Differential Stereoselectivity on Metabolism of Triphenylene by ...

2 downloads 10 Views 918KB Size Report
Differential Stereoselectivity on Metabolism of Triphenylene by. Cytochromes P-450 in Liver Microsomes from 3-Methylcholanthrene- and Phenobarbital-treated ...


Vol. 263, No. 1, Issue of January 5, pp. 98-105,1988 Printed in U.S.A.

Differential Stereoselectivity on Metabolism of Triphenylene by Cytochromes P-450in LiverMicrosomes from 3-Methylcholanthreneand Phenobarbital-treatedRats* (Received for publication, April 20, 1987)

Dhiren R. ThakkerS, CharlesBoehlertS, Seid MirsadeghiSS, Wayne Levinll, Dene E. Ryanll, Paul E. Thomasn, Haruhiko YagiII, Lewis K. PannellII, Jane M. Sayer/(,and Donald M. JerinaJI From the $Laboratory of Molecular Pharmacology, Division of Biochemistry and Biophysics, Center for Drugs and Biologics, 20892, the 7Laboratory of Experimental Carcinogenesis and Metabolism, Food and Drug Administration, Bethesda, Maryland Roche Institute of Molecular Biology, Nutley, New Jersey07110, and the IlLaboratory of Bioorganic Chemistry, National Institute of Diabetes, and Digestive and KidneyDiseases, National Institutesof Health, Bethesda, Maryland20892

Metabolism of triphenylenebyliver microsomes cytochrome P-450-dependent monooxygenases and epoxide from control, phenobarbital(PB)-treated rats and 3- hydrolase, are ultimate carcinogenic metabolites of a large methylcholanthrene(MC)-treatedrats as well as by a number of PAH (1, 2). The tetracyclic hydrocarbon triphenpurified system reconstituted with cytochrome P-45Oc in theabsence or presence of purified microsomal epox- ylene is a symmetric PAH with three identical bay regions ide hydrolase was examined. Controlmicrosomes me- and no K region, and it has thehighest resonance energy per tabolized triphenylene at a rate of 1.2 nmol/nmol of II-electron among all PAH (cf. Ref. 3). Although it is nontucytochrome P-450/min. Treatment of rats with PBor morigenic (4) and itsbay region diol epoxides are only weakly MC resulted in a 40% reduction and a %fold enhance- mutagenic toward bacterial and mammalian cells (5), oxidament in the rateof metabolism, respectively. Metabo- tive metabolism of triphenylene is of interest because of the lites consisted of the trum-1,2-dihydrodiol as well as hydrocarbon’s unique structural features.Due to its symme1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the try, triphenylene has a single double bond of the bay region dihydrodiol predominated (70 to 92%) under all incu- type available for metabolism. Many PAH arepoorly metabbation conditions. Incubation of racemic triphenylene olized in their bay regions by rat liver cytochromes P-450, and K region double bonds are often the predominant site of l,2-oxidewith microsomal epoxidehydrolaseproduced dihydrodiol whichwas highly enriched(80%)in metabolism for these PAH (6-10). We have investigated the the (-)-1R,2R-enantiomer. Experiments with “0-enmetabolism of triphenylene by liver microsomes fromcontrol riched water showed that attack of water was exclu- and induced rats as well as by apurified monooxygenase sively at the allylic 2-position of the arene oxide, indicating that the lR,2S-enantiomer of the oxide was system reconstituted with cytochrome P-45Oc,’ the isozyme with highest catalytic activity toward many PAH substrates preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that livermicrosomes (12). Studies with the reconstituted system were done with fromMC-treated rats produced almost exclusively and without addedepoxide hydrolase. (>go%) the lR,2S-enantiomerof triphenylene 1,2-0xMolecular orbital calculations have predicted that triphenide whereas liver microsomes from PB-treated rats ylene 1,P-oxide, the primary oxidative metabolite of triphenformed racemicoxide. The optically active oxide has a ylene that can be formed by the cytochromes P-450, should half-life for racemization of only -20 s under the in- undergo facile racemization (13). Indeed, attempts to prepare cubation conditions. This studymay represent the first attempt to address stereochemical consequences of a this oxide in optically pure form have resulted in a racemic 1,Z-oxide is product (3). If optically activetriphenylene rapidly racemizing intermediary metabolite. formed by the cytochromes P-450 in rat liver microsomes, it can undergo three separate transformations: (i) epoxide hyPolycyclic aromatic hydrocarbons (PAH)’ are environmen- drolase-catalyzed hydration to the trans-l,Z-dihydrodiol, (ii) talpollutantswithmutagenicand carcinogenic activities spontaneous isomerization to phenols, and (iii)racemization which result from metabolism to chemically reactive species. via an oxepin (Fig. 1).We have examined thestereoselectivity Bay region diol epoxides, formed by the combined action of of rat liver enzymes in the metabolic transformation of triphenylene to the areneoxide and the 1,2-dihydrodiol. These * The costs of publication of this article were defrayed in part by studies establish that the cytochromes P-450 in liver microthe payment of page charges. This article must therefore be hereby somes from MC-treated rats form predominantly the 1R,2Smarked “aduertisement” in accordance with 18 U.S.C. Section 1734 oxide,whereas the cytochromes P-450 in liver microsomes solely to indicate this fact. 5 National Research Council (Laboratory of Molecular Pharma- from PB-treated rats formracemic 1,2-oxide. The present cology, CDB, FDA) Research Associate. study also establishes that the rate of racemization of triThe abbreviations and trivial names used are: PAH, polycyclic phenylene 1,Z-oxide is approximately the same as the rateof aromatic hydrocarbons; triphenylene 1,2-dihydrodiol, (+)-trans-1,2dihydroxy-l,2-&hydrotriphenylene;MTPA, (-)-a-methoxy-a-(tri- isomerization to phenols (tlh 20 s). fluoromethy1)phenylacetic acid; MOA, (-)-menthyloxyacetic acid; HPLC, high performance liquid chromatography; PB, phenobarbital; MC, 3-methylcholanthrene; EI, electron impact; CI, chemical ionization.

