Carcinogenesis vol.11 no. 11 pp.2037-2045, 1990

Metabolism off nnnkrosomes

-3-metIfoylcIfoolaimtliiireinie by r a t liver

Magang Shou and Shen K.Yang1 Department of Pharmacology, F.Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20889-4799, USA 'To whom correspondence and reprint requests should be addressed

Introduction 3-Methylcholanthrene (3MC*), a potent mutagen and carcinogen, is metabolized to both non-toxic and mutagenic/carcinogenic products by mammalian drug-metabolizing enzyme systems (1—5). The C) and Cj positions of 3MC are major sites of oxidative metabolism, resulting in the formation of primarily 1-OH-3MC and 2-OH-3MC, and to a minor extent, 3MC-l-one, 3MC-2-one and 3-methylcholanthrylene (1,3,6—12). Tentative identification of 3-hydroxymethylcholanthrene (3-OHMC) as a metabolite of 3MC has been reported (2,6,7,9,13). A recent study indicated that the amount of 2-OH-3MC formed in the metabolism of 3MC is substantially higher than that of 1-0H-3MC and the •Abbreviations: 3MC, 3-methylcholanthrene; 3-OHMC, 3-hydroxymethylcholanthrene, 3MC-2-one 9,10-dihydrodiol, 9,10-dihydroxy-9,10-dihydro3MC-2-one; 2-OH-3MC 9,10-dihydrodiol, 9,10-dihydroxy-9,10-dihydro2-OH-3MC; 3MC tfms-l,2-diol, trans-l,2-dihydroxy-3-methylcholanthrene: other derivatives of BA and 2-OH-3MC are similarly abbreviated; G-6-P, glucose 6-phosphate; BA, benz[o]anthracene; /{-DNBPG-C, covalently bonded chiral stationary phase (/f)-/V-(3,5-dinitrobenzoyl)phenylglycine; CD, circular dichroism; PB, phenobarbital; B[a]P, benzo[a]pyrene; AUC, area under the chromatographic peak; solvent A, ethanol/acetonitrile (2/1, v/v).

© Oxford University Press

Thakker et al. (9,12) identified two diastereomeric 1-0H-3MC trans-9,10-dihydrodiols of unknown absolute stereochemistry as the most abundant metabolites of 1-OH-3MC by liver microsomes form 3MC-treated rats. Of these, the one designated as 1-OH-3MC rra«5-9,10-dihydrodiol-a was more active in both S. typhimurium TA100 and Chinese hamster V79 cells than the one designated as 1-OH-3MC trans-9,10-dihydrodiol-* (4). 1-OH-3MC fran.y-9,10-dihydrodiol-a was found to be as active as 3MC as a tumor initiator on mouse skin and more than twice as active as a carcinogen in newborn mice, whereas 1-0H-3MC trans-9,10-dihydrodiol-b was only a relatively weak tumor initiator 2037

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Products formed in the metabolism of 2S-hydroxy-3-methylcholanthrene (2S-OH-3MC) by liver microsomes prepared from phenobarbital-treated rats were isolated by sequential use of reversed-phase and normal-phase HPLC. Metabolites of 2S-OH-3MC were characterized by UV-visible absorption, mass and circular dichroic spectra, and chiral stationary phase HPLC analyses. The metabolites that had been identified were 2S-hdyroxy-3-hydroxymethylcholanthrene (25-OH-3-OMMC), 3MC-2-one, 3MC-2-one 9,10-dihydrodiol, 8-hydroxy-2S-Q]rI-3MC, a pair of stereoisomers 3MC (trans)-lR,2R-diol and (cis)-lS,2R-dio\ in a ratio of ~ 11:89, a pair of diastereomers 2S-OH-3MC 9#,10fl-dihydrodiol and 2S-OH-3MC 9S,10S-dihydrodiol in a ratio of - 9 : 1 , and a pair of diastereomers 25-OH-3MC llfl,12#-dihydrodiol and 2S-OH-3MC HS,12S-dihydrodiol in a ratio of ~ 77:23. A few tentatively identified minor metabolites were 3-OHMC trans-lR,2R-dio\, 10-hydroxy-2S-QH-3MC, a 9,10dihydrodiol derived from 3MC c«-lS,2/?-diol, and a 11,12dihydrodiol and two diastereomeric 9,10-dihydrodiols derived from 2S-OH-3-OHMC. Since the racemic 2-OH-3MC is a known potent carcinogen and 2S-OH-3MC is the most abundant metabolite of 3MC, some of the 2S-OH-3MC metabolites identified in this study may be further converted to proximate and ultimate carcinogens which may contribute to the overall carcinogenicity exhibited by 3MC.

