the olefinic double bond in the 1,2-position is prevented. Previous investigations have shown that metabolically formed DBA-3P-dihydrodiol is highly enriched in its R,Renantiomer (8,10,11),which is activated to much stronger bacterial mutagens than the corresponding S,S-isomer (34). Therefore, it will be interesting to determine the stereoselectivity in the metabolic conversion of enantiomeric DBA-3P-dihydrodiol and to eluciate the role of the stereoisomers of the bisdihydrodiols in the genotoxicity of DBA. The relevance of the metabolic pathways elucidated in this study for the genotoxic activity of DBA has been furthermore ascertained by determination of the DNA binding in vitro (36,38,39)and in vivo (40-42). Thus the postulated reactivity of the ultimate mutagens of the three bisdihydrodiols was confirmed by the extent of DNA binding in mouse skin (42),which rose from no detectable binding in the case of DBA-3,4:12,13-bisdihydrodiol to weak binding in the case of DBA-3,4:8,9-bisdihydrodiol, reaching its highest value in the case of DBA-3,4:10,11bisdihydrodiol. Furthermore, DBA and DBA-3,4-dihydrodiol yielded the same pattern of DNA adducts when incubated with rat liver microsomes (38, 39) or when applied to mouse skin in vivo (40),demonstrating anew the biological significance of the M-region dihydrodiol of DBA as precursor of its genotoxic metabolite(s). In both metabolizing systems DNA adducts based on bay-region dihydrodiol oxides were detected (38-40) and could be characterized (39),yet, especially in vivo, their amount was surpassed by more polar adducts (40). Remarkably,

this could be shown not only with mouse skin (40)but also with human skin in culture (41). Finally, it was shown in vitro (36)and in vivo (42)that these polar DNA adducts are formed from DBA-3,4:lO,ll-bisdihydrodiol through further metabolic activation. Taking the results of the metabolism, mutagenicity, and DNA binding studies together, it is reasonable to assume that the major metabolic pathway of DBA leading to genotoxic events is characterized by the sequential attack of cytochrome P450 a t the 3,4-, 10,ll-, or 8,9- (or 1,2-) position of the PAH, culminating in the formation of DBA3,4:10,1l-bisdihydrodiol1,Zoxide as the ultimate mutagenic metabolite. This does not, however, exclude the possibility that dihydrodiol oxides or other metabolites could play a presumably less important role in the genotoxicity of DBA. The metabolism of DBA-3P-dihydrodiol to bisdihydrodiols is not unique among the PAH; it was also observed with the M-region dihydrodiols of benz[alanthracene (32) and, benzo[clphenanthrene (33). In the case of these just weakly carcinogenic PAH the bypassing of the dihydrodiol oxide pathway leads to less genotoxic metabolites, while in the case of the carcinogenic DBA the formation of bisdihydrodiols provides the basis for stronger mutagenic bisdihydrodiol oxides responsible for the pronounced genotoxicity of this PAH.

Acknowledgment. We express our appreciation to S. Kuthan for excellent technical assistance as well as to Drs. H. Frank and F. Setiabudi for providing some synthetic derivatives of dibenz[a,h]anthracene. The help of M. Eider, S. Gebhard, and A. Vierengel in performing the mass, fluorescence, and NMR spectra, respectively, is gratefully acknowledged. Thanks are also due to Dr. G. Jennings for proofreading the rough draft and to I. Bohm for skillful help in the preparation of the manuscript. We are grateful for financial support by the Deutsche Forschungsgemeinschaft (Grant SFB 302).

References (1) Hoffmann, D., and Wynder, E. L. (1976) Environmental respiratory carcinogenesis. In Chemical Carcinogens (Searle, C. E., Ed.) ACS Monograph 173, pp 324-365, American Chemical Society, Washington, DC. (2) Dipple, A. (1976) Polynuclear aromatic carcinogens. In Chemical Carcinogens (Searle, C. E., Ed.) ACS Monograph 173, pp 245-314, American Chemical Society, Washington, DC. (3) Kennaway, E. L. (1930) Further experiments on cancer-producing substances. Biochem. J.24,497-504. (4) Heidelberger, C. (1975) Chemical carcinogenesis. Annu. Rev. Biochem. 44, 79-121. (5) Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D.M. (1985) Polycyclic aromatic hydrocarbons: Metabolic activation to ultimate carcinogens. In Bioactiuation of Foreign Compounds (Anders, M.W., Ed.) pp 177-242, Academic Press, Orlando. (6) Cavalieri,E. L., and Rogan, E. G. (1990) Radical cations in aromatic hydrocarboncarcinogenesis.Free Radical Res. Commun. 11,7747. (7) Flesher, J. W., and Myers, S. R. (1990) Bioalkylation of benz[a]anthracene as a biochemical probe for carcinogenic activity. Lack of bioalkylation in a series of six noncarcinogenic polynuclear aromatic hydrocarbons. Drug Metab. Dispos. 18, 163-167. (8) Nordqvist, M., Thakker, D. R., Levin, W., Yagi, H., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1979) The highly tumorigenic 3,4-dihydrodiol is a principal metabolite formed from dibenzo[a,hlanthracene by liver enzymes.Mol.Pharmacol. 16,643655. (9) MacNicoll, A. D., Burden, P. M., Rattle, H., Grover, P. L., and Sims, P. (1979) The formation of dihydrodiols in the chemical or enzymic oxidation of dibenz[a,c]anthracene, dibenz[a,hlanthracene and chrysene. Chem.-BioL Interact. 27, 365-379.

