Chem. Res. Toricol. 1992,5, 576-583
576
Examination of Diols and Diol Epoxides of Polycyclic Aromatic Hydrocarbons as Substrates for Rat Liver Dihydrodiol Dehydrogenase Lynn Flowers-Geary,? Ronald G. Harvey,$ and Trevor M. Penning'lt Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, and The Ben May Institute, University of Chicago, Chicago, Illinois 60637 Received March 4, 1992 Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) can suppress the formation of anti-diol epoxides that arise from the metabolic activation of PAH by oxidizing their precursor trans-dihydrodiols to o-quinones [Smithgall, T. E., et al. (1988) J. Biol. Chem. 263,1814-18201. DD has also been found to reduce the mutagenicity of benz[alanthracene (*)-anti-8,9-dihydrodiol l0,ll-epoxide [(&)-anti-BADE]in the Ames test (Glatt, H. R., et al. (1982) Science 215,1507-1509), suggesting that anti-diol epoxides are substrates for this enzyme. In this study, attempts have been made to demonstrate directly that diol epoxides of PAH are substrates for homogeneous DD. Spectrophotometric assays indicate that high concentrations of the stable anti-diol epoxides, naphthalene (i)-anti-1,2-dihydrodiol3,4-epoxide (10 mM) and (*)-anti-BADE (20 pM; limit of solubility) were not oxidized by micromolar concentrations of enzyme. By contrast, 20 pM (f)-trans-7,8-dihydroxy-7,8-dihydrobenzo[alpyrene[(&)-trans-BP-diol] was oxidized by 50fold less enzyme. Using a reverse-phase high-performance liquid chromatography (RP-HPLC)could be almost completely oxidized by DD in the presence based assay [1,3-3Hl-(~:)-trans-BP-diol of NADP+. Using a similar assay, [1,3-3Hlbenzo[a]pyrene (*)-anti-7,8-dihydrodiol 9,lOepoxide [(f)-anti-BPDEI, and unlabeled (&)-syn-BPDE and (&)-anti-BADE were tested as substrates for DD. Incubations were performed in the presence of 2.3 mM NADP+ in either potassium phosphate (pH 7.0) or glycine (pH 9.0) buffer, and reactions were terminated by the addition of 2-mercaptoethanol(2 mM) to trap unreacted diol epoxides as thiol ether adducts. RP-HPLC analysis of the reaction mixtures using diode array detection showed that they contained only hydrolysis products (tetraols) and thiol ether adducts of diol epoxides. No loss of enzyme activity was found after incubation of the enzyme with the diol epoxides. The results indicate that PAH diol epoxides are not substrates for DD and that they do not inactivate the enzyme. However, when (*)-anti-BADE (20 pM)was incubated with bovine serum albumin or DD alone, its rate of hydrolysis and formation of its thiol ether adduct were prevented in a concentration-dependent manner. The decrease in mutagenicity of this diol epoxide in the presence of DD, therefore, may be due to its sequestration by protein.
Introduction (*)-anti-BPDE polycyclic aromatic hydrocarbons (PAH)' are environmental pollutants that require metabolic activation to exert their carcinogenic effects (1).In the case of benzo[a]pyrene, this pathway involves initial epoxidation catalyzed by cytochrome P-450 to yield the 7(R),8(S)-epoxideand hydration by epoxide hydrolase to yield the 7(R),8(R)-dihydrodiol[(-)-trans-7,8-dihydroxy7,8-dihydrobenzo[a]pyrene;(-)-trans-BP-diol], a proximate carcinogen (1, 2). Secondary epoxidation of (*)trans-BP-diol results primarily in the formation of the (*)-anti-diol epoxide [(f)-anti-78,8a-dihydrox~9~,lOaepoxy-7,8,9,10-tetrahydrobenzo[alpyrene; (k)-anti-BPDEl (3, 4) which alkylates DNA (5-7). (*)-anti-BPDE is a potent mutagen (8,9) and is by far the most tumorigenic metabolite of benzo[alpyrene (10, 11). (*I-anti-BPDE is, therefore, regarded as an ultimate carcinogen, and similar anti-diol epoxides are ultimate carcinogens for other PAH (12-16). Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) is one enzyme that could suppress the levels of PAH anti-diol * To whom requests for reprints should be addressed. t f
University of Pennsylvania School of Medicine. University of Chicago.
epoxides. Addition of the homogeneous enzyme to an Ames test for benzo[alpyrene (Salmonella typhimurium + TA98/TA100 + microsomal activation system)resulted in a significant reduction of revertants (17). On the basis of earlier work which showed that DD catalyzed the oxidation of benzene dihydrodiol to catechol (181, it was concluded that the mutagenicity of benzo[alpyrene was suppressed because DD oxidized the 7(R),8(R)-dihydrodi01, formed in situ, to the corresponding innocuous catechol. In related experiments,DD was found to significantly reduce the mutagenicity of benz[alanthracene (*)-anti8,9-dihydrodiol l0,ll-epoxide [(*)-anti-BADE1 in the Ames test (19). These results suggested that anti-diol epoxide ultimate carcinogens may also be substrates for DD. However, milligram quantities of DD were used in these Ames tests; therefore, these studies were conducted using nonphysiological amounts of the enzyme. In addition, PAH trans-dihydrodiols and PAH anti-diol epoxides were not examined as substrates for DD. We have shown that PAH non-K-region trans-dihydrodiols (proximate carcinogens) are substrates for homogeneous DD. Moreover, the enzyme displayed the appropriate stereoselectivity in oxidizing the proximate carcinogens formed metabolically from benzo[al pyrene,
0893-228x/92/2705-0576$03.00/00 1992 American Chemical Society
Diol Epoxides and Dihydrodiol Dehydrogenase
Chem. Res. Toxicol., Vol. 5, No. 4,1992 577
chrysene, 5-methylchrysene, benz[alanthracene, and 7,12Chart I. Structures of Potential Dihydrodiol Dehydrogenase Substrates dimethylbenz[a]anthracene (20,21). In characterizing the products of these reactions, it was found that the corresponding catechols were formed transiently and underwent rapid air oxidation to form reactive o-quinones which could be trapped as thiol ether adducts with 2-mercaptoethanol. Since similar reactions occur with cysteine and gluI I tathione, DD may initiate a chain of reactions which results OH OH (~)-an6benzo[a]pyrenein the formation of glutathionyl conjugates of PAH o7.8-dihydrodioi-9,lO-epoxide quinones (22,23). For benzo[a] pyrene, the glutathionyl conjugate of benzo[alpyrene-7,8-dione would be the metabolite of interest. In our earlier work PAH anti-diol epoxides were not examined as substrates for DD. Recently, homogeneous DD was also shown to reduce the mutagenicity of benzene (i)-anti-1,2-dihydrodiol3,4epoxide in the Ames test (24). In addition, it was shown 1 I OH OH that benzene (&)-anti- and benzene (f)-syn-l,2-dihy(~)-syn-benzo[a]pyrene(~)-antbbenz[a]anlhracenedrodiol 3,4-epoxides were oxidized by DD to unknown 7,8-dihydrodioi-9.IO-epoxide 8,9dihydrodioi-l0,11-epoxide products (possibly ketone triols). However, Oesch and his associates (25) have now shown that, using HPLCOmnisolve grade and were purchased from Bodman Chemicals based assays, anti-BADE and the trans-BP-diol are not (Aston, PA). THF was distilled from CaH2 before use. Deusubstrates for homogeneous DD. Because of these conterated solvents and TMS were obtained from Aldrich (Milflicting results, it became important toaddress the question waukee, WI). Androsterone was purchased from Steraloids (Wilton, NH). (&)-anti-BADEand the cis and trans hydrolysis of whether known ultimate carcinogens (bay-region antiproducts of (&)-anti-and (&)-syn-BPDE,r-7,t-8,9,10-,r-7,t-8,9,cdiol epoxides) are substrates for homogeneous DD. In lo-, r-7,t-8,c-9,10-, and r-7,t-8,10-~-9-tetrahydrotetrols, were addition, it was important to confirm our earlier obserobtained from the NCI Chemical Carcinogen Repository, Midwest vations that (A)-trans-BP-diol was a substrate for DD. Research Institute (Kansas City, MO). [1,3-3H]-(f)-anti-BPDE In this paper PAH diol epoxides (Chart I) were examined (365mCi/mmol)and [ 1,3-3H]-(&)-trans-BP-diol (764 mCi/mmol) directly as substrates for purified rat liver DD. Our results were obtained from Chemsyn Science Laboratories (Lenexa, KS). indicate that diol epoxides are not substrates for DD under Caution: All PAH are potentially hazardous and should conditions in which (f)-trans-BP-diol is almost completely be handled in accordance with “NIHGuidelines for the Laboratory Use of Chemical Carcinogens”. consumed. A preliminary report of these results has Synthesis of Polycyclic Aromatic Hydrocarbons. Benappeared (26).