According to a recently recommended nomenclature system for cytochromes P-450 (11), cytochrome P-45Oc is encoded by the rat P450IA1 gene.


Metabolism of Triphenylene















FIG.1. Enzymatic formation of triphenylene lR,ZS-oxide

I’ METABOLITES {proteinO.mrng/ml)

by cytochrome P-45Oc and subsequent enzymatic and nonenzymatic transformations MATERIALS ANDMETHODS3 RESULTS

Metabolism of Triphenylene by Rat Liver Cytochromes P450 and Epoxide Hydrolase-Chromatographic separation of the metabolites of [3H]triphenylene formed by liver microsomes from MC-treated rats is shown in Fig. 2. Triphenylene 1,2-dihydrodiol and the two phenols, 1- and 2-hydroxytriphenylene, were identified as metabolites by their cochromatography with the authentic standards (Fig. 2) and by the identity of their UV spectra with those of the synthetic standards. Mass spectra (CI-NH,) gave the required molecular ions at m/t 263 (M+ + 1) for the dihydrodiol and at m f z 245 (M+ 1)for the two phenolic metabolites. Quantitation of metabolites formed by liver microsomes from control, PB-treated, or MC-treated rats as well as by cytochrome P-45Oc in the absence or presence of epoxide hydrolase is given in Table I. At a low conversion of the substrate (94%) over a wide range of concentrations (0.005 to 0.1 nmol of Phenobarbital Treated Rats 10 cytochrome P-450/ml) in the presence of 1 nmol of epoxide hydrolase (Fig. 4). These results suggest that epoxide hydrolase is limiting inmicrosomes for the metabolism of triphen5withdecreasing ylene 1,2-oxide. A titrationexperiment amounts of epoxidehydrolase at a fixed concentration of cytochrome P-45Oc (20 pmol/ml) shows that epoxide hydrolase becomes limiting when the ratio of epoxide hydrolase to PROTEIN CONC. (mg/ml) cytochrome P-45Oc is less than 12.5 to 1 (Fig. 5). Although an FIG. 3. Total metabolism of ['Hltriphenylene by liver mi- increase inmicrosomal protein concentrationdoes not change PB-treated (A-A), or MCcrosomes from control (-), treated (W) rats as a function of microsomal protein the ratio of epoxide hydrolase to cytochromes P-450, a deconcentration. Incubation conditions are as described under "Ma- crease in the rate of metabolic formation of the 1,2-oxide/ terials and Methods." Total metabolism was calculated as described nmol of cytochrome P-450 at high protein concentrations in the legend to Table I. without concurrentdecrease in the rate of its hydration would account for the higher percentages of the 1,2-dihydrodiol microsomes; 5 KM 1- and 2-hydroxytriphenylene caused 38 formed at higher protein concentrations. and 59% inhibition, respectively, of the metabolism of triInterestingly, at the same microsomal protein concentraphenylene by these microsomes. Phenols of other PAH are tions, considerably higher relative amounts of triphenylene also inhibitory to cytochromes P-450 (6,23-26). However, the 1,2-dihydrodiol are formedbyliver microsomes fromMCresults presented here suggest that factors other than inhibi- treated rats than by microsomes from PB-treated rats (Fig. tion by metabolites play a significant role in the decreased 4). Since livermicrosomes fromPB-treatedratscontain rate of metabolism of triphenylene at high microsomalprotein higher amounts of epoxide hydrolase than do themicrosomes concentrations. The relationship between rate of metabolism from MC-treated rats (27), the above results could be exof triphenylene and substrate concentration was examined at plained if opposite enantiomers of triphenylene 1,2-oxide a microsomalprotein concentrationof 1mg/ml. With increase predominate when triphenylene is metabolized by liver microin substrate concentration from 50 to 300 FM, the rate of somes from PB- or MC-treated rats and if the enantiomer of metabolism increased upt o maximum values of 1.30 and 0.55 the 1,2-oxide predominantly formedby liver microsomes from nmol of products/nmol of cytochrome P-450/min at 200 ~ L M PB-treatedratsis a poor substrate of epoxidehydrolase. substrate for liver microsomes from control and PB-treated Indeed, in a recent study (28), it was shown that a higher

