Formation of Glucuronic Acid Conjugates of 7,12-Dimethylbenz(A)- Anthracene Phenols in 7,12-Dimethylbenz(A)Anthracene-Treated Hamster Embryo Cell Cultures1

[CANCER RESEARCH 38, 3432-3437, October 1978] 0008-5472/78/0038-OOOOS02.00 Formation of Glucuronic Acid Conjugates of 7,12-Dimethylbenz(a)- anthracene Phenols in 7,12-Dimethylbenz(a)anthracene-treated Hamster Embryo Cell Cultures1

William M. Baird,2 Ruth Chemerys, Ching Jer Chern, and Leila Diamond

The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104

ABSTRACT carcinogenic hydrocarbon BP3 in hamster embryo cells consisted of glucuronic acid conjugates of 3-hydroxy- Secondary cultures of hamster embryo cells exposed to benzo(a)pyrene and 9-hydroxybenzo(a)pyrene; Cohen and 0.5 nmol [G-3H]7,12-dimethylbenz(a)anthracene (DMBA) Moore (7) found that BP phenol-glucuronic acid conjugates per ml medium metabolized more than 90% of the DMBA are the major water-soluble metabolites of BP in short-term within 48 hr. Samples of medium were extracted with organ cultures of hamster lung. chloroform, methanol, and water. The chloroform phases Some weakly carcinogenic polycyclic aromatic hydrocar contained about one-third of the DMBA metabolites; the bons such as benzo(a)anthracene and chrysene become major chloroform-extractable metabolite was 8,9-dihydro- potent carcinogens if they are methyl-substituted in certain 8,9-dihydroxy-7,12-dimethylbenz(a)anthracene. /3-Glucu- positions (reviewed in Ref. 14). When the metabolism of the ronidase treatment of the aqueous methanol-soluble me potent carcinogen DMBA was investigated in microsomal tabolites converted almost one-half of them to chloroform- incubation mixtures, it was found that ring oxidation prod soluble metabolites, of which more than 80% were identi ucts similar to those of unsubstituted hydrocarbons and fied as phenolic derivatives of DMBA. Similar metabolite methyl group oxidation products were both formed (e.g., profiles were obtained by treating the medium with ÃŸ- see Refs. 4, 13, 19, 28, 30, 39, 41, and 45). Both organic glucuronidase before chloroform extraction. solvent- and water-soluble metabolites are formed from Separation of the methyl group-hydroxylated deriva DMBA in early-passage cultures of mouse and hamster tives of DMBA from the phenolic derivatives was accom embryo cells (12, 20, 27, 38), and the major organic solvent- plished by high-pressure liquid chromatography. Small soluble metabolites have been identified as DMBA-8,9-diol amounts of hydroxymethyl derivatives were detected only and 7-OHMe-12-MeBA (20, 38). The water-soluble metabo in the chloroform-extractable material, whereas DMBA lites had not been identified. phenols were the major component of the /J-glucuroni- This study shows that glucuronic acid conjugates of dase-released material. These results indicate that the DMBA phenols represent a major portion of the water- major pathway of DMBA metabolism in hamster embryo soluble DMBA metabolites formed in hamster embryo cells cells is oxidation of the aromatic rings and not oxidation and that most of the DMBA metabolites formed result from of the methyl groups. oxidation of the aromatic ring rather than of the methyl groups. INTRODUCTION MATERIALS AND METHODS Determination of the metabolic pathways of polycyclic aromatic hydrocarbons is essential to the understanding of Chemicals and Radiochemicals. DMBA was purchased how some of these chemicals induce transformation, mu from Schuchardt GmBH, West Germany. 7-OHMe-12-MeBA, tation and cytotoxicity in cells in culture and cancer in 12-hydroxymethyl-7-methylbenz(a)anthracene, 7,12-dihy- animals (e.g., see Refs. 11, 14, 18, 23, 24, 26, and 29). Cells droxymethylbenz(a)anthracene, and c/s-5,6-dihydro-5,6- in culture and tissue homogenates have been useful in dihydroxy-7,12-dimethylbenz(a)anthracene were the gener these determinations because hydrocarbon oxidation prod ous gift of Dr. Anthony Dipple of the Frederick Cancer ucts such as dihydrodiols. phenols, quiÃ±ones, and even Research Center, Frederick, Md. Dr. Joseph Tomaszewski sulfate conjugates can be analyzed by chromatography of the Frederick Cancer Research Center kindly provided after extraction of these metabolites into organic solvents the samples of 3-OHDMBA, 4-OHDMBA, and [3H]DMBA-8,9- (e.g., see Refs. 6, 8. 11, 15, 23, 36. and 40). However, a diol. large portion of the hydrocarbon metabolites produced by [G-3H]DMBA (specific radioactivity, 12 Ci/mmol) and [12- many cells remains in the aqueous phase after such extrac 14C]DMBA (specific radioactivity, 21 mCi/mmol) were pur tion (10, 15, 25). Recently, we (1) showed that a major chased from Amersham/Searle Corp., Arlington Heights, portion of the water-soluble metabolites formed from the