Experimental Procedures Materials. @-NADP+and NAD+ were obtained from Pharmacia-LKB Biotechnology (Piscataway, N J ) a n d Boehringer-Mannheim (Indianapolis, IN). 2-Mercaptoethanol was purchased from Pierce (Rockford, IL). Crystallized BSA was obtained from Armour Pharmaceutical Co. (Kankakee, IL). All solvents were either HPLC grade or EM Science ~~
zene (*)-trans-dihydrodiol was synthesized according to Smithgall and Penning (27). (&)-anti-NDE was synthesized as described (28). (&)-trans-BP-dioland (&)-anti-and (&)-synBPDEs were synthesized by the method of Fu and Harvey (29, 30). High-field lH-NMR data for thiol ether adducts and tetraols were obtained on a Bruker AM-500 spectrometer (Bruker, U.K., Ltd., Coventry, U.K.) equipped with an ASPET 3000 Computer operating at 500.13 MHz. lH chemical shifts are reported relative to TMS. Synthesis of (&)-lo+[(2-Hydroxyethyl)thio]-7&8~,9c-trihydroxy-7,8,9,1O-tetrahydrobenzo[a]pyrene. (*)-anti-BPDE (1 mg; 3.3 pmol) was dissolved in 1 mL of T H F and added to T H F (9 mL) containing 2 mM 2-mercaptoethanol. The reaction mixture was stirred at room temperature for 45 min. T H F and excess 2-mercaptoethanol were removed under vacuum to yield 0.7 mg of thiol ether adduct: lH-NMR (500 MHz, acetone-ds) 6 2.90 (q, 2 H, CH20), 3.05 (q,2 H, CHzS), 3.75 (m, 1H, H9), 4.37 (dd, 1H, HS), 5.05 (d, 1H, H10; JSJO 7.5 Hz), 5.25 (d, 1H, H7; 57,~ = 7 Hz),8.&8.3 (m,6H,Hl-H5,Hl2),8.44(s,lH,H6),8.58 (d, 1 H, H11). Synthesis of (&)-1 IC-[ (2-Hydroxyethyl)thio]-8&9a,l0c-tri-
1 Abbreviations: PAH,polycyclicaromatic hydrocarbons;BSA,bovine serum albumin;RP-HPLC, reverse-phasehigh-performanceliquid chromatography; T M S , tetramethylsilane;THF,tetrahydrofuran;DMSO, dimethyl sulfoxide;EDTA,ethylenediaminetetraaceticacid;T L C , thin1H nuclear magnetic resonance; DD, layer chromatography; 1H-NMR, dihydrodioldehydrogenase [trans-l,2-dihydrobenzene-l,2-diol dehydrogenase ( E C 1.3.1.20)]; androsterone, 3a-hydroxy-5a-androstan-17-one; benzene (f)-tram-dihydrodiol,(&)-trans-l,2-dihydroxy-3,5-cyclohexadiene;naphthalene (+)-trans-1,2-dihydrodiol, (*)-trans-l,2-dihydroxy1,2-dihydronaphthalene;benzo[a]pyrene (+)-trans-7,8-dihydrodiolor (+)-trans-BP-diol, (+)-trans-7,&dihydroxy-7,8-dihydrobenzo[alpyrene; benzene (+)-anti-1,2-dihydrodiol3,4-epoxide, (+)-18,2a-dihydroxy-3a,4aepoxy-bcyclohexene;benzene (&)-syn-1,2-dihydrodiol3,4-epoxide, (&)l~,2a-dihydroxy-3~,4@-epoxy-5-cyclohexene; naphthalene (i)-anti-1,2dihydrodiol 3,4-epoxideor (+)-anti-NDE,(*)-1/3,2a-dihydroxy-3a,4a- hydroxy-8,9,10,1l-tetrahydrobenz[a]anthracene.(*)-antiBADE (1mg; 3.6 pmol) was dissolved in 1mL of DMSO (10% epoxy-1,2,3,4-tetrahydronaphthalene;benzo[alpyrene (&)-anti-7,8dihydrodiol9,lO-epoxideor (+)-anti-BPDE,(t)-anti-78,8a-dihydroxyfinal; v/v) and added to 9 mL of glycine buffer (50 mM; pH 9.0) 9a,lOa-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; benzo[alpyrene (+)containing 2 mM 2-mercaptoethanol. The reaction mixture was syn-7,8-dihydrodiol9,lO-epoxide or (+)-syn-BPDE, (&)-syn-7&8a-distirred a t room temperature for 30 min. The product (retention hydroxy-9~,10@-epoxy-7,8,9,lO-tetrahydrobenzo[alpyrene; benzraltime = 29.5 min) was purified by semipreparative RP-HPLC anthracene (&)-anti-8,9-dihydrodioll0,ll-epoxideor (&)-anti-BADE, (f)-anti-8~,9a-dihydroxy-l0a,lla-epoxy-8,9,10,11-tetrahydrobenz[al- using a Partisil ODs-M9 column (10 pm; 12 mm X 50 cm; Whatanthracene;BP-tetraol 1,r-7,t-8,9,10-tetrahydroxy-7,8,9,lO-tetrahydro- man Labsales, Inc., Hillsboro, OR). The product was eluted from benzo[al pyrene;BP-tetraol2,r-7,t-8,9,~-10-tetrahydroxy-7,8,9,1~tetrahydrobenzo[a]pyrene;BP-tetraol3,r-7,t-8,~-9,10-tetrahydroxy-7,8,9,10-tet- the column using a 70-min linear gradient of 6O-80% methanolrahydrobenzo[alpyrene;BP-tetraol4,r-7,t-8,10,c-9-tet.rahydroxy-7,8,9,10- water (v/v) with a solvent flow rate of 2 mL/min and was concentrated to dryness under vacuum:lH-NMR (500 MHz, tetrahydrobenzo[alpyrene; BA-tetraol, 88,9a,lOa,118-tetrahydroxy8,9,10,1l-tetrahydrobenz[alanthracene;thiol ether adduct of (+)-antiacetone-&) d 2.85 (9, 2 H, CHzO), 3.00 (4, 2 H, CH&, 3.85 (m, BPDE,(*)-lOe-[(2-hydroxyethyl)thiol-7~,8a,9e-trihydroxy-7,8,9,lO-tet- 1 H, HlO), 4.31 (dd, 1 H, H9), 4.56 (d, 1 H, H11; J i 0 , i i = 9 Hz), rahydrobenzo[alpyrene;thiol ether adduct of (f)-anti-BADE,(*)-lie7.6-8.2 (m, 6 H, H2-H7), 8.78 (d, [(2-hydroxyethyl)thio]-8~,9a,l0c-trihydroxy-8,9,lO,ll-tetrahydrobenz[al-4.95 (d, 1 H, H8;J8,s= 7 Hz), anthracene. 1 H, Hl), 8.99 (s, 1 H, H12).