Metabolism of Triphenylene


TABLE111 Enantiomeric composition of the 1,2-dihydrodiol formed from triphenykne and (+)-triphenykne 1,e-orideby rat liver enzymes


P-45oc t Epoxide Hydrolase



3-Melhv Treated

Enzymesa %


\ Phenob. Treated



[3H]Triphenylenesubstrate 95 5 13 Control microsomes 11 84 16 PB microsomes 9 92 8 MC microsomes Cytochrome P-45Oc + hydrolase 23 85 15 (+)-Triphenylene1,2-oxide substrate PB microsomes 18 80 20 For metabolism of [3H]triphenylene, protein concentrations of liver microsomes fromcontrol andPB-treated rats was 1 mg/ml and of liver microsomes from MC-treated rats was 0.125 mg/ml. CytochromeP-45Oc and epoxidehydrolasewereused at 0.1 and 1 p~ concentrations, respectively,in the reconstituted system. For metabolism of racemic triphenylene 1,2-oxide, liver microsomes from PBtreated rats were used at a protein concentrationof 0.25 mg/ml. *Total metabolism of the unlabeled (kbtriphenylene 1,2-oxide was determined by integration of the 1,Z-dihydrodiol andphenol peaks (254 nm). Authentic triphenylene l,2-dihydrodiol and phenols were used to prepare a standard curve. Triphenylene was used as an internal standard. ‘The retention times of the diastereomeric bis-esters of MTPA are in parentheses. The chromatographic conditions are described under “Materials and Methods.” Absolute configurations have been assigned to the enantiomers of the triphenylene 1,2-dihydrodiol(3).













“ 0

100 pmol P-450clml


FIG.4. Percentage of triphenylene 1,2-dihydrodiol formed as a function of protein concentration by liver microsomes PB-treated (A-A), or MC-treated from control (W), (a”0) rats and by cytochrome P-4SOc plus epoxide hydrolase Incubation conditions areas described under “Mate-


rials and Methods.”