1 Supported in part by Grants CA 19948, CA 08936. CA 16685, and CA 10815 from the National Cancer Institute, Department of Health, Education 3 The abbreviations used are: BP, benzo(a)pyrene; DMBA, 7,12-dimethyl- and Welfare. benz(a(anthracene; DMBA-8,9-diol, 8,9-dihydro-8,9-dihydroxy-7,12-dimeth- 2 To whom requests for reprints should be addressed, at the Wistar ylbenz(a)anthracene; 7-OHMe-12-MeBA. 7-hydroxymethyl-12-methylbenz- Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. (a)anthracene; 3-OHDMBA, 3-hydroxy-7,12-dimethylbenz(a)anthracene; 4- 19104. OHDMBA, 4-hydroxy-7,12-dimethylbenz(a)anthracene; TLC, thin-layer chro Received April 10, 1978; accepted July 18. 1978. matography; HPLC. high-pressure liquid chromatography.

3432 CANCER RESEARCH VOL. 38

Preparation and Treatment of Cells. First- or second- hamster embryo cell cultures exposed for 48 hr to 0.5 nmol passage Syrian hamster embryo cells were prepared and [3H]DMBA per ml medium were analyzed first by TLC. The grown in 75-sq cm plastic flasks or 490-sq cm plastic roller chloroform-soluble metabolites and unchanged DMBA bottles as described previously (1, 2). Three to 4 days after (40% of the radioactivity originally present in a sample of plating, one-tenth volume of medium containing [3H]DMBA medium) were extracted as described in "Materials and Methods." The aqueous methanol-soluble material was in 1% dimethyl sulfoxide was added to each flask to give a final concentration of 0.5 nmol [3H]DMBA per ml medium. then treated with ,8-glucuronidase to cleave DMBA-glucu- Roller bottles were treated with 0.01 volume of medium to ronic acid conjugates, and the metabolites released by this give a final concentration of 1.2 nmol [14C]DMBA per ml enzyme were extracted into chloroform. A control sample medium. of aqueous methanol-soluble material was incubated for a Isolation of DMBA Oxidation Products. At the times similar period without enzyme and then extracted with specified samples of medium were removed from the cul chloroform. The 3 chloroform phases were then chromato ture vessels and primary oxidation products were extracted graphed on a silica gel TLC sheet developed in into the chloroform phase of a mixture of chloroform, benzene:ethanol (19:1) (Chart 1). methanol, and water (2). The aqueous methanol phase was The chloroform-extractable material from the original evaporated to dryness, dissolved in 0.5 ml of 0.2 M sodium medium (Chart 1a) contained, from left to right: an uniden acetate buffer (pH 4.5), and incubated with 2000 Fishman tified peak at the origin; a large peak of RK identical with units of 0-glucuronidase from bovine liver (type B-10; that of DMBA-8,9-diol, the compound previously reported Sigma Chemical Co., St. Louis, Mo.) at 37Â°for 1 hr as to be a major metabolite of DMBA in these cells (20); a peak described previously (1). The primary oxidation products of of R,, similar to the marker of 7-OHMe-12-MeBA; and un DMBA released by this treatment were then extracted into changed DMBA. The chloroform extract of the control chloroform by the procedure described previously. The sample of aqueous methanol-soluble materials contained amount of radioactivity in each phase was determined by only 1 peak at the origin (Chart 10, dotted line), whereas placing 0.1-ml samples in 7-ml polyethylene vials (Amer- the ÃŸ-glucuronidase-treated sample (Chart 1b, solid line) sham/Searle Corp.), adding 4 ml scintillant [TT-21 (York- contained a large peak of material with a RK similar to that town Research, Hackensack, N. J.) or Instagel (Packard of 7-OHMe-12-MeBA and a small peak with a RK similar to Instrument