578 Chem. Res. Toxicol., Vol. 5, No. 4, 1992 Synthesis of (*)-8~,9a,lOa,11/3-Tetrahydroxy-8,9,10,ll-tetrahydrobenz[a]anthracene. (*)-anti-BADE (1mg; 3.6 pmol) was dissolved in 1mL of DMSO (10% final; v/v) and added to 9 mL of 2 M HC1. The reaction mixture was stirred a t 37 "C for 30 min. After neutralization with 2 M NaOH, the product was extracted with 4 X 10 mL of ethyl acetate. The organic extracts were dried over MgSOd. Concentration under vacuum gave a yellowish oil which was dissolved in acetonitrile and purified by semipreparative RP-HPLC using a Partisil ODS-M9 column. The product (retention time = 34.0 min) was eluted from the column using a 70-min linear gradient of 5040% methanolwater (v/v) and a solvent flow rate of 2 mL/min and was concentrated to dryness under vacuum: 'H-NMR (500 MHz, acetone-&) 6 3.95 (d, 1H, H9), 4.05 (d, 1H, HlO), 4.71 (d, 1 H, H8), 4.80 (d, 1 H, H l l ) , 7.6-8.3 (m, 6 H, H2-H7), 8.75 (d, 1 H, Hl), 8.83 (s, 1 H, H12). Stereochemical assignment of the tetraol was determined by comparing the chemical shifts and coupling constants to those for the trans and cis hydrolysis products of (*)-anti-BPDE (31). The tetraol was determined to be the trans hydrolysis product of the diol epoxide due to the relatively large coupling constants ( J S ,=~6.4 Hz and Jg,n = 6.7 Hz). P u r i t y of P A H Substrates. The purity of (f)-trans-BPdiol, (A)-anti-NDE,and the hydrolysisproducts of (*)-anti- and (*)-syn-BPDE was confirmed by RP-HPLC (purity >99% ). The purities of (A)-anti-BPDE, (A)-syn-BPDE,and (&)-anti-BADE were checked prior to use by measuring the formation of their thiol ether adducts with 2-mercaptoethanol (final concentration of 2 mM) in 50 mM glycine buffer (pH 9.0) at 25 "C. In each case, the reaction mixture was analyzed after 30 min by RP-HPLC (see later for conditions). Analyses showed the presence of a single peak that comigrated with authentic synthetic standards of thiol ether adducts indicating that the diol epoxides were in excess of 99% pure. This reaction has previously been described for analysis of the purity of diol epoxides of chrysene (32). Source of Dihydrodiol Dehydrogenase. DD was purified to electrophoretic homogeneity from rat liver cytosol according to the published procedure (33). In this tissue, dihydrodiol and 3a-hydroxysteroid dehydrogenase (EC 1.1.1.50) are the same enzyme (33,34). The specific activity of the purified enzyme was 1.97 pmol of androsterone oxidized/(min.mg) and 0.62 pmol of benzene (A)-trans-dihydrodiol oxidized/(min-mg) when assayed spectrophotometrically a t 25 "C in the presence of either 75 pM androsterone and 2.3 mM NAD+a t pH 7.0 or 1mM benzene (f)-trans-dihydrodiol and 2.3 mM NADP+ at pH 9.0. The enzyme was stored at a concentration of 3.82 mg/mL in 400 pL of 20 mM potassium phosphate buffer (pH 7.0) containing 1mM EDTA, 1 mM 2-mercaptoethanol, and 30% glycerol a t -70 O C . Before use, the enzyme was dialyzed overnight against three changes of 10mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA to remove glycerol and 2-mercaptoethanol. Spectrophotometric Assay for Measuring either transDiol o r Diol Epoxide Oxidation Catalyzed by Dihydrodiol Dehydrogenase. The oxidation of diol epoxides was monitored in 1.0-mL systems containing 50 mM glycine buffer (pH 9.0), 2.3 mM NADP+, and either 10 mM (&)-anti-NDE or 20 pM (&)anti-BADE (limit of solubility), plus varying amounts of homogeneous enzyme (1.9-114.6 pg; 0.05-3.1 pM enzyme). Compounds were dissolved in DMSO, and the final concentration of organic solvent in the assay was 8%. Reactions were monitored by following the formation of the reduced pyridine nucleotide at 340 nm; e = 6270 M-l cm-l in a 1.0-cm light path in a Beckman Model DU-7 UV/vis spectrophotometer at 25 "C. Control incubations included benzene (&)-trans-dihydrodiol (1 mM), naphthalene (&)-trans-1,2-dihydrodiol(1mM), and (&)-transBP-diol (20 pM), which are known substrates for the enzyme (20). No change in the absorbance was observed in the absence of enzyme or when either substrate or nucleotide was incubated alone with the enzyme. Specific activities are expressed as nmol of substrate oxidized/(min.mg).
Flowers-Geary et al.