enantiomer was predominantly formed (84-95%) by all three microsomal preparationsand by cytochrome P-45Oc plus epoxidehydrolase. Furthermore,hydration of the racemic oxide, catalyzed by epoxide hydrolase, also resulted in the formation of predominantlythe(-)-(lR,2R)-dihydrodiol (go%, Table 111). Provided that the attack of water is predominantly orexclusively at the allylic 2-position of triphenylene 1,2-oxide during its enzymatic hydration, as hasobserved been for all other benzo-ring arene oxides examined (22) except naphthalene 1,2-oxide (29), these results indicate that the lR,2S-enantiomer of triphenylene oxide is preferentially hydrated by epoxide hydrolase in the racemic mixture. To determine the positiona t which the attack of water takesplace duringenzymatichydration of triphenylene 1,2-oxide, the racemic oxide was incubated with liver microsomes from PBEPOXIDE HYDROLASE (nM) treated rats in 180-enriched water. Over 70% metabolism of FIG. 5. Percentage of triphenylene 1,2-dihydrodiol formed the substrate to dihydrodiol ensured that at least some of by cytochrome P-4SOc (20 nM) plus epoxide hydrolase as a each enantiomer of the oxide had been hydrated by epoxide function of epoxide hydrolase concentration. Incubation conditions and quantitation of the dihydrodiol are described under “Ma- hydrolase. The 1R,2R- and lS,2S-enantiomers (4:l) of the dihydrodiol were resolved, and each enantiomer was subjected terials and Methods.” to acid-catalyzed dehydration tophenols. Mass spectral analyratio of dihydrodiol t o phenol is obtained upon metabolism of sis of the separated phenols from the (lR,2R)-dihydrodioI naphthalene by liver microsomes from MC-treated ratscom- showed that the l80label was present exclusively in 2-hypared to itsmetabolism by liver microsomes from PB-treated droxytriphenylene (TableIV). Similarly, almost all of the l80 rats. Furthermore, it was shown that,while liver microsomes label (-90%) was foundin2-hydroxytriphenylene formed from MC-treated rats formed almost exclusively naphthalene from the lS,2S-enantiomer (TableIV). These results require that the 1R,2R- and lS,2S-enantiomers of triphenylene 1,2lR,BS-oxide, liver microsomes from PB-treated rats formed dihydrodiol are formedfrom1R,2Sand lS,2R-oxides, resignificantamounts of the lS,PR-oxidewhichwasapoor substrate forepoxide hydrolase in comparison with the 1R,2S- spectively, by allylic attack of water at the2-position. Stereoselectively of Cytochromes P-450 in Rat Liver Microoxide. Stereoselectivity of Epoxide Hydrolase-The enantiomer somes-For determination of enantiomeric composition of the composition (Table 111) of triphenylene 1,2-dihydrodiol 1,2-oxide formed fromtriphenylene by liver microsomes from hydrocarbon was incubated formed from triphenylene under various conditions was de- PB- and MC-treated rats, the of trichloropropene oxide termined by chromatographic separationof its diastereomeric with themicrosomes in the presence bis-esters with MTPA. Cochromatography of the late eluting (to block the enzymatic hydration of the triphenylene oxide diastereomerwiththebis-MTPAester derived fromthe formed), and theoxide was trapped by the thiolate group of (-)-(lR,2R)-dihydrodiol (3) indicated that the (-)-1R,2RN-acetyl-L-cysteine. Enantiomeric composition of several ar2.5

RATIO [Epoxide Hydrolase/Cyt. P-450~1 5.0 7.5

Metabolism of Triphenylene



TABLEIV Determination of regioselectivity of epoxide hydrolase toward triphenylene 1,a-oxide by mass spectrometry Triphenylene derivative

Relative intensity"





(M + 2)' mol '80/mol


Triphenylene 1,2-dihydrodiol0.398 68.48 100 From lR,2R-dihydrodiol 1-HOTP' 100 2-HOTP 57.20 100 From lS,2S-dihydrodiol 1-HOTP 100 2-HOTP 100

k, = 1 . 8 ~

t x = 200sec

k, = 1 . 8 ~

", 0.75


tx = 20sec

2.88 6.49 60.16

0.006 0.355



0.041 0.367

kr = 1.8 x 10-l sec-1

t x = Psec

Relative intensities represent average values. ' "0 content was calculated after correcting for the natural isotopic abundance. ' 1- and 2-HOTP, 1-and 2-hydroxytriphenylene. a

ene oxide metabolites hasbeen determined by trapping them with the thiolate group of N-acetyl-L-cysteine or glutathione (22, 28, 31). As shown in the equation below, the oxide can spontaneously isomerize t o phenols, and/or racemize before it is trapped.

n qn _._"