enzymatically formed 2-OH-3MC is highly enriched in the 25-enantiomer (14). Carcinogenic activities of 3MC, 1-OH-3MC, 2-OH-3MC, 3MC1-one and 3MC-2-one were tested by three weekly s.c. injections into mice; all four 3MC derivatives were active carcinogens, but less so than 3MC itself (2). Repeated applications of the same set of compounds to the skin of mice showed that 3MC and 2-OH-3MC were highly active, 3MC-2-one was moderately active, 1-OH-3MC weakly active and 3MC-l-one inactive (3). Tumorinitiating activity tests on mouse skin (5) gave essentially the same results: 3MC, 2-OH-3MC and 3MC-2-one were of approximately equal activity, 1-OH-3MC was weakly active and 3MC-l-one was inactive. In tests for the induction of pulmonary and hepatic tumors in newborn mice (5), it was found that 3MC and 3MC-2-one were the most active in inducing pulmonary tumors, 2-OH-3MC was slightly less active, 1-OH-3MC showed marginal activity and 3MC-l-one was inactive. In the induction of hepatic tumors in newborn male mice, 3MC, 2-OH-3MC and, to a lesser extent, 3MC-2-one were active. 3MC fra/w-9,10-dihydrodiol was active as a tumor initiator on mouse skin but no more active than 3MC itself, other dihydrodiols of 3MC were virtually inactive (5,15). 3MC rraws-9,10-dihydrodiol was also found to be highly active in the induction of sister chromatid exchanges in Chinese hamster ovary cells (16), to induce high mutagenic activity in Salmonella typhimurium TA100 and Chinese hamster V79 cells and malignant tranformation foci in M2 mouse fibroblasts (17). 3MC and a racemic 3MC fra/w-9,10-dihydrodiol exhibited high tumorinitiating activity on the skin of SENCAR mice; both compounds were more active than a racemic 3MC anrt-9,10-diol-7,8epoxide (18). In that study (18), 3MC rra/w-9,10-dihydrodiol was also found to be — 2 times more potent as a mutagen than 3MC toward Chinese hamster V79 cells in a cell mediated mutagenicity assay. King et al. (19,20) identified two 9,10-dihydrodiols formed in the metabolism of 3MC by liver microsomes from 3MCtreated rats; one was a 3MC 9,10-dihydrodiol and the other was proposed to be a 9,10-dihydrodiol derived from either 1-0H-3MC or 2-OH-3MC. Both 9,10-dihydrodiols were further metabolized by rat liver microsomes to react with DNA in vitro (19). Since 25-OH-3MC was found to be the most abundant rat liver microsomal metabolite of 3MC (14), the second 9,10-dihydrodiol reported by King et al. (19,20) was probably a 25-OH-3MC 9,10dihydrodiol.

M.Shou and S.K.Yang

Materials and methods Materials 3MC, NADP + , glucose 6-phosphate (G-6-P), and G-6-P dehydrogenase were purchased from Sigma Chemical Co. (St Louis, MO). 3MC-2-one was synthesized according to procedures described by Cavalieri et al. (3) and Sims (6). A racemic 2-OH-3MC was prepared by reduction of 3MC-2-one with NaBH4 in methanol Monohydroxylated derivatives of benz[a]anthracene (BA), 3MC 11,12-quinone, 3MC cis-\ 1,12-dihydrodiol were obtained from the Chemical Repository of the National Cancer Institute. These authentic compounds were purified by reversedphase HPLC prior to UV-visible absorption spectral measurement. 3MC trans-11,12-dihydrodiol was synthesized from 3MC CM-11,12-dihydrodiol as previously described (27). 3MC trans-\ ,2- (cl.l1B.2S

ABSORB ANCE (

dipole in the longitudinal axis of the p-nitrobenzoate chromophore intersects at near the midpoint of that of the BA chromophore (14), we were concerned that the exciton splitting resulting from the interaction between the two transition dipoles may not be interpreted in a simple manner to provide a definitive answer on the abolsute configuration of the enantiomeric 2-OH-3MC. The absolute configuration of the enantiomeric 2-OH-3MC has been elucidated by a method independent of that previously described (14) and this is described below. Enantiomeric pairs of 3MC trans-1,2-diol and 3MC cis-\ ,2-diol can be separated by HPLC using a fl-DNBPG-C column (21). The absolute configuration of a 3MC trans- 1,2-diol enantiomer, following conversion to ato-/?-N-,A/-dimethylarninobenzoate,has been established by the exciton chirality CD method (21). The metabolites contained in peaks 11 and 16 (AUC ratio 11:89) of Figure 1 had UV-visible absorption and mass spectra and retention times identical to those of the authentic 3MC trans- 1,2-diol and 3MC cis- 1,2-diol respectively. The metabolically formed trans- 1,2-diol and cis- 1,2-diol co-eluted with the more strongly retained 3MC lR,2R-dio\ and the less strongly retained 3MC 15,2/?-diol respectively, on a /?-DNBPG-C column (Figure 3). It should be pointed out that due to the addition of a chiral center at C|, the hydroxyl group with 25 designation in an enantiomeric 2-OH-3MC is changed to 2R (and vice versa) in the enantiomeric 3MC trans- and cis-1,2-diols (see illustration at the top of Figure 3). Since the absolute configurations of the metabolically formed 1,2-diols have been established (21), the 2-OH-3MC enantiomer from which the 1,2-diols are derived is deduced to be the 25-enantiomer (Figure 3). These results are consistent with those described in the earlier report (14) indicating that the 2-OH-3MC enantiomer which has a positive CD Cotton band centered around 258 run (Fig. 2) is the 25-enantiomer. In

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Fig. 3. Chiral stationary phase HPLC separation of enantiomeric 3MC irons- 1,2-diols (left chromatogram) and 3MC cis- 1,2-diols (right chromatogram). Retention times of 3MC trans- 1,2-diol (—, left chromatogram) and 3MC era-1,2-diol (—, right chromatogram), formed in the metabolism of 25-OH-3MC, are indicated. A fl-DNBPG-C column was used and the mobile phase was 10 and 7% (v/v) of solvent A (ethanol/acetonitrUe, 2/1, v/v) in hexane respectively, at a flow rate of 2 ml/min. Absolute configurations of enantiomeric 3MC trans-1,2-diol and 3MC cis- 1,2-diol have been established in an earlier study (21). Structure and stereochemistry of products formed by C|-hydroxylation of 25-OH-3MC are shown at the top of the figure.

the earlier study (21), the absolute configuration of the 2-OH-3MC enantiomer was determined by analysis of the CD spectrum of a p-nitrobenzoate.