-;--I
/
Time (min) F i g u r e 1. Linearity of dihydrodiol dehydrogenase-catalyzed oxidation of (f)-trans-BP-diolwith time and protein. [ 1,3-3H](&)-trans-BP-diol(20pM; 100 OOOcpdnmol)was incubated with DD (19 pg for time dependence or 0-30 pg for protein dependence) and 2.3 mM NADP+ in 50 mM glycine buffer (pH 9.0) at 37 "C. Samples were analyzed by RP-HPLC as described under Experimental Procedures. The initial velocity was calculated from the linear portion of the data as shown. No oxidation of the diol occurs in the absence of enzyme or cofactor. RP-HPLC Assay for Measuring either trans-Diol o r Diol Epoxide Oxidation Catalyzed by Dihydrodiol Dehydrogenase. Assays were conducted in 0.1 mL of 100 mM potassium phosphate (pH 7.0) or 50 mM glycine (pH 9.0) buffer containing 2.3 mM NADP+ and one of the following: 20 pM [1,3-3H]-(*)trans-BP-diol (100 OOO cpm/nmol), [ 1,3-3H]-(f)-anti-BPDE (100 OOO cpm/nmol), or 20 pM (*)-anti-BPDE, (*)-syn-BPDE, or (f)-anti-BADE. The substrates were dissolved in DMSO, and the final organic solvent concentration was 8%. Reactions were initiated by the addition of homogeneous enzyme (19-47.5 pg; 5.0-12.5 pM enzyme) and incubated over a 2-h time course a t 37 OC. At each time point unreacted diol epoxides were trapped by the addition of 2-mercaptoethanol (final concentration of 2 mM), and thiol ether adduct formation was then allowed to proceed for 30 min a t 25 OC. Control incubations were performed in the absence of either NADP+, purified enzyme, or 2-mercaptoethanol. Reaction mixtures were extracted with ethyl acetate (two 0.2-mL aliquots). Extracts were pooled and evaporated to dryness, and the residues were redissolved in methanol (50 pL) for subsequent RP-HPLC analysis. At each time point the thiol ether adducktetraol ratio as determined by RP-HPLC gave an estimate of the diol epoxide remaining. RP-HPLC analyses were conducted by injecting aliquots (20 pL) onto a Zorbax Ultrasphere-ODS (10 pm; 4.6 mm X 25 cm; Dupont, Wilmington, DE) reverse-phase column. Compounds were separated using a 70-min linear gradient of 6040% methanol-water (v/v). The solvent flow rate was 0.5 mL/min, and the chromatographic system was operated at ambient temperature. The eluent was monitored by absorbance at 254 nm using a Perkin-Elmer Model LC-480 diode array detector. PAH were quantified by comparison of retention times and peak areas to authentic synthetic standards [i.e., (*)-trans-BP-diol, tetraol hydrolysis products of (f)-anti-BPDE, (A)-syn-BPDE,(*)-antiBADE, and thiol ether adducts of (*)-anti-BPDE and (&)-antiBADE]. When radiolabeled compounds were used as substrates, fractions were collected every minute and added to 5 mL of Ecolite (ICN Biomedical, Inc., Irvine, CA). The amount of radioactivity in each fraction was determined by liquid scintillation spectrometry with a Tracor Model 43 scintillation counter having a 53 % efficiency for tritium.
Results and Discussion Oxidation of PAH trans-Dihydrodiols by Dihydrodiol Dehydrogenase. Aromatic hydrocarbon transdihydrodiols ranging in ring size from benzene t o benzo[a1pyrene can be oxidized by homogeneous dihydrodiol dehydrogenase. Oxidation occurs at maximum efficiency (VmJJKm) at pH 9.0 with NADP+ as cofactor (20,21).
Diol Epoxides and Dihydrodiol Dehydrogenase 7500
L0
T I
A
enzyme amount compound benzene (&)-trans-dihydrodiol naphthalene (*)- trans-l,2dihydrodiol (*)- trans-BP-diol (*)-anti-NDE (*)-anti-NDE (f)-anti-BADE (f)-anti-BADE
7508 3000
2500
L0
5000 2500
7508 5000
0
2500 0
concn 1mM 1 mM
(Peg)
20 pM 10 mM 10 mM 20 pM 20 pM
1.9 1.9 114.6 1.9 114.6
1.9 1.9
initial rate [nmol/ (mimmg)] 365.0
7.2 6.0 ND* ND ND ND
a Oxidation of benzene (*)-trans-dihydrodiol, naphthalene (&)tr~ns-1,2-dihydrodiol,(&)-trans-BP-diol,(*)-anti-NDE, and (&)anti-BADE in 50 mM glycine buffer (pH 9.0) was monitored by measuring the change in absorbance of the pyridine nucleotide at 340 nm under the conditions stated in Experimental Procedures. Initial rates shown are the mean values calculated from at least three independent determinations; in all cases the SE was less than 10% of the mean. * Not detectable.
7508
t
Table I. Spectrophotometric Assay of PAH trans-Dihydrodiol and anti-Diol Epoxide Oxidation Catalyzed by Homogeneous Dihydrodiol Dehydrogenase*
5000 2500
L0
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 579
10 20
30
40
50
60
70
TIME (mln)
Figure 2. RP-HPLC assay of [1,3-aH]-(*)-tram-BP-diol oxidation catalyzed by homogeneous dihydrodiol dehydrogenase. [1,3-3H]-(&)-tram-BP-diol (20 pM; 100 OOO cpm/nmol) was incubated with DD (19 pg) in 50 mM glycine buffer (pH 9.0) at 37 "C in the presence and absence of NADP+ (2.3 mM), and samples were analyzed by RP-HPLC as described under Experimental Procedures. Radiochromatogram of the complete system, (*)-tram-BP-diol, NADP+, and enzyme at zero time (panel A); radiochromatogram of the incubation mixture, (*)tram-BP-diol and enzyme, minus NADP+, incubated for 2 h a t 37 "C (panel B); radiochromatogram of the complete system, (+)-tram-BP-diol, NADP+, and enzyme, incubated for 30 min at 37 O C (panel C); radiochromatogram of the complete system, (*)-trans-BP-diol, NADP+,and enzyme, incubated for 2 h a t 37 "C (panel D). These reactions can be monitored with ease by measuring the formation of NADPH at 340 nm using trans-dihydrodiols [benzene (*)-trans-dihydrodiolandnaphthalene (*)-trans-l,2-dihydrodioll that are transparent at this wavelength, and these results are replicated here. The relatively slow oxidation of (*I-trans-BP-diol can also be monitored using the same technique (Table I). This assay has been criticized since trans-BP-diol also contributes to the absorbance at 340 nm. Because of this concern the oxidation of [1,3-3Hl-trans-BP-diol was also monitored using a TLC-based assay (27). In this TLC assay the diol was completely oxidized only when NADP+ and DD were both present. No diol was consumed in the presence of either NADP+ or DD alone, and the diol was completely stable to the conditions of the assay. Estimates of initial velocities based on the TLC assay were of the same order as those observed spectrophotometrically, indicating that the spectrophotometric method is reliable. To substantiate further that trans-BP-diol is a substrate for DD, [1,3-3Hl-trans-BP-diol was incubated with DD in the presence of NADP+ in either phosphate or glycine buffer over 6 h. At each time point the radioactivity was