Co., Downers Grove, III.)] and counting in Pack that of 12-hydroxymethyl-7-methylbenz(a)anthracene. Sim ard Tri-Carb liquid scintillation counters. Counting efficien ilar results were obtained when an identical chromatogram cies were determined by the use of automatic external was developed in benzene. standard ratios. Markers of 3-OHDMBA and 4-OHDMBA were not com TLC. The chloroform phases were dried over sodium pletely resolved from the 7-OHMe-12-MeBA marker in either sulfate, evaporated to low volumes under nitrogen, and solvent system; thus the identity of the material associated chromatographed on silica gel chromagram sheets (No. with the radioactivity in that area of the chromatogram 13179 without fluorescent indicator; Eastman Kodak Co., could not be established by TLC. Therefore, chloroform Rochester, N. Y.) developed in benzeneiethanol (19:1) or extracts of media samples from a similar experiment were benzene (4). Markers of the DMBA metabolites described in chromatographed by HPLC as described in "Materials and the charts were chromatographed with all samples and Methods". The UV absorbance scan of a chromatogram of visualized by UV. Chromatograms were then cut into 1-cm DMBA markers (Chart 2, fop) shows the DMBA phenols to squares or 0.5- x 1-cm strips and placed in counting vials, be clearly resolved from the hydroxymethyl derivatives. The eluted with 1 ml methanol for at least 6 hr, and counted as radioactivity profile of a chromatogram of a chloroform described previously. extract (55% of the radioactivity originally present in the HPLC. HPLC of DMBA metabolites was by a modification sample of medium) of a sample of medium from [3H]DMBA- of the method of Yang and Dower (45). Chloroform phases treated cells revealed some early-eluting material, a large from extractions done as described previously were dried peak of the same retention time as that of DMBA-8,9-diol over sodium sulfate, evaporated to dryness, and dissolved (19% of the radioactivity originally present in the sample of in methanol. After filtration through a fluoropore filter (0.5- medium), a very small amount of monohydroxymethyl deriv /Â¿mpore; Millipore Corp., Bedford, Mass.), the solution was atives, some DMBA phenols, and unmetabolized DMBA evaporated to approximately 40 /Â¿I,and 20 /nl were chro (19% of the radioactivity originally present in the sample of matographed at room temperature on a 4.6-mm x 25-cm medium) (Chart 2a). The major chloroform-soluble metab Spherisorb octadecyl silane (5 ^m) reverse-phase column olite was subsequently shown to be DMBA-8,9-diol by on a Model 312 HPLC (Altex Scientific Inc., Berkeley, isolating the chloroform-soluble metabolites from Calif.), with a 50-min methanol:water linear gradient from [14C]DMBA-treated cells and chromatographing them with 3:2 to 9:1 followed by 5 min at 9:1 with a flow rate of 1 ml/ a marker of [3H]DMBA-8,9-diol in the Chromatographie min. The absorbance of UV markers was monitored contin systems described in Charts 1 and 2. The major [14C]DMBA uously at 254 nm. One hundred ten fractions (0.5 min each) metabolite cochromatographed with [3H]DMBA-8,9-diol in were collected in scintillation vials and counted as de both systems. The aqueous methanol-soluble metabolites scribed previously. were treated with /3-glucuronidase and extracted with chlo roform; the chloroform-soluble material (35% of the radio RESULTS activity originally present in the sample of medium) was chromatographed by HPLC (Chart 2b). More than 75% of The hydrocarbon metabolites in the medium of secondary the /3-glucuronidase-released material chromatographed