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Metabolism of 2S-hydroxy-3-methylcholanthrene

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Oxidation of C2-hydroxyl group The metabolite contained in peak 18 was identified as 3MC-2-one. It had UV—visible absorption and mass spectra and a retention time on reversed-phase HPLC identical to those of the authentic 3MC-2-one (see ref. 27 for UV-visible absorption spectrum). This confirms the earlier finding by Sims (3) who reported that 3MC-2-one is a metabolite of 2-OH-3MC. In principle, 3-OHMC-2-one may be formed by oxidation of the C2-hydroxyl group of 2-OH-3-OHMC and/or hydroxylation of the C3-methyl group of 3MC-2-one. 3-OHMC-2-one should have a UV—visible absorption spectrum similar to that of 3MC-2-one and is expected to be eluted between 30 and 33 min in Figure 1. However, normal-phase HPLC and UV-visible absorption spectral analyses of metabolites eluted between 30 and 33 min in Figure 1 did not reveal the presence of 3-OHMC-2-one.

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RETENTION TIME (min) Fig. S. Normal-phase HPLC separation of the two major components contained in peak 5 of Figure I. The mobile phase was 15% (v/v) of solvent A in hexane, at a flow rate of 2 ml/min. Identities of metabolites contained in the chromatographic peaks are shown. Evidence for the identification of these two metabolites is described in the text.

Oxidation of C3-methyl group The metabolite contained in peak 14 of Figure 1 was the most abundant metabolite of 25-OH-3MC and was identified as 25OH-3-OHMC. It has a UV-visible absorption spectrum (Figure 4) essentially the same as that of 2-OH-3MC, a M + at mlz 300 and characteristic fragment ions at mlz 282 (loss of H2O) and 265 (loss of H2O and OH). 3MC, 3-OHMC, 1-OH-3MC, 2-OH-3MC, 1-OH-3-OHMC, 2-OH-3-OHMC and the trans1,2-diol and the c/j-l,2-diol derived from both 3-MC and 3-OHMC all have the same absorption characteristics because of their common BA chromophore. The second hydroxyl group

Oxidation at the M-region 9,10-double bond The M-region is defined, in the context of the bay-region theory of polyaromatic hydrocarbon carcinogenesis (30), as the double bond (such as the 9,10-double bond of 2-OH-3MC or 3MC) at which the procarcinogenic dihydrodiol is formed (33). There were six 9,10-dihydrodiols detected as metabolites of 25-OH-3MC. These were: 3MC-2-one 9,10-dihydrodiol (peak 9), 25-OH-3MC 95,105-dihydrodiol (peak 7), 25-OH-3MC 9fl,10fl-dihydrodiol (the major component in peak 5, which also contained a K-region 11,12-dihydrodiol, see below), two 9,10-dihydrodiols probably derived from 25-OH-3-OHMC (metabolites contained in peaks 1 and 3), and a 9,10-dihydrodiol (peak 4 of Figure 1) probably derived from 3MC (cis)-lS,2/?-diol. Peak 5 contained two metabolites, which were separated by normal-phase HPLC (peaks 5a and 5b in Figure 5). Peak 5b had UV —visible absorption and CD spectra (Figure 6) identical to that of 25-OH-3MC 9/?,10/?-dihydrodiol reported earlier (25). The UV-visible absorption spectrum of peak 5b was also similar to that of 3MC 9,10-dihydrodiol (11,34). The major metabolite contained in peak 5 and the predominant component contained in peak 7 had retention times on reversed-phase HPLC (Figure 1) and UV-visible absorption spectra (Figure 6) identical to those of the two diastereomeric 2-OH-3MC 9,10-dihydrodiols derived by NaBH4 reduction of the 3MC-2-one 9,10-dihydrodiol (25) 2041

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Fig. 4. UV—visible absorption (—, methanol) and CD ( , cone. 10 A294/ml, methanol; *256/A294 = 5.7 millidegrees) spectra of the metabolite (2S-OH-3-OHMC) contained in peak 14 of Figure 1.

of metabolite peak 14 was not located at the C, position, because the retention time of metabolite peak 14 on reversedphase HPLC (Figure 1) was different from those of 3MC fra/w-l,2-diol (peak 11) and 3MC c«-l,2-diol (peak 16). Furthermore, if the second hydroxyl group were located at an aromatic ring position (i.e. the hydroxyl group is phenolic), the UV-visible absorption spectrum would have been different from that of 2-OH-3MC (or 3MC). The CD spectrum of this metabolite ($256^294 = 5.7 millidegrees, Figure 4) had Cotton effect similar to those of 25-OH-3MC (Figure 2B). Thus the metabolite contained in peak 14 was established to be 25-OH-3-OHMC. The minor metabolite contained in peak 8 was tentatively identified as 3-OHMC trans-\,2-d\o\. It had a UV-visible absorption spectrum (not shown) similar to that of 2-OH-3-OHMC (see Figure 4). When 3MC trans-\,2-d\o\ was incubated with rat liver microsomes and cofactors, a metabolite (identified as 3-OHMC rra/u-l,2-diol, unpublished results) with a retention time identical to that of peak 8 was detected. Metabolite peak 8 was unlikely to be derived from the minor metabolite 3MC trans- 1,2-diol (peak 11), but rather from microsome-catalyzed ?ran.y-hydroxylation at C| of 25OH-3-OHMC.

M.Shou and S.K.Yang

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Fig. 8. (A) UV-visible absorption spectra of 3-OH-BA (—, in methanol; an authentic compound) and 9-OH-2S-OH-3MC ( , in methanol; a product derived from acid-catalyzed dehydration of a 2-OH-3MC 9,10-dihydrodiol). (B) UV-visible absorption spectra of 4-OH-BA (—, in methanol; an authentic compound) and 10-OH-2S-OH-3MC ( , in methanol; a product derived from acid-catalyzed dehydration of a 2SOH-3MC 9,10-dihydrodiol). The latter is identical to that of the metabolite contained in peak 13 of Figure 1.