200 TIME (sect

FIG. 6. Calculated time courses for racemization of optically pure triphenylene 1,Z-oxide when it is being formed from triphenylene at a rate of 0.016 pM/s and simultaneously isomerizing to phenols at a rate of 0.035 s-l in 0.1 M potassium phosphate buffer (pH 7.4). The time courses were calculated for

rates of racemization (2kJ equal to '/loth ( t , = 200 s) and 10 times (tmh= 2 s) the experimentally observed rate of isomerization (k; = 0.035 s-', tsh= 20 s). The curves were generated by solving (30) the rate equations: dOX,/dt = c - k,OX, - kiOXl + k,0X2 and dOX,/dt Triphenylene 5 [oxide]Rs [oxide]sR = k,OXl - kiOX2- k,OX,, where OX, and O X , are the concentrations Ikl lkl of the two enantiomers of triphenylene 1,2-oxide, c is the rate of phenols phenols oxidation of triphenylene, and k; and 2k, are the rates of isomerization and racemizations, respectively. Values obtained experimentally with Kinetic analysis indicated that if only one of the enantiomers liver microsomes from MC-treated rats are indicated by solid circles.


of the arene oxide is formed metabolically, it will racemize as a function of time until a plateau is reached a t a value that will depend on the relative rates of racemization ( 2 k J uersus isomerization (k, = 0.035 s-l) (Fig. 6). This plateauis reached within 2 min even at a relatively slow rate of racemization (Zk, = 0.0035 s-'). Hence, incubations were allowed to proceed for 2.5 min before the oxide(s) was trapped with N-acetyl-Lcysteine. Reaction of racemic triphenylene 1,2-oxide with Nacetyl-L-cysteine results in two minor adducts (I and 11) and two major adducts (I11 and IV)(Figs.7 and 8A). All four adducts have UV spectra which are similar to that of the trans-1,Z-dihydrodiol. Mass spectra (CI-NH3) of the major adducts 111and IV show a fragment at m/z = 245 (M H' N-acetylcysteine). The NMR spectrum (300 MHz, DzO) of the major adduct IV is as follows: 6 2.01 (3H, s, -COCH3), 2.72 (lH, dd, S-CH.CHb), 2.91 (lH, dd, S-CH,CfrIb), 4.02 ( l H , dd, Hz), 4.19 ( l H , dd, -CH), 5.69 ( l H , d, HI), 6.39 ( l H , dd, H d , 7.57 OH, d, H A 7.71 (4H, m, H6,7,1~,11), 8.33 (ZH, m, H , , J , 8.79 (2H, m, H8J; J1,2= 1.4 Hz, J2,3= 5.7 Hz,J3,4= 9.8 Hz, JcH-cH. = 7.8 Hz, J C H - C H ~ = 4.5 Hz, and JcH,H, (gem) = 13.8 Hz. The above values of J1,z and J2.3 indicate that the hydroxyl and the thioethergroups are trans-diaxial, which is consistentwiththe previouslyobserved transaddition of thiolate to epoxides. The chemical shift ofH2 at 4.02 ppm is 0.42 ppm upfield compared to thatof HP in triphenylene1,Zdihydrodiol (measured in CD3COCD,); whereas the chemical shift of H1inthe above adduct (5.69 ppm) is 0.28 ppm downfield compared to that of the HI in triphenylene 1,2dihydrodiol (5.41 ppm). The upfield movement in the chemical shift of Hz in adduct IV compared to Hz in triphenylene 1,2-dihydrodiol suggests that the major adduct IV (and presumably the other major adduct 111) was formed by allylic attack of the thiolate group as has been the case for several benzo-ring arene oxides (22, 28, 31). The assignment of the of thioether moiety at C-2 is based on the NMR spectra thiolate andalkoxide adducts of benzene oxide for which the proton on the carbon atom bearing thehydroxyl substituent is at lower field relative to the proton on the carbon atom


a OH

R - S H , OH;





major I111

minor I II 1

major I I V )

minor I I )




FIG. 7. Structures of the N-acetyl-L-cysteine adducts formed from enantiomer of triphenylene 1,a-oxide. Minor (I and 11) and major (I11 and IV) trans-thiol adducts are formed from each oxide (cf. Fig. 8) in the presence of N-acetyl-L-cysteine and base by attack of the thiolate at the benzylic 1-position and the allylic 2position, respectively. Preferential attack of thiolate at the allylic position over benzylicposition has been previouslydemonstrated (29) for arene oxides of naphthalene and anthracene. R-SH, N-acetyl-Lcysteine.