OCTOBER 1978 3433

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5 10 15 l 5,000- DISTANCE (cm) Chart 1. TLC of the primary [3H]DMBA metabolites in the medium of [3H]DMBA-treated cell cultures and those primary metabolites released after ,A treatment with ,i-glucurorndase Secondary hamster embryo cells were exposed to 0.5 nmol [3H]DMBA per ml medium for 48 hr, and the medium 10 20 30 40 50 was analyzed as described below. The chloroform extracts were chromato- MINUTES graphed on a silica TLC sheet developed in benzene.ethanol (19:1). and the Chart 2 HPLC of /3-glucuronidase-released ['HJDMBA metabolites. Cells radioactivity in each 0.5-cm fraction was counted as described in "Materials were treated and samples were prepared as described for Chart 1 ; samples and Methods." Top, locations of DMBA metabolite markers chromato- were chromatographed as described in "Materialsand Methods."a, Original graphed with these samples, a, chloroform phase (containing 40% of the radioactivity originally present in a 0.4-ml sample of medium) after chloro- chloroform extract of medium (containing 55% of the radioactivity originally present in a 0.4-ml sample of medium); b, chloroform phase prepared by form-methanol extraction of the original medium; b, chloroform phases from extraction of the aqueous methanol phase obtained in a after incubation chloroform methanol extraction of the aqueous methanol phases described with ÃŸ-glucuronidase (containing 35% of the radioactivity originally present in a after incubation with ;t-glucuromdase (containing 43% of the radioactivity in a 0.4-ml sample of medium). The UV absorbance trace of the DMBA originally present in a 0.4-ml sample of medium) ( ) or incubation without metabolite markers is shown at the fop. Abbreviations are as in Chart 1. enzyme (containing 15% of the radioactivity originally present in a 0.4-ml sample of medium) ( ). Solid arrow, origin .broken arrow, solvent front. DMBA-5,6-diol. c/s-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene; The phenolic nature of the ÃŸ-glucuronidase-released 7,12-DiOHMeBA, 7,12-dihydroxymethylbenz(a)anthracene; 12-OHMe-7- MeBA, 12-hydroxymethyl-7-methylbenz(a (anthracene. DMBA metabolites was confirmed by another method (10). A sample of the water-soluble DMBA metabolites was treated with ÃŸ-glucuronidase and then was extracted with with a marker of 3-OHDMBA, and 20% chromatographed 3.5 volumes of hexane:acetone (6:1). The hexaneiacetone with a marker of 4-OHDMBA; there was also an unidentified phase (62% of the material) was divided in 2; one-half was early-eluting peak that was also present in a chromatogram extracted with an equal volume of water, and the other half of a chloroform extract (12% of the radioactivity originally was extracted with 1 M sodium hydroxide. More than 82% present in a sample of medium) of a control incubation of the hexane-soluble, 0-glucuronidase-released metabo (without enzyme) of the aqueous methanol-soluble metab lites was extracted into the sodium hydroxide, but less than olites. 2% was extracted into the water.

3434 CANCER RESEARCH VOL. 38

For determination of whether the same metabolites could from cells exposed to [14C]DMBA released mainly DMBA be released by treating the medium with ÃŸ-glucuronidase phenols. before extraction and whether DMBA metabolites escaped The nature of the /y-glucuronidase-released metabolites detection because of selective loss of tritium, the following of cells exposed to [3H]DMBA for various lengths of time procedures were done. Samples of medium from hamster was examined by TLC (Table 1). At each time point Â¡i- embryo cells exposed to 1.2 nmol [12-14C]DMBA per ml for glucuronidase treatment released more than 25% of the 48 hr were incubated with and without rt-glucuronidase, total DMBA metabolites as DMBA phenols. and after extraction with chloroform:methanol the chloro The water-soluble metabolites were further characterized form phases were analyzed by HPLC (Chart 3). Both control by chromatography on 1-mm-thick silica gel plates de and ÃŸ-glucuronidase-treated samples contained a peak of veloped in ethyl acetate:methanol:water:formic acid DMBA, a large peak of DMBA-8,9-diol, a peak of material at (100:25:20:1) (Chart 4). The control sample contained 2 the origin, and a small peak of unidentified material at 19 major product peaks, the larger of which was absent in a min. However, the ÃŸ-glucuronidase-treated sample also sample treated with ÃŸ-glucuronidase and extracted with contained a large peak of retention time identical with that chloroform prior to chromatography. A similar sample in of 3-OHDMBA and a small peak of retention time identical cubated with /i-glucuronidase in the presence of 33 HIM with that of 4-OHDMBA. Thus, as in the tritiated sample saccaro-1,4-lactone (an inhibitor of /3-glucuronidase) was described in Chart 2, ÃŸ-glucuronidase treatment of medium identical with the control. Thus the larger peak in these samples is composed of glucuronic acid conjugates of DMBA derivatives.