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Fig. 7. Structure and absolute configuration of two pairs of enantiomeric 2-OH-3MC 9,10-dihydrodiols contained in peak 5b of Figure 5 (the major component of peak 5 of Figure 1) and peak 7 of Figure 1. These four 9,10-dihydrodiols are derived from metabolism of a 2-OH-3MC with a (2S)/(2R) enantiomer ratio of - 9 9 : 1 .

respectively. 3MC-2-one 9,10-dihydrodiol is the principal metabolite formed in rat liver microsomal metabolism of 3MC-2-one (25). The metabolite contained in peak 7 of Figure 1 was further purified by normal-phase HPLC and had a UV-visible absorption spectrum identical to that of the 9,10-dihydrodiol contained in peak 5b of Figure 5 (see Figure 6). Its retention time on reversed-phase HPLC (Figure 1) was identical to that of 25-OH-3MC 95,105-dihydrodiol reported earlier (25). Only two diastereomeric 9,10-dihydrodiols, highly enriched either in the 9R, 10#-enantiomer or the 95,105-enantiomer, can be derived from 2-OH-3MC containing a (2S)/(2R) enantiomer ratio of — 99/1 (Figure 7). Hence the structures of the major component in peak 5 and the predominant component in peak 7 of Figure 2042

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1 was established to be 25-OH-3MC 9R, 10/?-dihydrodiol and 25OH-3MC 95,105-dihydrodiol respectively. Based on the AUC of peaks 5 and 7 (~ 93/7) in Figure 1 and those of the two major components separated on normal-phase HPLC (~ 34/66; Figure 5), the relative amount of 25-OH-3MC 9R, lOfl-dihydrodiol and 25-OH-3MC 95,105-dihydrodiol was found to be - 9 / 1 . The metabolite contained in peak 9 of Figure 1 had a retention time and UV—visible absorption and mass spectra identical to those of the most abundant metabolite (3MC-2-one 9,10dihydrodiol) formed in rat liver microsomal metabolism of 3MC-2-one (25). Since 3MC-2-one was one of the major metabolites (peak 18 in Figure 1), the formation of 3MC-2-one 9,10-dihydrodiol was to be expected. The predominant components contained in the minor metabolite peaks 1 and 3 of Figure 1, following purification by normal-phase HPLC, had UV-visible absorption characteristics (not shown) identical to those of 25-OH-3MC 9fl,10/?-dihydrodiol (Figure 6). The metabolite contained in peak 1 had M + at mlz 334 by mass spectral analysis. On the basis of their short retention times, these polar metabolites were tentatively identified as 9,10-dihydrodiols derived from the most abundant metabolite 25OH-3-OHMC (peak 14). The minor metabolite contained in peak 4 of Figure 1 had UV-visible absorption characteristics (not shown) identical to those of 25-OH-3MC 9R, 10/?-dihydrodiol (Figure 6). It had a retention time identical to the 9,10-dihydrodiol derived by incubation of 3MC (cw)-15,2/?-diol (unpublished results). Hence the metabolite contained in peak 4 in Figure 1 was tentatively identified as a 3MC cis-l,2-dio\:trans-9,10-dihydrodiol derived

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Fig. 6. UV-visible absorption (—, methanol) and CD ( , cone. 1.0 A 26g /ml, methanol; *267^A268 = 9.7 milhdegrees) spectra of the metabolite (2S-OH-3MC 9/?,10/J-dihydrodiol) contained in peak 5b of Figure 5. The metabolite contained in peaks 1, 3, 4 and 7 of Figure 1 had UV—visible absorption spectra similar to that of peak 5b of Figure 5.

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250 300 350 WAVELENGTH (nm) Fig. 9. UV—visible absorption spectrum of the metabolite contained in peak 5a of Figure 5 (••••) and CD spectra of 3MC ll/?,12/?-dihydrodiol (—, methanol; #250/A274 = ~ 1 3 - 7 rrullidegrees, (ll/?,12fl)/(llS,12S) = 98/2; data taken from ref. 36), 2S-OH-3-MC lltf,12/?-dihydrodiol (metabolite contained in peak 5a of Figure 5; , cone. 1.0 A273/ml, methanol: *247/A273 = -12.7 millidegrees), and 2S-OH-3MC 115,125-dihydrodiol (metabolite contained in peak 6 of Figure 1; —-, cone. 1.0 A273/ml, methanol, *24S^A273 = 10.4 and *272'A273 = 119 milligrees). UV-visible absorption spectra of metabolite contained in peaks 2 and 6 are closely similar to that of peak 5a of Figure 5.

