Prostaglandin Endoperoxide Synthetase-Dependent Cooxidation of (Â±)- 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 (Â±)- 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 (Â±)-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 (Â±)-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). MarneÂ«et al. (11 ) reported that BP-7,8-diol with those from (Â±)-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 (Â±)-7/8,8a-dihydroxy-9Â«,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 (Â±)-7ÃŸ,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, (Â±)-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. (Â±)-7/Ã®,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, (Â±)-7/5,8a-dihydroxy-9/Ã¯,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-tetrahydroxy-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 25Â° 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 37Â°. 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 /Â¿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 37Â°. 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 /Ã®-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). been described previously (18). Plastic Petri dishes (100 mm; Falcon Plastics, Oxnard, Calif.) were seeded with 105 cells (passages 9 to 12) in 10 ml of Eagle's basal medium containing 10% fetal calf serum RESULTS (Grand Island Biological Co., Grand Island, N. Y.), penicillin (100 units/ Metabolism. Initial studies were designed to determine the ml), and streptomycin (100 ^g/ml). Cells were maintained at 37Â°in a time dependence of BP-7,8-diol cooxidation in cultured cells. humidified atmosphere of 5% CO2 in air and grown to confluence (7 to At time points less than 48 hr, very little metabolism (<5%) in 8 days). the presence or absence of arachidonic acid was observed. For the metabolism studies, cells were fed with 8 ml of fresh media 4 days after reaching confluency (106 cells/plate). Twelve hr later, 4 After time periods greater than 72 hr, cell nutritional factors ml of media were removed from each plate to prepare a stock solution were an experimental problem. Metabolic activity was greatest containing the appropriate amounts of [3H]BP-7,8-diol (1, 5, 20, or 50 at 72 hr, and this time point was chosen for all further experi Â¡J.M).Eachincubation contained [3H]BP-7,8-diol, approximately 3.5 x ments. The addition of arachidonic acid to the cells did not 106 dpm/plate. This stock solution was then used to replace the 4 ml alter the time course of BP-7,8-diol oxidation. left on each plate. To initiate the experiment, appropriate plates were BP-7,8-diol was metabolized by confluent C3H/10TV2 CL8 treated with indomethacin (100 Â¿IM),arachidonic acid (100 /ÃŒM),indo in the absence of added arachidonic acid. The major organic methacin and arachidonic acid, or ethanol (control). The reaction was extractable metabolites were the cis 1- and frans 1-tetrols, terminated at the appropriate time point by removing the media from the plates and placing both at â€”60Â°. Metabolic Assays. Media samples were thawed then extracted with Table 1 3x3 volumes of ethyl acetate. Organic extracts were evaporated to Distribution of BP-7, 8-diol and products for various treatment conditions dryness under N2, and the residue was resuspended in 0.5 ml methanol. At a BP-7,8-diol concentration of 5 /IM, HPLC analysis of all organic extractable material (see Chart 1) and subsequent analysis of water-soluble materials were Cells were thawed and scraped from plates with a rubber policeman performed to account for all of the diolÂ° initially added to the cells (20 nmol at after treatment with 2 ml Triton X-100 detergent (0.1 %). This aqueous this particular concentration). The water-soluble materials were primarily glucu- cell suspension was then extracted as above. Total water-soluble and ronide conjugates of BP-7.8-diol as described in the text. organic extractable radioactivity were then determined by standard cells)ProductsUnidentified nmol /dish (~106 liquid scintillation techniques for both media and cells, using a Packard Model 3390 liquid scintillation spectrometer and Aquasol scintillation +AA0.06 â€¢AAtI0.08 Â±0.25b2.76 cocktail (New England Nuclear, Boston, Mass.). (voidvolume)Tetrols Â±0.017.09 Â±0.021 Organic extract samples were analyzed by injecting 50 /il onto a Waters high-performance liquid Chromatograph, using a Model U6K BPDEITetrolsfrom Â±0.960.58 Â±0.400.57 .64 Â±0.320.37 injector, a Model 6000 A solvent delivery system, and an RCM-100 BPDEIIUnmetabolizedBP-7,8-diolWaterfrom Â±0.083.11 Â±0.152.87 Â±0.1015.91 radial compression unit with a 10-jum Cia-reverse phase cartridge (Waters Associates, Inc., Milford, Mass.). Samples were eluted iso- Â±1.2912.69 Â±0.240.41 Â±1.652.10 cratically with 60% methanol for 17.5 min and 100% methanol for 12.5 min. All solvents were of HPLC grade (Fisher Scientific Co., Fairlawn, solubleDiol0.86 Â±1.03Diol Â±0.46Diol Â±0.06 N. J.). Eluant fractions were collected at 30-sec intervals on a Gilson a Diol, BP-7,8-diol (5 /UM);AA. arachidonic acid (100 JIM);I, indomethacin fraction collector (Middleton, Wis.) at a flow rate of 1 ml/min. (100/iM); BPDE I, BP-diol-epoxide I; BPDE II, BP-diol-epoxide II. BP-tetrol standards were prepared from [14C]BP-diol-epoxides I and 6 Mean Â±S.D.of 3 determinations.