DISCUSSION Glucuronic acid conjugates of BP derivatives are formed in rodent tissues, in hamster embryo cell cultures, and in microsomal incubation mixtures that contain appropriate substrates (1, 7, 16, 22, 34, 35). As with BP almost all the ÃŸ- glucuronidase-released DMBA metabolites in hamster em bryo cells were phenols. The organic solvent extracts of the culture medium contained other DMBA oxidation products; the major one was DMBA-8,9-diol as reported in previous studies of DMBA metabolism in hamster cells (20). Appar ently, however, these DMBA derivatives are not conjugated I200- to glucuronic acid. Hamster cells are able to form glucu ronic acid. Hamster cells are able to form glucuronic acid conjugates of BP dihydrodiols (1, 7), but in microsomal IOOO- incubation mixtures BP diols were a much poorer substrate for UDP glucuronosyltransferase (EC 2.4.1.17) than were

S 800- BP phenols (34, 35). tr Table 1 bJ CL Metabolism of DMBA by hamster embryo cells 600 At the times specified samples of medium were removed from four 75-sq cm flasks of second-passage hamster embryo cells treated with 0.5 nmol [3H]DMBA per ml medium (15.0 nmol/flask) 400- on the fourth day after seeding. 0-Glucuronidase-treated samples were incubated with 2000 units of /3-glucuronidase for 2 hr, whereas samples of untreated medium were similarly incubated 200- without enzyme. All samples were extracted and chromatographed as described for Chart 1. Each value represents the mean of the 4 samples Â±S.D. More than 97% of the radioactivity was recovered I I as DMBA at 0 hr, and after 48 hr of incubation without cells more than 95% was recovered as DMBA. IO 20 30 40 50 DMBA metabolites (nmol/flask) at: MINUTES Chart 3. HPLC of ["CJDMBA metabolites released by /i-glucuronidase 6hr 24 hr 48 hr treatment of medium from ["C]DMBA-treated cells. Chloroform extracts of 0.4-ml samples of medium from roller bottles of secondary hamster embryo Total DMBA metabo cells treated with 1.2 nmol ["C]DMBA per ml for 48 hr were prepared, lites chromatographed, and counted as described in Chart 2. Top, locations of ÃŸ-Glucuronidase- 4.8 Â±0.6 12.1 Â±1.0 13.2 Â±0.3 DMBA metabolite markers chromatographed under these conditions. O. treated medium chloroform phase (containing 87% of the radioactivity originally present in a Untreated medium 4.7 Â±0.2 11.7 Â±0.8 13.3 Â±0.5 0.4-ml sample of medium) of a chloroform:methanol extract of a sample of medium diluted with buffer and treated with 0-glucuronidase; â€¢¿.chloroform DMBAphenols/S-Glucuronidase-treated phase (containing 60% of the radioactivity originally present in a 0.4-ml sample of medium) of a chloroform:methanol extract of a sample of medium diluted with buffer and incubated as described above but without enzyme. mediumUntreated Abbreviations are as in Chart 1. medium1.80.94.31.44.71.3

