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Prostaglandin Endoperoxide Synthetase-Dependent Cooxidation of (±)- Frans-7,8-Dihydroxy-7,8-Dihydrobenzo(A)Pyrene in C3H/10T1/2 Clone 8 Cells

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Prostaglandin Endoperoxide Synthetase-Dependent Cooxidation of (±)- Frans-7,8-Dihydroxy-7,8-Dihydrobenzo(A)Pyrene in C3H/10T1/2 Clone 8 Cells [CANCER RESEARCH 42. 2628-2632, July 1982] Prostaglandin Endoperoxide Synthetase-dependent Cooxidation of (±)- frans-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene in C3H/10T1/2 Clone 8 Cells Jeff A. Boyd, J. Carl Barrett, and Thomas E. Eling1 Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 ABSTRACT 23). The first, fatty acid cyclooxygenase, catalyzes the bis- dioxygenation of arachidonic acid to prostaglandin G2, an (±)-frans-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene (BP- unstable cyclic endoperoxide. The second component, pros 7,8-diol), the proximate form of the carcinogen benzo(a)- taglandin hydroperoxidase, then catalyzes the reduction of pyrene, is cooxidized during the oxidation of arachidonic acid prostaglandin G2 to the corresponding alcohol, prostaglandin to prostaglandins by prostaglandin endoperoxide synthetase H2. Prostaglandin H2 is subsequently hydrolyzed to the various (PES). This enzyme can oxidize BP-7,8-diol to the reactive prostaglandins, thromboxane, and prostacyclin. Numerous xe- intermediate (±)-7/8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10- nobiotics may serve as reducing cofactors for the prostaglandin tetrahydrobenzo(a)pyrene, which binds covalently to macro- hydroperoxidase activity, being "cooxidized" in the process. molecules, is mutagenic in bacterial test systems, and forms In addition to prostaglandin G2, other peroxides can serve as 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene substrates to initiate the reaction since, unlike fatty acid cy (BP-tetrol) isomers. We have examined the cooxidation of BP- clooxygenase, the hydroperoxidase component is relatively 7,8-diol in an intact cell culture system of C3H/10T1/2 clone 8 nonspecific (4, 12, 15). mouse embryo fibroblasts, in which both the mixed-function PES cooxidizes various carcinogens and other chemicals in oxidase and PES systems are present. When BP-7,8-diol is vitro. BP-7,8-diol, the proximate form of the environmental incubated for 72 hr with approximately 106 confluent cells, carcinogen BP, is metabolized by PES to a reactive interme high-performance liquid chromatography analysis of the or diate in microsomal systems from various tissues, including ganic extractable products reveals all four pairs of BP-tetrols, human lung (19). Marne«et al. (11 ) reported that BP-7,8-diol with those from (±)-7/8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10- is metabolized to a mutagenic metabolite(s) by PES, as mea tetrahydrobenzo(a)pyrene predominating. The addition of ar sured in an Ames bacterial test system. We have recently achidonic acid (100 /iM) produced a 2- to 3-fold increase in the confirmed their findings and reported that bay-region diols of formation of BP-tetrols from (±)-7/8,8a-dihydroxy-9«,10a- other polycyclic aromatic hydrocarbons are also metabolized epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, while the metabo to mutagenic products using PES as an activating system in lism to BP-tetrols from (±)-7ß,8a-dihydroxy-9/?,10/8-epoxy- the Ames test (6). 7,8,9,10 tetrahydrobenzo(a)pyrene was unchanged. The ad Racemic BP-7,8-diol is converted by PES to primarily cis 1- dition of the PES inhibitor indomethacin (100 JUM)completely and trans 1-BP-tetrols, those formed through BP-diol-epoxide eliminated this stimulation. Cell transformation assays were 1(10, 21 ). BP-diol-epoxide I is considerably more mutagenic to carried out under the same conditions. The addition of arachi mammalian cells than is BP-diol-epoxide II (7, 9) and is consid donic acid resulted in a 10-fold increase in foci formation, while ered to be the ultimate carcinogenic form of BP. In addition, indomethacin inhibited the increase in foci formation by 70%. the PES-dependent cooxidation of acetaminophen (2) and These results suggest that cooxidation of BP-7,8-diol to reac several renal carcinogens (24-26) and /V-demethylation of a tive intermediates by PES can occur in an intact cell system if variety of aromatic amines (8, 20) have been reported. Thus, stimulated with arachidonic acid. In addition to mixed-function PES-dependent cooxidation of a variety of carcinogens is oxidase-dependent activation of carcinogens, the cooxidation possible. However, this PES-mediated activation of chemicals of chemicals to reactive metabolites during prostaglandin bio has been demonstrated primarily in cell-free biochemical as synthesis may also play a role in carcinogenesis. says with microsomal preparations, but not in intact cell assays. The goals of this study are 3-fold: (a) we wish to demonstrate INTRODUCTION that PES-dependent cooxidation of BP-7,8-diol can occur in PES2 consists of 2 distinct activities, which