Metabolism of Dibenz[aa,h]anthracene-3,4-diol Platt, K. L., and Reischmann, I. (1987)Regio- and stereoselective metabolism of dibenz[a,h]anthracene: Identification of 12 new microsomal metabolites. Mol. Pharmacol. 32, 710-722. Mushtaq, M., Weems, H. B., and Yang, S.K. (1989)Stereoselective formations of enantiomeric K-region epoxideand trans-dihydrodiols in dibenz[a,hlanthracene metabolism. Chem. Res. Toxicol. 2, 8493. Wood, A. W., Levin, W., Thomas, P. E., Ryan, D., Karle, J. M., Yagi, H., Jerina, D.M., and Conney, A. H. (1978)Metabolic activation of dibenzo[a,hl anthracene and its dihydrodiols to bacterial mutagens. Cancer Res. 38, 1967-1973. Platt, K. L., and Oesch, F. (1988)Metabolic pathways of dibenz[a,h]anthracene and their enzymatic control in relation to bacterial mutagenicity. Rev. Biochem. Toxicol. 9, 185-224. Buening, M. K., Levin, W., Wood, A. W., Chang, R. L., Yagi, H., Karle, J. M., Jerina,D. M., and Conney, A. H. (1979)Tumorigenicity of the dihydrodiols of dibenzo[a,h]anthracene on mouse skin and in newborn mice. Cancer Res. 39, 1310-1314. Lecoq, S.,Chalvet, O., Strapelias, H., Grover, P. L., Phillips, D. H., and Duquesne, M. (1991)Microsomal metabolism of dibenz[a,clanthracene, dibenz[a,hlanthracene and dibenz[ajlanthracene to bis-dihydrodiols and polyhydroxylated products. Chem.-Biol. Interact. 80,261-279. Oesch, F., Stillger, G., Frank, H., and Platt, K. L. (1982)Improved synthesesof (*)-trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene and of (*)-trans-l,2-dihydroxy-l,2-dihydrodibenz[a,h]anthracene. J. Org. Chem. 47,568-571. Platt, K. L., and Oesch, F. (1983)Efficient synthesis of non-Kregion trans-dihydro diols of polycyclicaromatic hydrocarbons from o-quinones and catechols. J. Org. Chem. 48,265-268. LaBudde, 3. A., and Heidelberger, C. (1958)The synthesis of the mono- and dihydroxy derivatives of 1,2,5,6-dibenzanthraceneexcreted by the rabbit and of other hydroxylated dibenzanthracene derivatives. J. Am. Chem. SOC. 80, 1225-1236. Lee, H.M., and Harvey, R. G. (1980)Synthesis of biologicallyactive metabolites of dibenz[a,hlanthracene. J. Org. Chem. 45,588-592. Yagi, H.,Thakker, D. R., Hernandez, O., Koreeda, M., and Jerina, D. M. (1977)Synthesis and reactions of the highly mutagenic 7,8diol 9,lO-oxides of the carcinogen benzo[a]pyrene. J. Am. Chem. SOC.99, 1604-1611. (21) Frank, H. (1986) Potential metabolites of dibenz[a,hlanthracene-Synthesis, separation of racemates and determination of absolute configuration, Ph.D. Thesis, University of Mainz. (22) Platt, K. L., and Oesch, F. (1982)Synthesis of non-K-region orthoquinones of polycyclic aromatic hydrocarbons from cyclic ketones. Tetrahedron Lett. 23, 163-166. (23) Setiabudi, F., Oesch,F., andPlatt, K. L. (1987)Radioactivelylabelled epoxides. Part V. Tritium labelled K-region oxides and transdihydrodiols of pyrene, benzo[alpyrene and dibenz[a,hlanthracene. J.Labelled. Compd. Radiopharm. 24, 979-986. (24) Schmassmann, H.,Sparrow, A., Platt, K., and Oesch, F. (1978) Epoxide hydratase and benzo[alpyrene monooxygenase activities in liver, kidney and lung after treatment of rats with epoxides of widely varying structures. Biochem.Pharmacol. 27, 2237-2245. (25) Lowry, 0.H.,Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)Protein measurement with the Folin Dhenol reagent. J.Biol. &em. 193, 265-275. (26) . . Omura. T.. and Sato. R. (1964) The carbon monoxide-binding pigment of liver microsomes. 11. Solubilization, purification, a n i properties. J.Biol. Chem. 239,2379-2385. (27) Ames, B. N., McCann, J., and Yamasaki, E. (1975)Methods for detecting carcinogens and mutagens with the Salmonelldmammalian-microsome mutagenicity test. Mutat. Res. 31, 347-363. (28) Platt, K. L., Reischmann, I., Flenner, C., and Oesch, F. (1986) Isolation, separation and quantification of metabolites of the I