JULY 1982 2629

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. Cooxidation of BP-7,8-diol in C3H/ÃŒOT/2Cells derived from BP-diol-epoxide I, as seen in Table 1. Only small Table 2 amounts of cis 2- and frans 2-tetrols, formed from BP-diol- Effect of BP-7,8-diol concentration on amounts of total organic extractable BP- epoxide II, were detected. In addition, a small unidentified and tetrol products Organic extract samples from both media and cells were analyzed by HPLC very polar metabolite eluted with the void volume of the column as described in "Materials and Methods." Total amounts of cis 1- and trans 1- (Chart 1). The addition of the PES inhibitor indomethacin did tetrols (derived from BP-diol-epoxide I) and cis 2- and trans 2-tetrols (from BP- not significantly alter the metabolism of BP-7,8-diol by con diol-epoxide II) were calculated. cells)Treatment nmol/dish(~106 fluent cells. Ia1.15from BPDE The majority of the radioactivity was not extractable by ethyl conditions1 II0.09from BPDE acetate, indicating the possible formation of water-soluble me Â±0.026 MMdiol1 Â±0.01 tabolites, either glucuronides or glutathione conjugates of BP- I1 I'M diOl â€¢ 1.10 Â±0.01 0.10 Â±0.04 7,8-diol or its oxidative products. After digestion of the aqueous fiM diol + AA 1.19 Â±0.01 0.09 Â±0.01 layer with /J-glucuronidase (essentially free of sulfatase), 151 /IM diol + AA + 1.30 Â±0.022.76 0.11 Â±0.010.58 greater than 95% of the remaining radioactivity was extractable fiM diol Â±0.96 Â±0.08 with ethyl acetate. This organic extractable material was ana 5 Â¡iMdiol+ 1 2.69 Â±0.98 0.61 Â±0.04 5 /IM diol + AA 7.09 Â±0.40 0.57 Â±0.15 lyzed by HPLC, and only unmetabolized BP-7,8-diol was found. 1205 JIMdiol + AA + 1.64 Â±0.3210.01 0.37 Â±0.103.27 Thus, the water-soluble metabolites are primarily glucuronide conjugates of BP-7,8-diol. JIMdiol + 0.27 Â±1.15 20 Â¡IMdiol+ 1 9.89 Â±0.03 2.63 Â±0.01 The addition of arachidonic acid to the cells significantly 20 /UMdiol + AA 17.58 Â±0.95 4.26 Â±0.77 stimulated the oxidation of BP-7,8-diol (Chart 1). The concen 20150 JIMdiol + AA + 8.88 Â±1.8314.43 2.59 Â±0.374.62 trations of arachidonic acid and indomethacin which gave UMdiol Â±3.38 Â±0.71 optimal stimulation and inhibition of prostaglandin biosynthesis 50 UMdiol + 1 12.78 Â±2.26 5.10 Â±1.94 were determined for a previous study (1 ). As seen in Table 1 50 JIMdiol + AA 11.65 Â±1.52 3.60 Â±0.01 3.33 Â±0.03 and Chart 1, the formation of cis 1- and trans 1-tetrols (from 50 fiM diol + AA + 1Tetrols 9.66 Â±1.32Tetrols a BPDE I, BP-diol-epoxide I; BPDE I . BP-diol-epoxide II; diol, BP-7,8-diol; I. BP-diol-epoxide I) was significantly elevated. Little or no in Indomethacin; AA, arachidonic acid. crease in the formation of cis 2- and trans 2-tetrols (from BP- 6 Mean Â±S.D. of 3 determinations. diol-epoxide II) was observed. Furthermore, this increase in oxidation products of BP-7,8-diol was accompanied by a de increase in BP-tetrols formed through BP-diol-epoxide I. The crease in water-soluble glucuronide conjugates of the parent addition of indomethacin with the arachidonic acid completely compound. The addition of indomethacin and arachidonic acid eliminated this stimulation. At a concentration of 20 /J.M,stim to the confluent cells resulted in inhibition of the effect observed ulation of BP-tetrol formation with arachidonic acid and inhibi with arachidonic acid only. The increased formation of BP- tion with indomethacin were also observed, although the effect tetrols derived from BP-diol-epoxide I and concomitant de was slightly less than that observed at 5 /tw. Some stimulation crease in water-soluble glucuronide conjugates produced by of BP-tetrols formed through BP-diol-epoxide II occurred also arachidonic acid addition was not observed after addition of at 20 JKM,although total BP-tetrol formation was still greatly in arachidonic acid and indomethacin. favor of