~~OCTOBER 1978 3435~~

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. W. M. Baird et al. 4-hydroxy-7-hydroxy met hy1-12-methylbenz(a)ant tiracene 10,000 (unpublished results). The biological significance of the DMBA phenol glucuro- nides has not been established. Conjugation may serve to 8,000 detoxify those phenolic hydrocarbon derivatives that are cytotoxic (21, 32) by forming water-soluble products that can be removed from the cells. However, one phenol of BP is 6,000 a potent carcinogen for mouse skin (43), and enzymatic hydrolysis of a BP phenol glucuronide results in the forma tion of a DNA-binding BP derivative (31). Thus it is conceiv

3 4,000 O able that a DMBA phenol glucuronide formed in one tissue could be transported to another tissue and be cleaved to release a carcinogenic DMBA phenol. 2,000 The failure to detect large amounts of free 7-OHMe-12- MeBA in these cells is consistent with recent evidence that suggests that DMBA is bound to DNA in cell cultures (3, 33) i 5 I0 15 and in mouse skin (42) through a diol-epoxide derivative of I DISTANCE (cm) the parent hydrocarbon rather than through a metabolite of Chart 4. TLC of aqueous methanol-soluble metabolites of [3H]DMBA from 7-OHMe-12-MeBA, a compound proposed to be a proximate hamster embryo cells before and after /j-glucuronidase treatment. The aqueous methanol phases were prepared from 0.3-ml samples of medium as carcinogen of DMBA in some animal systems (e.g., see described in Chart 1 and were chromatographed on a Silica Gel G plate Refs. Sand 17). developed in ethyl acetate:methanol:water:formic acid (100:25:20:1). , Aqueous methanol phase (containing 47% of the radioactivity originally present in a 0.3-ml sample of medium); â€”¿,aqueous methanol phase ACKNOWLEDGMENTS (containing 21% of the radioactivity originally present in a 0.3-ml sample of medium) obtained after the aqueous methanol phase above was treated with The authors wish to thank Dr. Anthony Dipple and Dr. Joseph Tomasz /i-glucuronidase and reextracted with chloroform:methanol: , ewski (Frederick Cancer Research Center. Frederick, Md.) for providing the aqueous methanol phase (containing 43% of the radioactivity originally DMBA metabolite markers and for many helpful discussions during the present in a 0.3-ml sample of medium) obtained after incubation with ÃŸ- course of this work. glucuronidase as described above but in the presence of 33 mM saccharo- 1.4-lactone. Solid arrow, origin; broken arrow, solvent front. The following DMBA metabolite markers migrated with the solvent front: DMBA, 7-OHMe- REFERENCES 12-MeBa. 12-hydroxymethyl-17-methylbenz(a)anthracene, c/s-5,6-dihydro- 5,6-dihydrozy-7.12-dimethylbenz(a(anthracene, and 7,12-dihydroxymethyl- 1. Baird, W. M., Chern, C. J.. and Diamond. L. Formation of benz(a)anthracene. Benzo(a)pyrene-Glucuronic Acid Conjugates in Hamster Embryo Cell Cultures. Cancer Res., 37: 3190-3197, 1977. 2. Baird. W M., and Diamond. L. Effect of 7.8-Benzoflavone on the Formation of Benzo[a)pyrene-DNA-bound Products in Hamster Embryo Although the ÃŸ-glucuronidase-released DMBA derivatives Cells. Chem.-Biol. Interactions, 13: 67-75. 1976. cochromatographed with 3-OHDMBA and 4-OHDMBA by 3. Baird, W. M., and Dipple, A. Photosensitivity of DNA-bound 7,12-Dimeth- ylbenz(a)anthracene. Intern. J. Cancer. 20: 427-431. 1977. HPLC and in 2 TLC systems, they can be identified only as 4. Boyland. E., and Sims. P. Metabolism of Polycyclic Compounds: The DMBA phenols because other DMBA phenols may cochro- Metabolism of 7.12-Dimethylbenz(a)anthracene by Rat-Liver Homoge- matograph with them (J. Tomaszewski, personal communi nates. Biochem. J.. 95: 780-787, 1965. 