bearing the thioether substituent (32). The separation of Nacetyl-L-cysteine adducts with arene oxide formed by liver microsomes from MC- and PB-treated rats is shown in Fig. 8, B and C, respectively. Integration of the peaks indicates that for liver microsomes from MC-treated rats, adducts I1 and I11 are formed in 3-fold excess over adducts I and IV, respectively. Thus, adducts I1 and I11 are formed from the enantiomer of triphenylene 1,2-oxide which accounts for 75% of total areneoxide once the plateauis reached. Interestingly, when the oxide was trapped after 30 s of incubation, adducts I1 and I11 accounted for 83% of the total adducts. These results indicate that liver microsomes from MC-treated rats form almost exclusively a single enantiomer (>go%) of tri-

Metabolism of Triphenylene -


indicates that the lR,2S-oxide is hydrated by epoxide hydrolase in preference to the1S,2R-oxide. This is also consistent From with the observation that the 1,2-dihydrodiol formed by liver I 1 R 25 I OXIDE microsomes from PB-treated rats consists predominantly of the R,R-enantiomer (80%) despiteformation of nearly racemic oxide by these microsomes (Fig. 8C). The preferential hydration of the lR,BS-oxide, coupled with the observation that significantly lower relative amounts of the dihydrodiol 1 are formed by liver microsomes from PB-treated rats comB From oxlde formed by pared to the microsomes from MC-treated rats, implies that MC-Microsomes the lS,2R-enantiomer of triphenylene oxide must be formed in higher percentages by the microsomes from PB-treated rats. Determination of enantiomeric composition of the arene oxide formed by these microsomes at 2.5 min of incubation showed that while the two enantiomers of the 1,2-oxide are formed in the ratio of >3 to 1 by liver microsomes from MC1 treatedrats,they were formedinequal amounts by liver C From oxlde formed by Therefore, it follows that microsomes from PB-treated rats. PB-Mlcrosomes the predominant enantiomer of triphenylene1,2-oxide formed by liver microsomes from MC-treated rats must have lR,2Sabsolute configuration. These results are consistent with predictionbased onoursteric model for thebindingsite of cytochrome P-45Oc (33,34), the predominantisozyme in liver microsomes from MC-treated rats (27, 35). It remains to be determined whether the racemic oxide: formed by liver mi0 10 20 crosomes from PB-treated rats, is due to the lack of stereoselectivity of a specific isozyme of cytochrome P-450 in these TIME (rnin) FIG. 8. Chromatographic separation of adducts formed by microsomes or is due to opposite stereoselectivity of two or reaction of N-acetyl-L-cysteine with racemic triphenylene more isozymes. Cytochrome P-450b, which is selectively in1,2-oxide ( A ) , the oxide formed by liver microsomes from duced by P B (27), does not appear to be responsible for the MC-treated rats ( B ) , and the oxide formed by microsomes lack of stereoselectivity of these microsomes, since the purifrom PB-treated rats (C). Adducts I and I1 are presumed to be fied isozyme has negligible catalytic activity toward triphenformed by the attack of thiolate at the benzylic position of the oxide ylenewhen reconstituted (C0.2 nmol of products/nmol of and adducts 111 and IV are formed by allylic attack of the thiolate group. Chromatographic conditions are described under “Materials cytochrome P-450/min). Finally, the results of the trapping and Methods.” Resultsin the present study indicate that the adducts experiment also provided an estimate of the rate of racemiI1 and 111 (shaded) are formed from the lR,ZS-oxide. As expected, zation of triphenylene 1,Z-oxide. The oxide formed by liver sum of areas under peaks I and IV (50.7%)in chromatogram A is microsomes from MC-treated rats consisted of two enantioequal to the sum of areas under peaks I1 and 111 (49.3%) for the mers in the ratios of 83:17 and 75~25 when it was trapped by adducts derived from racemic oxide. N-acetyl-L-cysteine after 30 and 150 s of incubation, respectively. These results are consistent with the rate of racemiphenylene 1,2-oxide, which racemizesa t a rate approximately zation of triphenylene 1,2-oxide of 0.036 s-’, provided that equal to the rate of its isomerization(Fig. 6). In contrast, liver microsomes from MC-treated rats formed only one enwhen liver microsomes from PB-treated rats are used, adducts antiomer (Fig. 6). The actualvalue for the rate of racemization I1 + 111 and I + IV are formed in -1:l ratio. This indicates of triphenylene 1,2-oxide may turn out tobe somewhat lower that both the enantiomers of the 1,2-oxide are present in if the microsomes from MC-treated rats form the oxide with similar amountsonce the plateau is reached. less than 100% optical purity. Hence, the present study provides thefirst evidence thattherate of racemization of DISCUSSION triphenylene 1,2-oxide is almost as high as the rate of its Triphenylene, despite its lack of a K region and the presence isomerization to phenols (ty2= 20 s) and may also be the first of six identical bay region double bonds due t o symmetry, is metabolism study which attempts to address the stereochema moderately good substrate, relative to other PAH, for the ical consequences of a rapidly racemizingintermediate metabcytochromes P-450 in liver microsomes fromMC-treated rats. olite. It is metabolized a t about the same maximum ratebenzo[a] as pyrene and benzo[e]pyrene and at about half the rate as other Acknowledgments-We gratefully acknowledge the help of Ellen tetracyclic hydrocarbons such as benzo[c]phenanthrene and Kirshbaum in the preparation of this manuscript. benz[a]anthracene (cf. Ref. 1). Metabolites of triphenylene consist of the 1,2-dihydrodiol REFERENCES and a pair of phenols, all formed via the 1,2-oxide (Fig. 1). 1. Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. The (-)-lR,2R-enantiomer of the dihydrodiol predominates H., and Jerina, D. M. (1985) in Bioactiuation of Foreign Comwith microsomes and with the reconstituted system containpounds (Anders, M. W., ed) pp. 177-242, Academic Press, New York ing cytochrome P-45Oc and epoxide hydrolase. Experiments 2. Sims, P., and Grover, P. L.(1981)in Polycyclic Hydrocarbons and with “0-enriched water established that epoxide hydrolase Cancer (Gelboin, H. V., and Ts’o, P.0. P., eds) Vol. 3, pp. 117directs the attackof water almostexclusively at the allylic 2position in both enantiomers of the 1,2-oxide. Hence, the(-)‘Trapped oxide in the plateau region was racemic within experi1R,2R- and (+)-1S,2S-dihydrodiols are formed from 1R,2Smental error. Had the initially formed enantiomer mixture been 60:40, and lS,BR-oxides, respectively. Stereoselective hydration of this would have been trapped at a ratio of 55:45 in the plateau region racemic1,2-oxide predominantlytothe lR,2R-dihydrodiol using the rate constants described.