extracted and subjected to RP-HPLC analysis. Initial velocities based upon the disappearance of the diol were found to be 1.40 and 0.60 nmol/(min-mg) in glycine and phosphate buffers, respectively. Only the data obtained in glycine buffer is shown in Figure 1. These initial velocities are in accord with our previous estimates from the TLC-based assay r1.80 nmol/(min.mg)l (27). The enzymatic oxidation of (&)-trans-BP-diolwas linear with protein (Figure 1 inset), and subsequent assays were performed using protein concentrations on the linear portion of the plot. Under optimal conditions the radioactive diol was almost completely consumed in glycine buffer over a 2-h time period (Figure 2). These data reaffirm that DD will oxidize both the (+)-7(S),8(S)-and the (-)-7(R),8(R)-BP-diol (20). In the chromatograms shown,reaction mixtures were analyzed without extraction and the presence of a product is apparent in the solvent front. When the reactions were extracted and analyzed, no product peak was observed. This is consistent with our earlier findings that benzo[al pyrene-7,Sdione is the product of the reaction but readily forms buffer adducts which are nonextractable (22). No diol was consumed when it was incubated witheither cofactor or enzyme alone. An important feature is that DD will not transform the diol in the absenceof cofactor to yield the 7-phenol (Figure 2B) as previously described by Klein et al. (25). The establishment of a RP-HPLC assay for the oxidation of [1,3-3Hl-trans-BP-diol provides a useful control for reactions that will monitor the oxidation of PAH anti-diol epoxides. The inability of Klein et al. (25) to measure the DDcatalyzed oxidation of [1,3-3Hl-trans-BP-diol could be explained by a variety of factors. First, DD is purified by a route that is substantially different from ours (34). Second,their radiochemical assay was not optimized (i.e., only a single protein concentration was used over a short time course). Third, these workers used a cosolvent that was incompatible with complete solubilization of the substrate. Fourth, the ability of their purified DD to catalyze the formation of the 7-phenol in the absence of cofactor suggests contamination of their enzyme with a factor that can catalyze dehydration of trans-BP-diol and effectively remove the substrate from the reaction. Examination of Diol Epoxides as Substrates for Dihydrodiol Dehydrogenase: (A) Spectrophotomet-
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680 Chem. Res. Toxicol., Vol. 5, No. 4, 1992
Scheme I. Competing Reactions For Diol Epoxides: Trapping as Thiol Ether Adducts
I
-
1
TETRAOL 1
GHO W 'H KETONE TRIOL
OH
+
-
i
THIOL ETHER
OH
"4'
HO 6H
I
OH
TETRAOL 2
ric-Based Assays. To determine whether PAH antidiol epoxides were substrates for DD, (*)-anti-NDE and (f)-anti-BADEwere examined initially for severalreasons. First, both diol epoxides are extremely stable and undergo limited hydrolysis under the conditions employed [(*)anti-NDE ( t 1 / 2 >> 6 h in glycine or phosphate buffer) and (*)-anti-BADE (tip>> 2 hing1ycineorphosphatebuffer)l. Second, (*)-anti-NDE is highly soluble and can be used at millimolar concentrations. Third, (*)-anti-BADE was the diol epoxide whose mutagenicity was suppressed by DD in the Ames test (19). From the data presented (Table I), it is clear that, even in the presence of either very high diol epoxide concentrations, 10 mM (*)-anti-NDE or 20 pM (*)-anti-BADE (limit of solubility), or noncatalytic quantities of enzyme (3.06 pM), no enzymatic oxidation of these diol epoxides could be detected by measuring absorbance changes at 340 nm. It should be emphasized that the limit of detection of the spectrophotometric assay is 0.5 nmol/min and, therefore, it would have detected the turnover of 1/20000th of the (&)-anti-NDEsubstrate provided sufficient enzyme was present. These results indicate that PAH anti-diol epoxides are not substrates for DD under conditions which permit detection of transdihydrodiol oxidation. Examination of Diol Epoxides as Substrates for Dihydrodiol Dehydrogenase: (B) RP-HPLC-Based Assays. The RP-HPLC assay that was used to monitor the DD-catalyzed oxidation of [1,3-3H]-trans-BP-diol was adapted to monitor the oxidation of [1,3-3Hl-(*)-antiBPDE, (*)-syn-BPDE, and (&)-anti-BADE. Since antiand syn-BPDEs are known to be unstable in phosphate and glycine buffer (35),this property was exploited. Briefly, in the absence of enzyme these diol epoxideswould undergo time-dependent hydrolysis to the corresponding tetraols. At any given time, diol epoxide which was not hydrolyzed could be trapped as a thiol ether adduct with 2-mercaptoethanol. The thiol ether:tetraol ratio at that time would give the amount of diol epoxide remaining. Control incubations would yield a mixture of tetraols and thiol ether adduct only. By contrast, if the diol epoxides were substrates for DD, these reactions would yield a
mixture of tetraols, a diol epoxide thiol ether adduct and a new product (e.g., ketone triol; Scheme I). To ensure that these compounds could be detected, analyses were performed using a diode array detector, making it essentially impossible for any compound with a UV chromophore to go undetected. The results obtained in these experiments are presented in Figures 3-5 and are summarized below (Figures 4 and 5 are available as supplementary material). First, when (*)-anti-BPDE (Figure 3) was incubated in glycine buffer (pH 9.0) for 30 min at 25 OC, only tetraol hydrolysis products were detected. For (*)-anti-BPDE, the tetraols formed correspond to r-7,t-8,9,10- (BP-tetraol 1)and r-7,t-8,9,c-lO-tetrahydrotetrol(BP-tetraol2), the trans and cis hydrolysis products, respectively. Second, when (f)-anti-BPDE was incubated for 30 min at 25 OC in buffer containing 2-mercaptoethanol, a single peak was obtained which coeluted with an authentic synthetic thiol ether adduct. The detection of this single peak confirmed the purity of the diol epoxide substrate. Third, when (*)-anti-BPDE was incubated for b 2 h at 37 OC with DD and NADP+ and the reaction terminated at different times by the addition of 2-mercaptoethanol,the only PAH detected were either tetraols or thiol ether adducts. No products of enzymatic oxidation were found. Similar results were obtained for (*)-syn-BPDE and (*)anti-BADE irrespective of whether the incubations were conducted in glycine (pH 9.0) or potassium phosphate (pH 7.0) buffer (see Figures 4 and 5, supplementary material). In every instance the identity of the tetraols and thiol ether adducts was verified by coelution with synthetic standards. The data obtained for (*)-antiBPDE in glycine buffer is shown since these are the optimal conditions for trans-BP-diol oxidation. Further, the data obtained in the complete system after 2 h are shown to indicate that under prolonged incubation conditions with micromolar amounts of enzyme only tetraols and/or thiol ether adducts were detected. As shown in Table 11, when the reactions were replicated using larger amounts of enzyme to ensure that [1,3-3H]-trans-BP-diolwas completely consumed within 45 min, the same pattern was
Diol Epoxides and Dihydrodiol Dehydrogenase 0.050
t
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 581 Table 11. Rp-HPLC Assay of (*)-trans-BP-dioland (h)-anti-BPDEOxidation Catalyzed by Homogeneous Dihydrodiol Dehydrogenase. (*)- trans-BP-diolb (*)-anti-BPDEC
A
time % % thiol (min) remaining oxidized ether:tetraol 0 100 0 0.91 5 91.2 8.8 0.78 10 73.2 26.8 0.57 15 65.9 34.1 0.39 20 50.4 49.6 0.26 30 30.9 69.1 co.01 45 0.0 100 co.01
a [1,3-3Hl-(*)-trans-BP-diol or [1,3-3Hl-(*)-anti-BPDE (20 pM; 100 OOO cpm/nmol) was incubated for the indicated times with DD (19 pg) and NADP+ (2.3 mM) in 50 mM glycine buffer (pH 9.0) at 37 O C in a final volume of 0.1 mL. At the end of the incubation period, 2-mercaptoethanol (final concentration 2 mM) was added to samples containing (*)-anti-BPDE to trap unreacted diol epoxide. All samples were analyzed by RP-HPLC as described under Experimental Procedures. The percent (%) remaining values are the percent of radioactivity corresponding to trans-BP-diolfrom which the percent of BP-diol oxidized was calculated. Values are calculated from 2-3 determinations; in all cases the SE was less than 10% of the mean. The thiol ether:tetraol values are the ratios of the radioactivity associated with each of the products determined by RP-HPLC analysis. The percent (%) remaining values are the percent of radioactivity corresponding to the thiol ether adduct. Values are calculated from 2-3 determinations; in all cases the SE was less than 10% of the mean.