those formed through BP-diol-epoxide I. At a concen The cooxidation of BP-7,8-diol during prostaglandin biosyn tration of 50 Â¿IM,therewas no difference in BP-tetrol formation thesis was dependent on substrate concentration (Table 2). At with any of the treatment conditions. Formation of water-soluble a 1-juM concentration, there was virtually no difference in BP- glucuronide conjugates of unmetabolized BP-7,8-diol was also 7,8-diol oxidation when the PES substrate arachidonic acid or dependent on BP-7,8-diol concentration; again, the conjuga the PES inhibitor indomethacin was added. At a BP-7,8-diol tion product was inversely proportional to the amount of oxi concentration of 5 /Â¿M,however, activation of the PES system dation product formed. Arachidonic acid-dependent cooxida with exogenous arachidonic acid resulted in a 2- to 3-fold tion of BP-7,8-diol to BP-tetrols formed from BP-diol-epoxide I, inhibited by indomethacin, clearly occurred then only at BP- 7,8-diol concentrations of 5 and 20 Â¡J.M.Those BP-tetrols formed through BP-diol-epoxide I clearly predominated at all concentrations tested, and there was no significant difference between treatments with regard to those BP-tetrols formed through BP-diol-epoxide II. It should be noted that the concen tration dependence for metabolism in the presence and ab sence of exogenous arachidonic acid varied from one experi ment to another, apparently as a result of variability in cell populations. Cell Transformation. To determine the possible role of PES- mediated cooxidation in BP-7,8-diol-induced carcinogenicity, confluent C3H/10T1/2 CL8 cells were treated with BP-7,8-diol and arachidonic acid or indomethacin. After treatment for 72

Retention time(mtn) hr at confluency, the cells were subcultured and plated at a Chart 1. A representative HPLC chromatogram from an organic extract sam lower cell density to allow for fixation and expression of the ple. Peak 1, unidentified; Peaks 2 to 5, BP-tetrols as indicated; Peak 6, unme transformation process. The number of transformed foci (types tabolized BP-7.8-diol. Two chromatograms are superimposed to illustrate the metabolic stimulation produced by adding arachidonic acid (AA). at a 5-uM II and III) were scored after 6 weeks. concentration of BP-7,8-diol. DE I, BP-diol-epoxide I; DE II, BP-diol-epoxide II. Treatment of the cells for 72 hr with ethanol alone, arachi-

2630 CANCER RESEARCH VOL. 42

Table 3 creased metabolism of BP-7,8-diol to BP-diol-epoxide I in the Effects of PES manipulation on cell transformation presence of arachidonic acid was highly dependent on the The effects of AAa and I on BP-7.8-diol-induced cell transformation, as concentration of BP-7,8-diol used. Moreover, this concentra measured by number of transformed foci, were assessed at various concentra tions of BP-7.8-diol. tion dependence appeared to vary from one experiment to Foci/20 dishes (~103 cells/dish) another. BP-7,8-diol-induced cell transformation was not inhibited by BP-7,8-dioli> AA No. of foci indomethacin, suggesting that oxidation to reactive metabolites _c+++ occurs via the MFO system present in C3H/10T1/2 cells (5). The number of transformed cells, as indicated by focus for +11 mation, was significantly increased by the addition of arachi donic acid. Indomethacin in the presence of arachidonic acid +1 prevented this stimulation of focus formation. The discrepancy +1 in concentration dependence between transformation and me +55 + tabolism studies probably reflects the shift in dose-response relationship from one experiment to another, as noted in +5 "Results." These cell transformation results agree with our +5 metabolic data regarding the modulation of BP-diol-epoxide I + +000111135655 formation by initiating or inhibiting prostaglandin biosynthesis. AA, arachidonic acid (100 JIM); I, indomethacin (100 /IM). 