Chem. Res. Toxicol., Vol. 7, No. 1, 1994 97 carcinogenic polycyclic aromatic hydrocarbon dibenz[a,hlanthracene. Fresenius’ 2.Anal. Chem. 324, 357. (29) Oesch,F.,Kaubisch,N., Jerina,D. M.,andDaly, J. W. (1971)Hepatic epoxide hydrase. Structure-activity relationships for substrates and inhibitors. Biochemistry 10, 4858-4866. (30) Chou, M. W., Fu, P. P., and Yang, S. K. (1981)Metabolic conversion of dibenz[a,h]anthracene (+)-trans-1,2-dihydrodioland chrysene (+)-trans-3,4-dihydrodiol to vicinaldihydrodiolepoxides.h o c . Natl. Acad. Sci. U.S.A. 78,4270-4273. (31) Sayer, J. M., Yagi, H., van Bladeren, P. J., Levin, W., and Jerina, D. M. (1985) Stereoselectivity of microsomal epoxide hydrolase toward diol epoxides and tetrahydroepoxides derived from benz[alanthracene. J. Biol.Chem. 260, 1630-1640. (32) Thakker, D. R., Levin, W., Yagi, H., Tada, M., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1982) Stereoselective metabolism of the (+)-and (-)-enantiomers of trans-3,4-dihydroxy3,4-dihydrobenz[a]anthraceneby rat liver microsomes and by a purified and reconstituted cytochrome P450 system. J.Biol.Chem. 257,5103-5110. (33) Thakker, D. R., Levin, W., Yagi, H., Yeh, H. J. C., Ryan, D. E., Thomas,P. E., Conney,A. H., and Jerina,D. M. (1986)Stereoselective metabolism of the (+)-(S,S)-and (-)-(R,R)-enantiomera of trans3,4-dihydroxy-3,4-dihydrobenzo[clphenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P450 system. J. Biol. Chem. 261, 5404-5413. (34) Platt, K. L., Schollmeier, M.,, Frank, H., and Oesch, F. (1990) Stereoselective metabolism of dibenz[a,hlanthracene to transdihydrodiols and their activation to bacterial mutagens. Enuiron. Health Perspect. 88, 37-41. (35) Jerina, D. M., and Lehr, R. E. (1977)The bay-region theory: A quantum mechanical approach to aromatic hydrocarbon-induced carcinogenicity. In Microsomes and Drug Oxidations (Ullrich, V., Roots, I., Hildebrandt, A., Estabrook, R. W., and Conney, A. H., Eds.) pp 709-720, Pergamon Press, Oxford. (36) Fuchs, J., Mlcoch, J., Platt, K. L., and Oesch, F. (1993)Characterization of highly polar bis-dihydrodiolepoxide-DNA adducts formed after metabolic activation of dibenz[a,hlanthracene. Carcinogenesis 14,863-867. (37) Wood, A. W., Chang, R. L., Levin, W., Ryan, D. E., Thomas, P. E., Mah, H. D., Karle, J. M., Yagi, H., Jerina, D. M., and Conney, A. H. (1979)Mutagenicity and tumorigenicity of phenanthrene and chrysene epoxides and diol epoxides. Cancer Res. 39, 40694077. (38) Lecoq, S.,Sh6, M. N., Grover, P. L., Platt, K. L., Oesch, F., and Phillips, D. H. (1991)The in vitro metabolic activation of dibenz[a,hlanthracene, catalyzed by rat liver microsomes and examined by 32P-postlabelling. Cancer Lett. 57, 261-269. (39) Mlcoch, J., Fuchs, J., Oesch, F., and Platt, K. L. (1993)Characterization of DNA adducts at the bay region of dibenz[a,hlanthracene formed in vitro. Carcinogenesis 14,469-473. (40) Lecoq, S.,Sh6, M. N., Hewer, A,, Grover, P. L., Platt, K. L., Oesch, F., and Phillips, D. H. (1991)The metabolic activation of dibenz[a,hlanthracene in mouse skin examined by szP-postlabelling: minor contribution of the 3,4-diol l,a-oxides to DNA binding. Carcinogenesis 12, 1079-1083. (41) Lecoq, S.,Pfau, W., Grover, P. L., and Phillips, D. H. (1992)HPLC separation of 32P-postlabelled DNA adducts formed from dibenz[a,hlanthracene in skin. Chem.-Biol. Interact. 85, 173-185. (42) Carmichael, P. L., Platt, K. L., Sh6, M. N., Lecoq, S.,Oesch, F., Phillips,D. H., and Grover, P. L. (1993)Evidencefor the involvement of a bis-diol-epoxide in the metabolic activation of dibenz[a,h]anthracene to DNA-binding species in mouse skin. Cancer Res. 53, 944-948.