tetmol 1 1
7508:
D
1
L 20 30 40 7
'0
10
% % remaining oxidized 91 0 78 0 57 0 39 0 26 0 0 0 0 0
50
60
TIME (min)
Figure 3. RP-HPLC analysis of reactions containing [1,3-3H](t)-anti-BPDE,homogeneous dihydrodiol dehydrogenase, and cofactor. [1,3-3H]-(*)-anti-BPDE(20 pM; 100 OOO cpmhmol) was incubated with DD (19 pg) and NADP+(2.3 mM) in 50 mM glycine buffer (pH 9.0) at 37 "C in a final volume of 0.1 mL. At the end of the incubation period, unreacted diol epoxide was trapped by the additionof 2-mercaptoethanol(finalconcentration of 2 mM) for 30 min at 25 O C . Samples were analyzed by RPHPLC as described under Experimental Procedures. Tetraol standards (panel A); radiochromatogram of (*)-anti-BPDE incubated (30 min at 25 O C ) in buffer only (panel B); radiochromatogram of (*)-anti-BPDE incubated (30 min at 25 "C) in buffer in the presence of 2-mercaptoethanol(panel C); radiochromatogramof the complete system, (*)-anti-BPDE,NADP+, and enzyme, at zero time and quenched by the addition of 2mercaptoethanol (30 min at 25 O C ) (panel D); radiochromatogram of the complete system, (*)-anti-BPDE, NADP+, and enzyme, after incubation for 2 h at 37 O C and quenched by the addition of 2-mercaptoethanol(30 min at 25 "C)(panel E). observed. In addition, when the reaction mixtures were analyzed at earlier times (2-15 min), the only differences observed were in the thiol ether adducttetraol ratios. These data clearly show that as the thiol ether adduchtetraol ratio decreases (increase in diol epoxide hydrolysis), no anti-diol epoxide is oxidized under conditions in which the tram-BP-diol is consumed. To counter the argument that the rate of diol epoxide hydrolysis prevents the detection of enzymatic oxidation of diol epoxides, the data obtained with the stable anti-diol-epoxide, (&)-antiBADE, are revealing. This shows that after incubating this PAH with micromolar concentrations of DD and 2.3 mM NADP+ at pH 9.0 for 2 h, only three peaks are
observed. These peaks correspond to unreacted (&)-antiBADE, thiol ether adduct, and tetraol. When aliquots from all the reaction mixtures were analyzed by RP-HPLC without extraction, the only additional material that eluted was in the solvent front. This material had no PAH absorbance and, in the case of [1,3-3Hl-(&)-anti-BPDE,no radioactivity and in every instance corresponded to NADP(H). The formation of triols, which has been reported to occur nonenzymatically by the reaction of 400 pM NADPH with 20 pM (&)-antiBPDE (36),was not evident. This reaction is not expected since even if oxidation of the diol epoxides occurred, the amount of NADPH formed would be only 20 pM. It can be concluded that PAH diol epoxides examined in this study are not substrates for DD, which would support the recent findings of Klein et al. (25). Effects of PAH Diol Epoxides on Dihydrodiol Dehydrogenase Activity. Because diol epoxides alkylate macromolecules, it was conceivable that these compounds could inactivate DD and prevent enzymatic oxidation of diol epoxides. To examine this issue, (&)-anti-BPDE,(&)syn-BPDE, and (&)-anti-BADEwere evaluated as timedependent inactivators of the dehydrogenase in the presence of NADP+ over 24 h (Table 111). Under these conditions at least 42% of the diol epoxide, (&)-antiBADE, is not hydrolyzed. None of the diol epoxides were found to promote time-dependent inactivation. As a positive control, incubation of DD with (&)-trans-BP-diol and NADP+over 24 h led to complete enzyme inactivation, confirming that the product of this oxidation, benzo [a1pyrene-7,8-dione, causes an irreversible loss of enzyme activity (37). Effects of Contaminating trans-Dihydrodiols in Diol Epoxide Preparations. The observation that the benzene (&)-anti- and (f)-syn-1,2-dihydrodiol 3,4-ep oxides are oxidized by DD (24),whereas diol epoxides used in this investigation were not, requires an explanation. In our hands, contaminants in synthetically prepared (*I-
Flowers-Geary et al.
582 Chem. Res. Toxicol., Vol. 5, No. 4, 1992 Table 111. Effects of PAH Diol Epoxides on Dihydrodiol Dehydrogenase Activity enzyme activity remaining preincubation conditions' enzyme enzvme + (hLtrans-BP-diol enzyme + ifj-anti-BPDE enzyme + (f)-eyn-BPDE enzyme + (&)-anti-BADE
pmoli (min-mg) 1.42 NDc 1.46 1.34 1.25
%b
100
ND 103 94 88
PAH metabolites (20 pM) were preincubated for 24 h at 37 OC in 0.1-mL systems containing 19 pg of DD, 2.3 mM NADP+, 8% (v/v) DMSO, and 50 mM glycine buffer (pH 9.0). Aliquots from the preincubation mixture were diluted into 1.0-mL assay systems containing 100 mM potassium phosphate buffer (pH 7.0), 2.3 mM NAD+, and 4% (v/v) acetonitrile. Reactions were initiated by the addition of 75 pM androsterone, and DD activity was measured by monitoring the formation of NADH at 340 nm. In these measurements, the dilution factor into the final assay was 100-fold. Therefore, the initial velocities shown are a measure of the amount of enzyme activity remaining and are the mean values calculated from two determinations; in all cases the SE was less than 10% of the mean. Values expressed as mean percent (%) of control (enzyme alone). Not detectable.
anti-NDE were found to be responsible for initial positive results. Thus, significant rates of (A)-anti-NDEoxidation by DD could be measured but were found to be due to the presence of trace amounts of naphthalene (f)-trans-1,2dihydrodiol(99% pure, it is evident that extreme caution must be taken in the use of these compounds. Since trans-dihydrodiols are the usual synthetic precursors of the diol epoxides, it is probable that (*)-anti-and (f)syn-diol epoxides which are used in investigations are similarly contaminated unless they are rigorously purified. It is noteworthy that the specific activities for benzene turnover (*)-anti- and (&)-syn-l,2-dihydrodiol3,4-epoxide reported by Glatt et al. (24) were 20 times lower than that reported for benzene (*I-dihydrodiol, which would correspond to a 5 % contamination of the racemic diol epoxide with the racemic trans-dihydrodiol. Effects of ProteinConcentrationon (&)+"-BADE Hydrolysis and Thiol Ether Adduct Formation. It is not obvious why DD in the presence of NADP+ decreases the mutagenicity of (*)-anti-BADE in the Ames test (19). In our investigations it was evident that the concentration of enzyme affected the rate of diol epoxide hydrolysis and thiol ether adduct formation. To characterize these effects, (A)-anti-BADEwas incubated with BSA or DD in glycine buffer (pH 9.0) for 2 hat 37 OC and reactions were quenched with 2-mercaptoethanol. The reaction mixtures were analyzed by RP-HPLC, and the amounts of tetraol hydrolysis products, thiol ether adducts, and unreacted (*Ianti-BADE were quantified. As shown in Table IV, increasing concentrations of protein caused a decrease in tetraol and thiol ether adduct formation and a correspondingincrease in unreacted (*)-anti-BADE. It is noteworthy that, on a molar basis, DD is 50-fold more effective than BSA in preventing diol epoxide hydrolysis. These results indicate that (&I-anti-BADEcan be sequestered by DD, which in turn prevents either hydrolysis or thiol ether adduct formation. It is proposed that the decrease in (*)anti-BADE mutagenicity observed in the Ames test by Glatt et al. (19) may be due to protein binding which prevents ita interaction with DNA.