AFrom Racemic 1.2-oxide





Metabolism of Triphenylene

181, Academic Press, New York. 3. Boyd, D. R., Kennedy, D. A., Malone, J. F., O’Kane, G.A., Thakker, D. R., Yagi, H., and Jerina, D.M. (1987) J. Chem. SOC. Perkin Trans.I , 369-375 4. Dipple, A., Moshel, R. C., and Bigger, G. A. H. (1984) in Chemical Carcinogens, ACS Monograph 182 (Searle, C . E., ed) Vol. 1, pp. 41-163, American Chemical Society, Washington, D. C. 5. Wood, A. W., Chang, R.L., Huang, M. T., Levin, W., Lehr, R. E., Kumar, S., Thakker, D. R., Yagi, H., Jerina, D. M., and Conney, A. H. (1980) Cancer Res. 40, 1985-1989 6. Thakker, D. R., Levin, W., Yagi, H., Ryan, D., Thomas, P. E., Karle, J. M., Lehr, R., Jerina, D. M., and Conney, A. (1979) Mol. Pharmacol. 15,138-153 7. Macleod, M. C., Levin, W., Conney, A. H., Lehr, R. E., Mansfield, B. K., Jerina, D. M., and Selkirk, J. K. (1980) Carcinogenesis (Lond.) 1, 165-171 8. Ittah, Y., Thakker, D. R., Levin, W., Croisy-Delcey, M., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1983) Chem. Biol. Interact. 45, 15-28 9. Thakker, D. R., Levin, W., Yagi, H., Conney, A. H., and Jerina, D. M. (1982) in Biological Reactive Intermediates (Snyder, R., Parkes, D., Kocsis, J., Jollow, D., Gibson, G., and Witmer, C., eds) Vol. 11, Part A, pp. 525-539, Plenum Publishing Corp., New York 10. Steward, A.R., Kumar, S., and Sikka, H. C. (1986) Proc. Am. Assoc. Cancer Res. 2 7 , 111 11. Nebert, D. W., Adesnik, M., Coon, M. J., Estabrook, R. W., Ganzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Kemper, B., Levin, W., Philip, I. R., Sato, R., and Waterman, M. R. (1987) D N A (N. Y.) 6 , l - 1 1 12. Levin, W., Thomas, P. E., Reik, L. M., Wood, A. W., and Ryan, D.E. (1984) in IUPHAR 9th International Congress of Phnrmacology (Paton, W., Mitchell, J., and Turner,P., eds) Vol. 3, pp. 203-209, Macmillan Press Ltd., London 13. Boyd, D. R., and Stubbs, M. E. (1983) J . Am. Chem. SOC. 109, 2554-2559 14. Lehr, R. E., Taylor, C. W., Kumar, S., Mah, H. D., and Jerina, D. M. (1978) J. Org. Chem. 43, 3462-3466 15. Lu, A. Y. H., and Levin, W. (1972) Biochem.Biophys.Res. Commun. 46,1334-1339 16. Omura, T., and Sato, R. (1964) J. Biol. Chem. 2 3 9 , 2379-2385 17. Ryan, D., Thomas, P. E., and Levin, W. (1982) Arch. Bwchem. Biophys. 2 1 6 , 272-288 18. Yasukochi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 2 5 1 , 5337-5344