either from further metabolism of 3MC (c«)-15,2/?-diol (peak 16) or by (cw)-C,-hydroxylation of either 25-OH-3MC 9R,l0Rdihydrodiol (peak 5b) or 25-OH-3MC 95,105-dihydrodiol (peak 7). Since 25-OH-3MC 9/?,10fl-dihydrodiol (major component in peak 5 of Figure 1) was a major metabolite, its metabolic precursor 25-OH-3MC 9,10-epoxide is expected to be formed in the metabolism of 25-OH-3MC. In addition to being hydrated to form diastereomeric 9,10-dihydrodiols, 25-OH-3MC 9,10-epoxide is expected to partially isomerize to form 9-hydroxy25-OH-3MC and 10-hydroxy-25-OH-3MC (see UV-visible absorption spectra in Figure 8). 10-hydroxy-25-OH-3MC is expected to be the major isomerization product of 25-OH3MC 9,10-epoxide (31). The metabolite contained in peak 13 had a UV—visible absorption spectrum similar to that of 4-OHBA (Figure 8B) and different from those of other monohydroxyBAs. Acid-catalyzed dehydration of 25-OH-3MC 9R,IORdihydrodiol (major component in peak 5 of Figure 1) formed both 9-OH-25-OH-3MC (UV-visible absorption spectrum in Figure 8A) and 10-OH-25-OH-3MC (UV-visible absorption spectrum in Figure 8B), which had retention times identical to those of peaks 12 and 13 of Figure 1 respectively. However, the UV-visible absorption spectrum of metabolite peak 12 was not that of 9-OH-3MC and its identity was not established. Since 10-OH-25-OH-3MC (peak 13 of Figure 1) was a minor metabolite, 9-OH-25-OH-3MC was probably present in peak 12, but the amount might have been too small to be detected. Oxidation at the K-region 11,12-double bond The 11,12-double bond of 25-OH-3MC (or 3MC) is the most electron-rich K-region of the molecule. There were three K-

Discussion We have recently reported that the majority (70-90%, depending on the enzyme inducer used to treat the rats) of the hydroxylation products formed at the Q and C2 positions of 3MC by rat liver microsomes is 2-OH-3MC (14). Furthermore, 2-OH-3MC formed in the metabolism of 3MC by liver microsomes from untreated, PB-treated and 3MC-treated rats is highly enriched in the 25-enantiomer (enantiomeric excess 72—96%) (14). The substrate (25-OH-3MC) used in this study was isolated from a mixture of metabolites formed by incubation of 3MC with liver microsomes from 3MC-treated rats. Consistent with the results of an earlier report (14), the 2-OH-3MC formed in the metabolism of 3MC by liver microsomes from 3MC-treated rats was highly enriched in the 25-enantiomer (enantiomeric excess -98%). The absolute configuration of the major 2-OH-3MC enantiomer formed in the metabolism of 3MC has been established in this study by a method independent of that reported earlier (14). The results of this and the earlier study (14) provided 2043

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region 11,12-dihydrodiols detected as metabolites of 25OH-3MC. These were 25-OH-3MC 115,125-dihydrodiol (peak 6 of Figure 1), 25-OH-3MC lU?,12fl-dihydrodiol (peak 5a of Figure 5), and a minor metabolite (peak 2 of Figure 1) tentatively identified as a 11,12-dihydrodiol derived from 25-OH-3-OHMC. Peak 2 was further purified by normal-phase HPLC (Table I). This and peaks 5a (Figure 5) and 6 (Figure 1) had UV-visible absorption spectra (Figure 9) similar to that of an authentic 3MC trans- 11,12-dihydrodiol. Both peaks 5a and 6 had M + at mlz 318 and characteristic fragment ions at mlz 300 (loss of H2O) and 282 (loss of two H2O). There are two sets of CD Cotton bands in the CD spectrum of 3MC 1 \R, 12/?-dihydrodiol (Figure 9), the absolute configuration of which has been established earlier (35). Apart from redshifts by ~ 3 nm, the major CD Cotton effects of peak 5a of Figure 5 in the wavelength region ~ 230-255 nm were the same as those of 3MC llfl,12/?-dihydrodiol (Figure 9). It should be noted that 25-OH-3MC has positive CD Cotton bands centered at ~ 256 nm (Figure 2B). In the CD spectrum of metabolite peak 5a, the positive CD Cotton bands between ~ 255 and 275 nm owing to the additional 5-chiral center at C2 appeared to have cancelled the negative CD Cotton bands between —255 and 285 nm owing to the C n and C,2 chiral centers, resulting in small but positive CD Cotton bands (Figure 9). The CD Cotton effects of metabolite peak 6 in the wavelength region between ~ 230 and 252 nm were opposite in signs to those of the metabolite peak 5a (Figure 9). However, due to positive CD bands contributed by the 5-chiral center at C2, the CD Cotton effects in the wavelength region between - 2 5 5 and 275 nm became considerably larger in magnitude (Figure 9), i.e. the positive CD bands between ~ 255 and 275 nm owing to the additional 5-chiral center at C2 were additive to the positive CD bands between - 2 5 5 and 285 nm owing to the C u and C, 2 chiral centers. Taken together, the above results indicated that the major component contained in peak 5 (which is peak 5a of Figure 5) and the predominant component contained in peak 6 of Figure 1 were 25-OH-3MC llfl,12/?-dihydrodiol and 25-OH-3MC 115,125-dihydrodiol respectively. Based on the AUC of peaks 5 and 6 (~ 10:1) in Figure 1 and those of the two components in peak 5 separated by normal-phase HPLC (5a/5b: -34/66; Figure 5), the relative amount of 25-OH-3MC \\R,\2Rdihydrodiol and 25-OH-3MC 115,125-dihydrodiol was found to be -77/23.