Table IV. Effects of Protein Concentration on (A)-anti-BADE Hydrolysis and Thiol Ether Adduct Formation
conditions" control +BSA (14.7 nmol) +BSA (73.5 nmol) +BSA (147.0 nmol) +enzyme (0.5 nmol) +enzyme (2.0 nmol) +enzyme (3.0 nmol)
tetraol 30 35 25
% distribution thiol (*)-antiether adduct BADE
11 28 22 12
51 46 42 14 42 30 8
19 19 33 75 30 48 80
(f)-anti-BADE (20pM) was incubated for 2 h a t 37 O C in 50 mM glycine buffer (pH 9.0) with the amount of protein indicated. At the end of each incubation, unreacted diol epoxide was trapped with 2-mercaptoethanol (final concentration 2 mM) and the samples were analyzed by RP-HPLC as described under Experimental Procedures. Percentages were calculated from peak areas and are expressed as percent (%) relative to control [(*)-anti-BADE incubated in the absence of protein].
In summary, it has been suggested that DD will suppress levels of PAH anti-diolepoxides by two mechanisms. First, the enzyme could catalyze the oxidation of their transdihydrodiolprecursors. Second, the enzyme could catalyze the oxidation of PAH anti-diol epoxides. These studies confirm our previous findings that PAH trans-dihydrodi01s are substrates for DD and that trans-BP-diol is a substrate for the enzyme. This in turn provides an explanation for the Ames test data originally reported by Glatt et al. (17)which described the ability of DD to reduce the mutagenicity of benzo[alpyrene. In this system DD presumably oxidizes trans-BP-diol to benzo[alpyrene-7,8dione (22),and the reactive dione is then scavenged. When taken together with our previous findings on the broad substrate specificity of DD for non-K-region trans-dihydrodiols of PAH (20,21),it is concluded that this enzyme has the potential to suppress the formation of anti-diol epoxides derived from many carcinogenic PAH. In contrast, these studies also demonstrate that PAH diol epoxides are not substrates for DD. Reaction mixtures containing DD, NADP+,and either (&)-anti-BPDE,(k)-synBPDE, or (*)-anti-BADE in potassium phosphate (pH 7.0) or glycine (pH 9.0) buffer were found to contain only hydrolysis products (tetraols) and thiol ether adducts, indicating that no diol epoxide oxidation occurs. We provide evidence that (k)-anti-BADE is sequestered by protein (BSA or DD), thereby preventing hydrolysis to form tetraols and thiol ether adducts. This phenomenon may explain the observation that DD decreases the mutagenicity of this diol epoxide by preventing its interaction with bases within DNA.
Acknowledgment. This research was supported by NIH Grants CA 39504 and Research Career Development Award CA01335 to T.M.P.and by NIH Grant CA36097 and ACS Grant CN-22 to R.G.H. Portions of this work were supported by the High-Resolution NMR Facility in the Department of Biochemistry and Biophysics at the University of Pennsylvania. Supplementary Material Available: Figures 4 and 5, showingRP-HPLC analyses of reactions containing homogeneous dihydrodiol dehydrogenase, cofactor, and either (i)-syn-BPDE (Figure 4) or (i)-anti-BADE (Figure 5 ) (2 pages). Ordering information is given on any current masthead page.
References (1) Conney, A. H. (1982) Induction of microsomal enzymes by foreign
chemicals and carcinogenesisby polycyclic aromatic hydrocarbons.
Diol Epoxides and Dihydrodiol Dehydrogenase
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 583
Cancer Res. 42,4875-4917. F. (1982) Inactivation of a diol epoxide by dihydrodiol dehydroge(2) Gelboin, H. V. (1980) Benzo[alpyrene metabolism, activation and nase but not by two epoxide hydrolases. Science 215,1507-1509. carcinogenesis: Role and regulation of mixed function oxidases and (20) Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1986) Regiorelated enzymes. Physiol. Rev. 60, 1107-1166. and stereoepecificityof homogeneous 3a-hydroxyeteroid-dihydrdiol (3) Yang, S. K., McCourt, D. W., Roller, P. P., and Gelboin, H. V. (1976) dehydrogenase for trans-dihydrodiol metabolites of polycyclic Enzymatic conversion of benzo[alpyrene leading predominantly to aromatic hydrocarbons. J. Biol. Chem. 261,61844191. the diol-epoxider - 7 , t - ~ d i h y d r o x y - t - 9 , 1 ~ 0 ~ - 7 , ~ d ~ y d r ~ b e n z o [ a(21) ] - Smithgall, T. E., Harvey, R. G.,andPenning,T. M. (1988)Oxidation pyrene. Proc. Natl. Acad. Sci. U.S.A. 73, 2594-2598. of the tram-3,4-dihydrodiol metabolites of the potent carcinogen (4) Yang, S. K., Mushtaq, M., and Chiu, P. L. (1985) Stereoselective 7,12-dimethylbenz[alanthraceneand other benz[a]anthracene demetabolism and activation of polycyclic aromatic hydrocarbons. In rivatives by 3a-hydroxyeteroid-dihydrodioldehydrogenase: Effects Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) ofmethylsubstitutiononvelocity andstereochemicalcourseof transpp 19-34, ACS Monograph 283, American Chemical Society, dihydrodiol oxidation. Cancer Res. 48, 1227-1232. Washington, DC. (22) Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) (5) Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Spectroscopic identification of ortho-quinones as the products of Beland, F. A., Harvey, R. G., Kasai, H., Miura, I., and Nakanishi, polycyclic aromatic trans-dihydrodiol oxidation catalyzed by diK. (1976) Benzo[alpyrene-nucleic acid derivative found in vivo: hydrodiol dehydrogenase: Apotential route of proximate carcinogen Structure of a benzo[al pyrenetetrahydrodiol epoxide guanosine admetabolism. J. Biol. Chem. 263, 1814-1820. duct. J. Am. Chem. SOC.98,5714-5715. (23) Murty, V. S., and Penning, T. M. (1992) Characterization of mer(6) Jeffrey, A. M., Weinstein, I. B., Jennette, K. W., Grzeskowiak,K., capturic acid and glutathionyl conjugates of benzo[alpyrene-7,& Nakanishi, K., Harvey, R. G., Autrup, H., and Harris, C. (1977) dione by two-dimensional NMR. Bioconjugate Chem. 3,218-224. Structures of benzo[alpyrene nucleic acid adducts formed in human (24) Glatt, H., Padykula, R., Berchtold, G. A., Ludewig, G., Platt, K. L., and bovine bronchial explants. Nature (London) 269, 348-350. Klein, J., and Oesch, F. (1989) Multiple activation pathways of (7) Deutsch, J., Leutz, J. C., Yang, S. K., Gelboin, H. V., Chiang, Y. L., benzene leading to products with varying genotoxic characteristics. Vatais, K. P., and Coon, M. J. (1978)Regio- and stereoselectivity of Env. Health Perspect. 