6 Concentrations of BP-7.8-diol higher than 5 JIM were totally cytotoxic. Taken as a whole, these data indicate that the cooxidation of c â€”,absence of AA or I; t, presence of AA or I. BP-7,8-diol by PES to a reactive intermediate, BP-diol-epoxide I, can occur in an intact cell, resulting in cell transformation. donic acid, Â¡ndomethacin, or a combination of the latter 2 Investigations regarding the mechanism of BP-induced together failed to induce any transformed foci. Treatment with tumorigenesis have concentrated primarily on activation by the BP-7,8-diol alone induced a low level of transformation which cytochrome P-450-dependent MFO system. However, several increased in a dose-dependent manner from 1 to 5 Â¿IM.Higher target tissues for the carcinogenic effect of BP also contain concentrations (10 to 20 /Â¿M)werecytotoxic (<0.4% surviving relatively high levels of PES (19). Therefore, characterization fraction), and no or few cells survived the transformation assay. of the relative contribution of the MFO and PES systems in the Treatment of cells with 100 pM indomethacin had no effect on oxidation of BP and its derivatives, particularly in intact cells, the level of transformation induced by 1 or 5 Â¡J.MBP-7,8-diol. would seem of primary importance. Our results show that Indomethacin and arachidonic acid together increased the initiation of prostaglandin biosynthesis in the presence of MFO cytotoxicity of BP-7,8-diol by approximately 50%. activity results in increased BP-diol-epoxide I formation and Treatment with 1 /IM BP-7,8-diol plus arachidonic acid in cell transformation, suggesting a possible role for PES in BP- creased the number of foci per 20 dishes by 10-fold (Table 3). induced carcinogenesis. A wide variety of stimuli which lead to Addition of indomethacin to 1 /IM BP-7,8-diol plus arachidonic membrane perturbations may initiate prostaglandin biosyn acid reduced the stimulation of transformation by arachidonic thesis. Anaphylaxis, mechanical stimulation, and numerous acid by 70%, with no change in cytotoxicity. Indomethacin, chemical agents stimulate the biosynthesis of prostaglandins arachidonic acid, or a combination of the 2 increased the cytotoxicity of 5 fiM BP-7,8-diol by 50% but had no effect on ((membrane bound) the number of transformed foci per 20 dishes. AA , I phospholipose, mechanical stimulation, â€ž!"?, chemical? (TPA), hormones, pept.de?, inâ„¢SÂ» cell cycle DISCUSSION

Confluent monolayers of C3H/10TV2 CL8 mouse embryo fibroblasts metabolize BP-7,8-diol to oxidative products and water-soluble glucuronic acid conjugates. The primary oxida PGÂ«,TÂ«,PGI2 tion product of BP-7,8-diol appears to be BP-diol-epoxide I, since the c/s 1- and frans 1-tetrols were detected by HPLC. Small amounts of BP-diol-epoxide II were also produced. This oxidative metabolism is apparently mediated by the cytochrome DNA P-450-dependent MFO system (5). The PES inhibitor indo methacin did not alter the formation of BP-diol-epoxide I in the absence of arachidonic acid, suggesting little contribution of the PES system to BP-7,8-diol metabolism in nondividing con fluent cells. This is in agreement with previous work showing a low level of prostaglandin biosynthesis in nondividing confluent Tumori ? â€” - Transformation C3H/10TV2 cells (1). Initiation of prostaglandin biosynthesis by the addition of CELL arachidonic acid to the cells significantly increased the forma Chart 2. A proposed model for the effect of cellular PES stimulation on the tion of BP-diol-epoxide I, with a corresponding decrease in the metabolism of BP in the presence of cytochrome P-450-dependent MFO. AA, formation of glucuronide conjugates of BP-7,8-diol. Indometh arachidonic acid: TPA, 12-O-tetradecanoylphorbol-13-acetate; PGGi, prosta glandin G2; PGH2, prostaglandin H2; PGs, prostaglandins; Tx, thromboxane: acin inhibited this arachidonic acid-dependent effect. The in PG/2, prostacyclin; BPDE-I, BP-diol-epoxide I.