5. Boyland, E., Sims, P., and Huggins, C. Induction of Adrenal Damage cation). When BP phenols were chromatographed by a and Cancer with Metabolites of 7,12-Dimethylbenz(a)anthracene. Na similar HPLC procedure, only 2 peaks were observed, but ture, 207. 816-817, 1965. 6. Cohen. G. M., Haws, S. M., Moore, B. P., and Bridges, J. W. these were shown to contain a number of BP phenols (9, Benzo(a)pyrene-3-yl Hydrogen Sulphate, a Major Ethyl Acetate-extract- 37, 44). Thus exact identification of the DMBA phenols able Metabolite of Benzo(a)pyrene in Human. Hamster, and Rat Lung requires further analysis. Cultures. Biochem. Pharmacol., 25: 2561-2570, 1976. 7. Cohen. G. M., and Moore, B. P. The Metabolism of Benzo(a)pyrene, 7,8- The present results indicate that, similar to BP, DMBA is Dihydro-7,8-dihydroxybenzo(a)pyrene and 9,10-Dihydro-9,10-dihydrox- oxidized in hamster cells mainly on the aromatic ring and ybenzo(a)pyrene by Short-Term Organ Cultures of Hamster Lung. Bio not on the methyl groups. The possibility cannot be ex chem. Pharmacol.. 26: 1481-1487. 1977. 8. Cohen, G. M., Moore. B. P., and Bridges, J. W. Organic Solvent Soluble cluded that, once a methyl group on the molecule is Sulphate Ester Conjugates of Monohydroxybenzo(a)pyrenes. Biochem. hydroxylated, it is rapidly oxidized further to products such Pharmacol.,26: 551-553, 1977. 9. Croy, R. G., Selkirk. J. K.. Harvey, R. G.. Engel, J. F., and Gelboin, H. V. as carboxylic acids. This seems unlikely, for no substantial Separation of Ten Benzo(a)pyrene Phenols by Recycle High-Pressure amounts of either the monohydroxymethyl derivatives or Liquid Chromatography and Identification of Four Phenols as Metabo the dihydroxymethyl derivative have been detected. The lites. Biochem. Pharmacol..25. 227-230. 1976. 10. Diamond, L. Metabolism of Polycyclic Hydrocarbons in Mammalian Cell further possibility that hydroxymethyl derivatives are rapidly Cultures. Intern. J. Cancer, 8: 451-462, 1971. conjugated to some substrate other than glucuronic acid 11. Diamond, L., and Baird, W. M. Chemical Carcinogenesis in Vitro. In: G. H. Rothblat and V.J. Cristofalo (eds.). Growth, Nutrition and Metabolism also seems unlikely, for these cells can form glucuronic of Cells in Culture, Vol. 3. pp. 421-470. New York: Academic Press, Inc.. acid conjugates of 7-OHMe-12-MeBA. We found that, when 1977. water-soluble metabolites from cells exposed to 7-OHMe- 12. Diamond, L., Sardet, C., and Rothblat, G. H. The Metabolism of 7,12- 12-MeBA were treated with 0-glucuronidase, the primary Dimethylbenz(a)anthracene in Cell Cultures. Intern. J. Cancer, 3: 838- 849. 1968. metabolites released included some 7-OHMe-12-MeBA as 13. DiGiovanni. J.. Slaga. T. J., Berry. D. L.. and Juchau, M. R. Metabolism well as 2 derivatives of relative HPLC retention times con of 7,12-Dimethylbenz[a]anthracene in Mouse Skin Homogenates Ana lyzed with High-pressure Liquid Chromatography. Drug Metab. Disposi sistent with those reported by Yang and Dower (45) for 3- tion, 5: 295-301. 1977. hydroxy-7-hydroxymethyl-12-methylbenz(a)anthracene and 14. Dipple, A. Polynuclear Aromatic Carcinogens. In: C. E. Searle (ed.),

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~~OCTOBER 1978 3437~~

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. Formation of Glucuronic Acid Conjugates of 7,12-Dimethylbenz( a)-anthracene Phenols in 7,12-Dimethylbenz(a )anthracene-treated Hamster Embryo Cell Cultures

~~William M. Baird, Ruth Chemerys, Ching Jer Chern, et al.~~

~~Cancer Res 1978;38:3432-3437.~~

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