M.Shou and S.K.Yang

Three K-region 11,12-dihydrodiols are found as metabolites of 25-OH-3MC—two diastereomeric 25-OH-3MC 11,12dihydrodiols and one 25-OH-3-OHMC 11,12-dihydrodiol. In principle two diastereomeric trans- 11,12-dihydrodiols can be formed from 25-OH-3-OHMC. However, the amount of the second 25-OH-3-OHMC 11,12-dihydrodiol may be too small to be detectable. The amounts of these K-region dihydrodriols formed are considerably lower than those of 25-OH-3-OHMC or the 9,10-dihydrodiols derived from 25-OH-3MC. 25-OH-3MC 1IR. 12/?-dihydrodiol and 25-OH-3MC 115,125-dihydrodiol are formed in a ratio of -77/23. The 115,12/?-epoxide may be the major enantiomer formed by epoxidation at the K-region of 25OH-3MC by liver microsomes from 3MC-treated rats. In the metabolism of 3MC by liver microsomes from 3MC-treated rats, - 91 % of the 11,12-epoxide formed is the 115,12/J-enantiomer (37). Both the 115,12/?-epoxide and the 1 IR, 125-epoxide of 3MC are regioselectively hydrated by microsomal epoxide hydrolase to form a 3MC 11,12-dihydrodiol with a (HR,l2R)/( 115,125) enantiomer ratio of 85/15 (37). The (trans)- \R,2R-dio\ and (cis)-lS,2R-d\o\ are formed in a ratio of -11:89 in the metabolism of 25-OH-3MC by liver microsomes from 3MC-treated rats. Thus the stereoselective (ris)-C|-hydroxylation is more prevalent than the (trans)C,-hydroxylation (see illustration at the top of Figure 3). In the absence of the C2-hydroxyl group, rat liver microsomal metabolism of 3MC resulted in the preferential formation of 150H-3MC with an enantiomeric excess of 8-30%, depending on the enzyme inducer used to treat the rats (14). It should be pointed out that due to the addition of a chiral center at C1( the hydroxyl group with 25 designation in an enantiomeric 2-OH-3MC is changed to 2R (and vice versa) in the enantiomeric 3MC trans- and cis-1,2-diols (see illustration at the top of Figure 3). Thus the stereoselective hydroxylation at the Q position of 3MC is highly dependent on whether there is a hydroxyl group at C2. The carcinogenic activity of racemic 3MC cis-1,2-diol is weaker than that of racemic 1-OH-3MC (2). However, the 2044

carcinogenic activity of 3MC trans-l,2-diol has not been tested. Preliminary study in our laboratory indicated that a pair of diastereomeric 9,10-dihydrodiols are the predominant rat liver microsomal metabolites of 3MC trans- 1,2-diol (unpublished results), indicating that 3MC trans-\ ,2-diol may also be activated at the 7,8,9,10 benzo-ring. 3MC 9,10-dihydrodiol, a precursor of the bay region 9,10-diol-7,8-epoxide, was reported in one study to be a major rat liver microsomal metabolite of 3MC (11). In other studies, however, 3MC 9,10-dihydrodiol was either not found as a metabolite or a minor metabolite of 3MC (14 and references therein). By analyzing metabolite —DNA binding adducts derived from 3 MC-treated mouse embyro cells, Osborne et al. (13) reported that only a minor proportion (3-8%) was derived from the anr/-9,10-diol-7,8-epoxide of 3MC, some (11 -28%) were derived from the yyn-9,10-diol-7,8-epoxide of 3MC, and most of the adducts were derived from 9,10-diol-7,8-epoxides (anti and syn isomers) of 3-OHMC (37-51%) and 1-0H-3-0HMC (29-35%). In that study, Osborne et al. (13) prepared 3Hlabeled 3-OHMC by incubation of 3H-labeled 3MC with liver microsomes from Aroclor 1254-treated rats. It was reported (13) that 3-OHMC accounted for - 2 0 % of all the metabolites formed (based on the description that 3-OHMC accounted for 2% of all the radioactivity on the TLC plate, using a sample containing 90% of unmetabolized 3MC). Such high yield of 3-OHMC in 3MC metabolism was interesting since prior to that report (13), 3-OHMC had never been definitively identified as a rat liver microsomal metabolite of 3MC (6,7,9). Therefore, the role of 3-OHMC in the metabolic activation of 3MC should be reevaluated. Our earlier finding that 25-OH-3MC is the most abundant metabolite of 3MC (14) and the present finding that 25-OH-3-OHMC and two diastereomeric 25-OH-3MC 9,10-dihydrodiols are major metabolites of 25-OH-3MC are in constrast to the conclusions reported by Osborne et al. (13). Identification of metabolic pathways of 25-OH-3MC in this report and those of 3MC (14) and 3MC-2-one (25) should substantially enhance our understanding of the mechanism of metabolic activation of 3MC. The results reported to date on the metabolism of 3MC and its C, and C2 derivatives indicate that the procarcinogenic 9,10-dihydrodiols can be formed in the metabolism of 3MC, 1-OH-3MC, 1-OH-3-0HMC, 2-OH-3MC, 2-OH-3-OHMC, 3MC-l-one, 3MC-2-one, 3MC trans- 1,2-diol, 3MC cis-l,2-diol and possibly 3-OHMC. Tumorigenicity tests of these 9,10-dihydrodiols and their parent compounds should provide considerable insight into the roles of 7,8,9,10-benzo ring metabolism in the metabolic activation of the potent carcinogen 3MC. Acknowledgements We thank Henri Weems for mass spectral analysis. This work was supported by US Public Health Service grant CA29133. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the 'Guide for the Care and Use of Laboratory Animals', Institute of Animal Resources, National Research Council, DHEW publication no. (NIH) 78-23.

References 1. Sims.P. and Grover.P.L. (1981) Involvement of dihydrodiols and diol-epoxides in the metabolic activation of polycychc hydrocarbons other than benzo[a]pyrene. In Gelboin,H.V. and Ts'o.P.O P. (eds), Polycyclic Hydrocarbons and Cancer. Academic Press, New York, Vol. 3. pp 117—181. 2 Sims,P. (1967) The carcinogenic activities in mice of compounds related to 3-methylcholanthrene. Int. J. Cancer. 2. 505-508.