19. Ryan, D. E., Thomas, P. E., and Levin, W. (1980) J. Biol. Chem. 255,7941-7955 20. Dale, J. A., Dull, D. L., and Mosher, H.S. (1969) J. Org. Chem. 34, 2543-2549 21. Oesch, F., Kavbisch, W., Jerina, D.M., and Daly, J. W. (1971) Biochemistry 10,4858-4866 22. Boyd, D. R., and Jerina, D. M. (1985) in Small Ring Heterocytes (Hassner, A., ed) Part 3, pp. 197-282, John Wiley & Son, New York 23. Shen, A. L., Fahl, W.F., Wrighton, S. A., and Jefcoate, C.R. (1979) Cancer Res. 39,4123-4129 24. Fahl, W. F., Shen, A. L., and Jefcoate, C.R. (1978) Biochem. Bwphys. Res. Commun. 85,891-899 25. Thakker, D. R., Levin, W., Yagi, H., Ryan, D., Thomas, P. E., Karle, J. M., Lehr, R. E., Jerina, D. M., and Conney, A. H. (1979) Mol. Phurmacol. 19, 138-153 26. Vyas, K. P., Levin, W.,Yagi, H., Thakker, D.R., Ryan, D.E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1982) Mol. Phurmacol. 2 2 , 182-189 27. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1981) J. Biol. Chem. 2 5 6 , 1044-1052 28. van Bladeren, P. J., Sayer, J. M., Ryan, D. E., Thomas, P. E., Levin, W., and Jerina, D. M. (1985) J.Biol. Chem. 260,1022610235 29. van Bladeren, P. J., Vyas, K. P., Sayer, J. M., Ryan, D. E., Thomas, P. E.,Levin,W., and Jerina, D. M. (1984) J. Biol. Chem. 259,8966-8973 30. Knott, G. D. (1979) Comput. Programs Biomed. 10, 271-280 31. van Bladeren, P. J., Armstrong, R. N., Cobb, D., Thakker, D. R., Ryan, D. E., Thomas, P. E., Sharma, N. D., Boyd, D. R., Levin, W., and Jerina, D. M., (1982) Biochem. Biophys. Res. Commun. 106,602-609 32. Jeffrey, A. M., Yeh, H. J. C., Jerina, D. M., DeMarinis, R. M., Foster, C. H., Piccolo, D. E., and Berchtold, G. A. (1974) J. Am. Chem. Soc. 96,6929-6937 33. Jerina, D.M., Michaud, D. P., Feldmann, R. J., Armstrong, R. N., Vyas, K. P., Thakker, D. R., Yagi, H., Thomas, P. E., Ryan, D. E., and Levin, W. (198’2) in 5th International Symposium on Microsomes and Drug Oxidations, Microsomes, Drug Oxidations, and Drug Toxicity (Kato, R., and Sato, R., eds) pp. 195-201, Japan Scientific Society Press, Tokyo 34. Jerina, D. M., Sayer, J. M., Yagi,H., van Bladeren, P. J., Thakker, D. R., Levin, W., Chang, R. L., Wood, A.W., and Conney, A. H. (1985) in 6th International Symposium on Microsomes and Drug Oxidations (Brobis, A. R., Caldwell, J., DeMatteis, F., and Elcombe, R., eds) pp. 310-319, Taylor & Francis Ltd., London 35. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1983) J. Biol. Chem. 258, 4590-4598

Metabolism of Triphenylene


Suggest Documents