copurify (13, 15, an intact cell culture system; (o) and perhaps most importantly, we wish to correlate PES-dependent metabolism with cell trans ' To whom requests for reprints should be addressed. formation; (c) we wish to elucidate the conditions under which 2 The abbreviations used are: PES, prostaglandin endoperoxide synthetase; PES-dependent activation becomes a significant factor in the BP-7,8-diol, (±)-frans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene; BP, benzo- presence of cytochrome P-450-dependent MFO activity in (a)pyrene; cis 1-tetrol, 7/8,9,10-tetrahydroxy-7/8,9,10-tetrahydrobenzo- (a)pyrene; trans 1-tetrol, 7,10/8,9-tetrahydroxy-7,10/8,9-tetrahydrobenzo- oxidizing BP-7,8-diol and subsequent cell transformation. To (a)pyrene; BP-diol-epoxide I. (±)-7/î,8a-dihydroxy-9a.10a-epoxy-7,8,9.10- accomplish these goals, we chose the C3H/10T1/2 CL8 line of tetrahydrobenzo(a)pyrene; BP-diol-epoxide II, (±)-7/5,8a-dihydroxy-9/ï,10/i}- epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene; MFO, mixed-function oxidase; CL8, mouse embryo fibroblasts in which cell transformation can be clone 8; HPLC, high-performance liquid chromatography; BP-tetrol. 7,8.9,10- readily measured (17, 18), cytochrome P-450-dependent ac tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene; cis 2-tetrol, 7,9,10/8-tetrahy- tivity has been demonstrated (5,14), and PES activity has been droxy-7,9,10/8-tetrahydrobenzo(a)pyrene; trans 2-tetrol, 7,9/8,10-tetrahy- droxy-7,9/8,10-tetrahydrobenzo(a)pyrene. characterized (1 ). Received December 23, 1981 ; accepted March 30, 1982. PES activity in cultured cells is highly dependent on the 2628 CANCER RESEARCH VOL. 42 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. J. A. Boyd et al. culture conditions and rate of cell division. Confluent cells II by incubating separately at 25° for 24 hr in water (pH 7.0). The produce little or no prostaglandins, while cells in a logarithmic hydrolysis products were extracted with ethyl acetate, evaporated growth phase produce high amounts of prostaglandins (1). under N2, and dissolved in methanol as described above. These sam However, the addition of the prostaglandin precursor arachi- ples were used as BP-tetrol standards during HPLC analysis of un donic acid to confluent monolayers results in high prostaglan known organic extract samples. Water-soluble metabolites from both media and cell aqueous phases din formation, significantly greater than that observed in loga were subjected to /8-glucuronidase treatment. Samples were acidified rithmically growing cells (1 ). Peak levels of cytochrome P-450- to pH 5.0 and then incubated for 20 hr with an excess of /?-glucuroni- dependent activity have been observed to occur at confluency dase at 37°. Samples were extracted with ethyl acetate and analyzed (22). Our metabolism studies were therefore carried out with by HPLC as described above. confluent dishes and arachidonic acid addition to the cell Cell Transformation Assays. Cells were grown to confluency as culture medium in order to elicit maximal activities of both described for the metabolism studies. Four days after reaching con- cytochrome P-450-dependent MFO and PES. By addition of fluency, 8 ml of fresh media were added to the plates, and 4 ml of this indomethacin, a specific inhibitor of PES, the relative roles of were removed 12 hr later to prepare the stock solution and dilutions of BP-7,8-diol. The cells were then treated by adding 4 ml of media these activities can be elucidated. containing the indicated concentrations of BP-7,8-diol, indomethacin (100 fiM), arachidonic acid (100 /¿M),ethanol, or combinations of the MATERIALS AND METHODS above. The cells were treated for 72 hr, after which the cells were trypsinized Chemicals. [G-3H]BP-7,8-diol (395 mCi/mmol), unlabeled BP-7,8- by treatment with 0.1% trypsin (Grand Island Biological Co.) for 5 min diol, [7-14C]BP-diol-epoxide I (17.9 mCi/mmol), and [7-14C]BP-diol- at 37°. The cells were suspended in complete medium, counted on a epoxide II (17.9 mCi/mmol) were obtained from Dr. David Longfellow, Coulter Counter, and then plated at 500 cells/100-mm dish in 5 dishes Chemical Resource Section, Division of Cancer Cause and Prevention, for determination of cloning efficiency or at 1000 cells/60-mm dish in National Cancer Institute, Bethesda, Md. Arachidonic acid (99.0% 20 dishes for the cell transformation assay. The plates for cloning pure) was purchased from Nu-Chek-Prep, Inc. (Elysian, Minn.). Indo efficiency were fixed and stained after 10 days of growth, and the methacin and /î-glucuronidase (Escherichia coli, type VII) were pur number of surviving colonies was scored. The cells for cell transfor chased from Sigma Chemical Co. (St. Louis, Mo.). mation studies were grown as described (17); the plates were fixed Cell Culture and Treatment. Establishment and growth character and stained after 6 weeks and scored for transformed foci (types II and istics of the C3H/10T'/2 CL8 line of mouse embryo fibroblasts have III).
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