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an identical conclusion regarding the absolute configuration of the enantiomeric 2-OH-3MC. Because 2-OH-3MC is itself a potent carcinogen and 25OH-3MC is the most abundant metabolite of 3MC, the metabolic activation pathway(s) of 25-OH-3MC may play a role in the metabolic activation of 3MC. In this study, 16 metabolites have been detected as products formed by incubation of 2S-OH-3MC with rat liver microsomes. Among these metabolites, six are 9,10-dihydrodiols which may be further metabolized to form the biologically reactive bay region 9,10-diol-7,8-epoxides. Two diastereomeric 9,10-dihydrodiols are derived from 25-OH-3MC, 2S-OH-3MC 9R, lOfl-dihydrodiol and 25-OH-3MC 95,105dihydrodiol in a ratio of - 9 / 1 . These results suggest that the metabolic precursor 9,10-epoxide has a (95,10/?)/(9/?,105) enantiomer ratio of —9/1, because microsomal epoxide hydrolase-catalyzed reactions occur predominantly at the nonbenzylic positions (32,36). Two 9,10-dihydrodiols are probably derived from 25-OH-3-OHMC since the latter was the most abundant metabolite of 25-OH-3MC. 25-OH-3-OHMC is a newly recognized metabolite of 25-OH-3MC and its carcinogenic activity is as yet unknown. The fifth 9,10-dihydrodiol is derived from 3MC-2-one, which is a potent carcinogen (2,3,5) and one of the major metabolites of 25-OH-3MC. The sixth 9,10-dihydrodiol may be derived either from further metabolism of 3MC (cis)-\S,2R-dio\ or by (cw)-Cphydroxylation of 25OH-3MC 9fl,10K-dihydrodiol or 25-OH-3MC 95, 105-dihydrodiol.

Metabolism of 2S-hydroxy-3-methylcholanthrene 25. Shou.M. and Yang.S.K. (1990) 9,10-Dihydroxy-9,10-dihydro-3-methylcholanthrene-2-one: a principle metabolite of the potent carcinogen 3-methylcholanthrene-2-one by rat liver microsomes. Carcinogenesis, 11, 689-695. 26. Gardiner.E.M. and Stoming.T.A. (1984) The metabolism of 1-hydroxy- and 2-hydroxy-3-methylcholanthrene by liver microsomes: effect of enzyme inducing agents. Cancer Lett., 24, 103 — 110. 27. Yang.S.K., Prasanna.P., Weems.H.B., Jacobs.M.M. and Fu.P.P. (1990) Metabolism of the potent carcinogen 3-methylcholanthrylene by rat liver microsomes. Carcinogenesis, 11, 1195 — 1201. 28. Alvares,A.P., Schilling,G., Garbut.A. and Kuntzman.R. (1970) Studies on the hydroxylation of 3,4-benzpyrene by hepatic microsomes. Effect of albumin on the rate of hydroxylation of 3,4-benzpyrene. Biochem, Pharmacol., 19, 1449-1455. 29. Lowry,O.H., Rosebrough,N.J., Farr.A.L. and Randall.R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265—275. 30. Conney.A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycylic aromatic hydrocarbons. G.H.A.Clowes Memorial Lecture. Cancer Res., 42, 4875-4917. 31. Fu.P.P., Harvey,R.G. and Beland.F.A. (1978) Molecular oribital theoretical prediction of the isomeric products formed from reactions of arene oxides and related metabolites of polycylcic aromatic hydrocarbons. Tetrahedron, 34, 857-866. 32. Yang,S.K., McCourt.D.W., Leutz.J.C. and Gelboin.H.V. (1977) Benzo[a]pyrene diol epoxides: mechanism of enzymatic formation and optically active intermediates. Science, 196, 1199-1201. 33. Yang.S.K., Chou,M.W. and Fu.P.P. (1980) Metabolic and structural requirements for the carcinogenic potencies of unsubstituted and methylsubstituted polycyclic aromatic hydrocarbons. In Pullman,B., Ts'o,P.O.P. and Gelboin,H. (eds), Carcinogenesis: Fundamental Mechanisms and Environ-

mental Effects. D.Reidel, Dordrecht, Holland, pp. 143-156. 34. Jacobs,S.A., Cortez.C. and Harvey,R.G. (1983) Synthesis of potential proximate and ultimate carcinogenic metabolites of 3-methylcholanthrene. Carcinogenesis, 4, 519-522. 35. Yang.S.K., Mushtaq,M., Weems.H.B. and Fu,P.P. (1986) Chiral recognition mechanisms in the direct resolution of diol enantiomers of some polycyclic aromatic hydrocarbons by high performance liquid chromatography with chiral stationary phases. J. Liquid Chromatogr., 9, 473—492. 36. Lu.A.Y.H. and Miwa.G.T. (1980) Molecular properties and biological functions of microsomal epoxide hydrase. Annu. Rev. Pharmacol. Toxicol., 20, 513-531. 37. Yang.S.K., Weems.H.B. and Mushtaq.M. (1990) Chiral stationary phase HPLC separation of enantiomeric oxygenated derivatives of polycyclic aromatic hydrocarbons and application to metabolism studies In Frank.H., Holmstedt.B. and Testa.B. (eds), Chirality and Biological Activity. Alan R.Liss, New York, pp. 81-109. Received on June 21, 1990, revised on August 9, 1990; accepted on August 20, 1990.