82, 81-89. various forms of purified cytochrome P-450 in the metabolism of (25) Klein, J., Seidel, A., Frank, H., Oesch, F., and Platt, K. L. (1991) benzo[alpyrene and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[alRegiospecific oxidation of polycyclic aromatic dihydrodiols by rat pyrene as shown by product formation and binding to DNA. Proc. liver dihydrodiol dehydrogenase. Chem.-Biol. Interact. 79, 287Natl. Acad. Sci. U.S.A. 75, 3123-3127. 303. (8) Malaveille, C., Kuroki, T., Sims, P., Grover, P. L., and Bartach, H. (26) Flowers, L., Harvey, R. G., and Penning, T. M. (1990)Examination (1977) Mutagenicity of isomeric diol-epoxides of benzo[alpyrene of diol-epoxides of polycyclic aromatic hydrocarbons as substrates and benz[alanthracene in S. typhimurium TA98 and TAlOOand in for rat liver dihydrodiol dehydrogenase. Proc. Am. Assoc. Cancer V79 Chinese hamster cella. Mutat. Res. 44,313-326. Res. 31, Abstract 662. (9) Newbold, R. F., and Brookes, P. (1976) Exceptional mutagenicity (27) Smithgall, T. E., and Penning, T. M. (1986) Inhibition of transof benzo[alpyrene diolepoxidein culturedmammalian cells. Nature dihydrodiol oxidation by the non-steroidal anti-inflammatory drugs. (London) 261, 52-54. Car-inogenesis 7, 583-588. Buening, M. K., Wislocki, P. G., Levin, W., Yagi, H., Thakker, D. (28) Yagi, H., Hernandez, O., and Jerina, D. M. (1975) Synthesis of R., Akagi, H., Koreeda, M., Jerina, D. M., and Conney, A. H. (1978) (~)-7j3,8a-dihydroxy-98,10B-epoxy-7,8-9,lO-tetrahydrobenzo[a]Tumorigenicity of the optical enantiomers of the diastereomeric pyrene, a potential metabolite of the carcinogen benzo[a]pyrene benzo[a]pyrene 7,8diol-9,10-epoxidesin newborn mice: Exceptional with stereochemistryrelatedto the antileukemic triptolides. J.Am. activity of (+)-7j3,8a-dihydroxy-9a,lO~-epoxy-7,8,9,1O-tetrahyChem. SOC.97, 68814883. drobenzo[a]pyrene. Proc. Natl. Acad. Sci. U.S.A. 75, 5358-5361. (29) Fu, P. P., and Harvey, R. G. (1977) Synthesis of the diols and diSlaga, T. J., Braken, W. J., Gleason, G., Levin, W., Yagi, H., Jerina, olepoxides of carcinogenic hydrocarbons. Tetrahedron Lett. 24, D. M., and Conney, A. H. (1979) Marked differences in the skin 2059-2062. tumor-initiating activities of the optical enantiomers of the dia(30) Harvey, R. G., and Fu, P. P. (1978) Synthesis and reactions of diol stereomeric benzo[alpyrene 7,8-diol-9,10-epoxides. Cancer Res. 39, epoxides and related metabolites of carcinogenichydrocarbons. In 67-71 -. .-. Polycyclic Hydrocarbons and Cancer (Gelboin, H. V., and T'so, P. (12) Wood, A. W., Levin, W., Ryan, D., Thomas, P. E., Yagi, H., Mah, 0. P., Eds.) Vol. 1, pp 133-165, Academic Press, New York. H. D., Thakker, D. R., Jerina, D. M., and Conney,A. H. (1977)High (31) Yagi, H., Thakker, D. R., Hernandez, O., Koreeda, M., and Jerina, mutagenicity of metabolically activated chrysene-1,2-dihydrodiol: D. M. (1977) Synthesis and reactions of the highly mutagenic 7,8Evidence for bay region activation of chrysene. Biochem.Biophys. diol 9,lO-epoxidesof the carcinogen benzo[alpyrene. J.Am. Chem. Res. Commun.78, 847-854. SOC.99, 1604-1611. (13) Hecht, S. S., Radok, L., Amin, S., Huie, K., Melikian, A. A., Hoff(32) Melikian, A. A., Leszczynska, J. M., Amin, S., Hecht, S. S., Hoffman, D., Pataki, J., and Harvey, R. G. (1985) Tumorigenicity of mann, D., Pataki, J., and Harvey, R. G. (1985) Rates of hydrolysis 5-methylchrysene dihydrodiol epoxide: DNA adducts in newborn and extents of DNA binding of 5-methylchrysene dihydrodiol epmice and mouse skin. Cancer Res. 45, 1449-1452. oxides. Cancer Res. 45, 1990-1996. (14) Slaga,T. J.,Huberman,E.,Selkirk, J. K.,Harvey,R. G.,andBracken, (33) Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) W. M. (1978)Carcinogenicityandmutagenicityofbenz[alanthracene Purification and properties of a 3a-hydroxysteroid dehydrogenase diols and diol epoxides. Cancer Res. 38, 1699-1704. of rat liver cytosol and its inhibition by anti-inflammatory drugs. (15) Levin, W., Thakker, D. R., Wood, A. W., Chang, R. L., Lehr, R. E., Biochem. J. 222, 601411. Jerina, D. M., and Conney, A. H. (1978) Evidence that benz[alan(34) Vogel, K., Bentley, P., Platt, K. L., and Oesch, F. (1980) Rat liver thracene-3,4-diol-l,2-epoxideis an ultimate carcinogen on mouse cytoplasmic dihydrodiol dehydrogenase: Purification to apparent skin. Cancer Res. 38, 1705-1710. homogeneity and properties. J.Biol. Chem. 255,9621-9625. (16) Slaga, T. J., Gleason, G. L., DiGiovanni, J., Sukumaran, K. B., and (35) Yang, S. K., McCourt, D. W., Gelboin,H. V., Miller, J. R., and Roller, Harvey, R. G . (1979) Potent tumor-initiating activity of the 3,4P. P. (1977) Stereochemistry of the hydrolysis products and their dihydrodiol of 7,12-dimethylbenz[a]anthracenein mouse skin. acetonides of two stereoisomeric benzo[alpyrene 7,8-diol 9,lO-epCancer Res. 39, 1934-1936. oxides. J. Am. Chem. SOC.99, 5124-5130. (17) Glatt, H. R., Vogel, K., Bentley, P., and Oesch, F. (1979)Reduction (36) Yang, S. K., and Gelboin, H. V. (1976) Nonenzymatic reduction of of benzo[alpyrene mutagenicity by dihydrodiol dehydrogenase. Nature (London) 277, 319-320. benzo[a]pyrene diol-epoxidesto trihydroxypentahydrobenzo[a]py(18) Ayengar, P. K., Hayaishi, O., Nakajima, M., and Tomida, I. (1959) renes by reduced nicotinamide adenine dinucleotide phosphate. Enzymatic aromatization of 3,5-cyclohexadiene-l,2-diol.Biochim. Cancer Res. 36, 4185-4189. Biophys. Acta 33, 111-119. (37) Penning, T. M., Sharp, R. B., and Smithgall, T. E. (1989) Non-K(19) Glatt, H. R., Cooper, C. S., Grover, P. L., Sims, P., Bentley, P., region o-quinones as enzyme-generated inactivators of dihydrodiol Merdes, M., Waechter, F., Vogel, K., Guenthner, T. M., and Oesch, dehydrogenase. Biochemistry 28, 4505-4511.