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3.Cavalieri,E.. Roth.R., Althoff,J., Grandjean,C, Patil,K., Marsh,S. and Mclaughlin,D. (1978) Carcinogenicity and metabolic profiles of 3-methylcholamhrene oxygenated derivative at the 1 and 2 positions. Chem.Biol. Interactions, 22, 6 9 - 8 1 . 4. Wood,A.W., Chang.R.L., Levin.W., Thomas.P.E., Ryan.D., Stoming.T.A., Thakker.D.R., Jerina,D.M. and Conney.A.H. (1978) Metabolic activation of 3-methylcholanthrene and its metabolites to products mutagenic to bacterial and mammalian cells. Cancer Res. 38, 3398-3404. 5 Levin,W., Buening.M.K., Wood,A.W., Chang.R.L., Thakker.D.R., Jerina,D.M. and Conney,A.H. (1979) Tumorigenic activity of 3-methylcholanthrene metabolites on mouse skin and in newborn mice. Cancer Res., 39, 3549-3553. 6 Sims,P. (1966) The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates. Biochem. J., 98, 215-228. 7. Eastman.A and Bresnick,E. (1979) Metabolism and DNA binding of 3-methylcholanthrene. Cancer Res., 39, 4316-4321. 8. Tierney,B , Bresnick.E., Sims,P. and Grover.P.L. (1979) Microsomal and nuclear metabolism of 3-methylcholanthrene. Biochem. Pharmacol, 28, 2607-2610. 9. Thakker,D.R., Levin,W., Stoming.T.A., Conney.A.H. and Jerina.D.M. (1978) Metabolism of 3-methylcholanthrene by rat liver microsomes and a highly purified monooxygenase system with and without epoxide hydrase. In Jones,P.W. and Freudenthal,R.I. (eds), Carcinogenesis, Vol. 3: Polynuclear Aromatic Hydrocarbons. Raven Press, New York, pp. 253—264. 10 Stoming,T.A., Bornstein.W. and Bresnick,E. (1977) The metabolism of 3-methylcholanthrene by rat liver microsomes—a reinvestigation. Biochem. Biophys. Res. Commun., 79, 461-469. ll.Tierney.B., Hewer.A., Rattle.A., Grover.P.L. and Sims.P. (1978) The formation of dihydrodiols by chemcial or enzymatic oxidation of 3-methylcholanthrene. Chem.-Biol. Interactions, 23, 121-135. 12. Thakker.D.R., Levin,W., Wood,A.W., Conney.A.H., Stoming.T.A. and Jerina.D.M. (1978) Metabolic formation of l,9,10-trihydroxy-9,10-dihydro3-methylcholanthrene: a potential proximate carcinogen from 3-methylcholanthrene J. Am. Chem. Soc, 100, 645-647. 13. Osborne.M.R , Brookes,P., Lee.H. and Harvey,R.G. (1986) The reaction of a 3-methylcholanthrene diol epoxide with DNA in relation to the binding of 3-methylcholanthrene to the DNA of mammalian cells. Carcinogenesis, 7, 1345-1350. 14. Shou.M. and Yang.S.K. (1990) 1- and 2-hydroxy-3-methylcholanthrene: regioselective and stereoselective formations in the metabolism of 3-methylcholanthrene and enantioselective disposition in rat liver microsomes. Carcinogenesis, 11, 933-940. 15. Chouroulinkov.L, Gentil.A., Tiemey.B., Grover.P L. and Sims.P. (1979) The initiation of tumours on mouse skin by dihydrodiols derived from 7,12-dimethylbenz[a]anthracene and 3-methylcholanthrene. Int. J. Cancer, 24, 455-460. 16. Pal,K., Grover.P.L. and Sims.P. (1979) The induction of sister chromatid exchanges by dihydrodiols derived from 7,12-dimethylbenz[a]anthracene and 3-methylcholanthrene. Cancer Lett., 7, 4 5 - 4 9 . 17. Malaveille.C, Bartsch.H., Marquardt.H., Baker,S., Tiemey.B., Hewer.A., Grover.P.L. and Sims.P. (1978) Metabolic activation of 3-methylcholanthrene: mutagenic and transforming activities of the 9,10-dihydrodiol. Biochem. Biophys. Res. Commun., 85, 1568-1574. 18. DiGiovanni,J., Diamond.L., Prichett.W.P , Fisher.E.P. and Harvey,R.G. (1985) Tumor-initiating activity of the 9,10-dihydrodiol- and 9,10-dihydrodiol7,8-epoxide of 3-methylcholanthrene in SENCAR mice. Cancer Lett., 28, 223-228. 19 King.H W.S., Osborne.M.R. and Brookes.P. (1977) The metabolism and DNA binding of 3-methylcholanthrene. Int. J. Cancer, 20, 564-571. 20. King.H.W.S., Osborne.M.R. and Brookes.P. (1978) The identification of 3-methylcholanthrene-9,10-dihydrodiol as an intermediate in the binding 3-methylcholanthrene to DNA in cells in culture. Chem.-Biol. Interactions, 20, 367-371. 21. Shou.M. and Yang.S.K. (1990) Enantioselective aliphatic hydroxylation of racemic 1 -hydroxy-3-methylcholanthrene by rat liver microsomes. Chirality, 2, 141-149. 22. Vigny.P., Duquesne.M., Coulomb.C, Tierney.B., Grover.P.L. and Sims.P. (1977) Fluorescence spectral studies on the metabolic activation of 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene in mouse skin. FEBSLett., 82, 278-282. 23. Phillips,D.H., Grover.P.L. and Sims.P. (1978) The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains. Int. J. Cancer, 22, 487-494. 24. Copper.C.S., Vigny.P., Kindts.M., Grover.P.L. and Sims.P. (1980) Metabolic activation of 3-methylcholanthrene in mouse skin: fluorescence spectral evidence indicates the involvement of diol-epoxides formed in the 7,8,9,10-ring; Carcinogenesis, 1, 855-860.

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Metabolism of 2S-hydroxy-3-methylcholanthrene by rat liver microsomes.

Products formed in the metabolism of 2S-hydroxy-3-methylcholanthrene (2S-OH-3MC) by liver microsomes prepared from phenobarbital-treated rats were iso...
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