JULY 1982 2631

by causing arachidonic acid release from the cell membrane M. B., and Eling, T. E. A free radical mechanism of prostaglandin synthetase- dependent aminopyrene demethylation. J. Biol. Chem.. 256. 7764-7767, (16) and, hence, the cooxidation of chemical substrates. Fur 1981. thermore, the level of PES activity is highly dependent on the 9. Levin, W., Wood. A. W.. Yagi, H.. Dansette. P. M., Jerina, D. M., and Conney, rate of cell growth. One can therefore propose a model in A. H. Carcinogenicity of benzo(a)pyrene 4,5-, 7,8-, and 9,10-oxides on mouse skin. Proc. Nati. Acad. Sei. U. S. A., 73. 243-247, 1976. which the stimulation of prostaglandin biosynthesis, in the 10. Marni;Â». L. J., Johnson, J. T., and Bienkowski, M. J. Arachidonic acid- presence of MFO, leads to enhanced BP-induced tumorigen- dependent metabolism of 7,8-dihydroxy-7,8-dihydro-benzo(a)pyrene by ram esis (Chart 2). seminal vesicles. FEES Lett.. 706. 13-16, 1979. 11. Marnett, L. J., Reed, G. A., and Dennison, D. J. Prostaglandin synthetase The role of PES-dependent cooxidation of chemicals in the dependent activation of 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene to mu initiation of carcinogenicity or toxicity is unclear. Fischer ef al.3 tagenic derivatives. Biochem. Biophys. Res. Commun., 82: 210-216, 1978. 12. Marnett, L. J., Wlodawer, P., and Samuelsson, B. Co-oxygenation of organic report that PES inhibitors significantly reduce the number of substrates by the prostaglandin synthetase of sheep vesicular gland. J. Biol. rodent skin tumors produced by BP-7,8-diol. In another report, Chem., 250. 8510-8517, 1975. bladder tumors produced in rats by /V-[4-(5-nitro-2-furyl)-2- 13. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, O. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland micro- thiazolyl]formamide, a chemical carcinogen, are inhibited by somes. J. Biol. Chem., 257. 2629-2636. 1976. pretreatment with aspirin, a PES inhibitor (3). Together with the 14. Nesnow. S.. and Heidelberger, C. The effect of modifiers of microsomal data presented in this paper, there is a significant amount of enzymes on chemical oncogenesis in cultures of C3H mouse cell lines. Cancer Res., 36. 1801-1808, 1976. evidence to propose a possible role for PES in the activation of 15. Ohki, S., Ogino, N., Yamamato. S., and Hayaishi, O. Prostaglandin hydro- carcinogens and other xenobiotics to reactive intermediates in peroxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem., 254: 829-836, 1979. vivo. 16. Piper, P., and Vane. J. R. The release of prostaglandins from lung and other tissues. Ann. N. Y. Acad. Sci., 780: 363-385. 1971. REFERENCES 17. Reznikoff. C. A.. Bertram, J. S., Brankow, D. W., and Heidelberger, C. Quantitative and qualitative studies of chemical transformation of cloned 1. Ali, A. E., Barrett. J. C.. and Eling, T. E. Prostaglandin and thromboxane C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res., 33. 3239-3249. 1973. production by fibroblasts and vascular endothelial cells. Prostaglandins, 20: 667-677. 1980. 18. Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. Establishment and 2. Boyd. J. A., and Eling, T. E. Prostaglandin endoperoxide synthetase de characterization of a cloned line of C3H mouse embryo cells sensitive to pendent co-oxidation of acetaminophen to intermediates which covalently post-confluence inhibition of cell division. Cancer Res., 33: 3231-3238, bind in vitro to rabbit renal medullary microsomes. J. Pharmacol. Exp. Ther., 1973. 219.: 659-664, 1981. 19. Sivarajah, K., Lasker, J. M., and Eling, T. E. Prostaglandin synthetase- dependent co-oxidation of (Â±)-benzo(a)pyrene-7,8-dihydrodiol by human 3. Cohen. S. M.. Zenser, T. V., Murasaki, G., Fukushima, S., Mattammal, M. B., Rapp, N. S., and Davis, B. B. Aspirin inhibition of W-{4-<5-nitro-2-furyl)-2- lung and other mammalian tissues. Cancer Res., 41: 1834-1839, 1981. thiazolyljformamide-induced lesions of the urinary bladder correlated with 20. Sivarajah, K., Lasker, J. M., Eling, T. E.. and Abou-Donia. M. B. Metabolism of W-alkyl compounds during the biosynthesis of prostaglandins. Mol. Phar inhibition of metabolism by bladder prostaglandin endoperoxide synthetase. Cancer Res.. 41: 3355-3359, 1981. macol. 27: 133-141, 1982. 21. Sivarajah, K., Mukhtar, H., and Eling, T. E. Arachidonic acid-dependent 4. Egan, R. W., Gale. P. H., and Kuehl, F. A. Reduction of hydroperoxides in metabolism of (Â±)-frans-7,8-dihydroxy-7,8-dihydro-benzo(a)pyrene (BP- the prostaglandin biosynthetic pathway by a microsomal peroxidase. J. Biol. Chem., 254. 3295-3302. 1979. 7,8-diol)to 7,10/8,9-tetrols. FEBS Lett., 706: 17-20, 1979. 22. Strobel-Stevens, J. D., Reid, J. W., Taylor, K. B., and Sarrif, A. M. Activity 5. Gehly, E. B., Fahl, W. E., Jefcoate, C. R., and Heidelberger, C. The metabolism of benzo(a)pyrene by cytochrome P-450 in transformable and and kinetic properties of basal aryl hydrocarbon hydroxylase during prolif nontransformable C3H mouse fibroblasts. J. Biol. Chem., 254. 5041-5048, eration in the transformable C3H 10TVÃ•CL8 cell line. Chem.-Biol. Interact., 33:45-61, 1980. 1979. 6. Guthrie. J.. Robertson, l. G. C., Zeiger, E., Boyd, J. A., and Eling, T. E. 23. Van der Ouderaa. F. J., Buytenhek. M.. Nugteren, D. H.. and Van Dorp, D. Selective activation of some dihydrodiols of several polycyclic aromatic A. Purification and characterization of prostaglandin endoperoxide synthe tase from sheep vesicular glands. Biochim. Biophys. Acta, 487: 315-331, hydrocarbons to mutagenic products by prostaglandin synthetase. Cancer Res.. 42. 1620-1623, 1982. 1977. 24. Zenser, T. V., Mattammal, M. B., and Davis, B. B. Co-oxidation of benzidine 7. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M. Benzo(a)pyrene-7,8-dihydrodiol is more carcinogenic than benzo(a)pyrene by renal medullary prostaglandin cyclo-oxygenase. J. Pharmacol. Exp. in newborn mice. Nature (Lond.), 266. 378-380, 1977. Ther., 277: 460-464, 1979. 8. Lasker, J. M., Sivarajah, K., Mason. R. P., Kalyanaraman, B., Abou-Donia, 25. Zenser, T. V., Mattammal, M. B., and Davis, B. B. Co-oxidative metabolism of 2-amino-4(5-nitro-2-furyl)-thiazole by prostaglandin hydroperoxidase. J. Lab. Clin. Med., 96: 425-432, 1980. 3 S. M. Fischer, G. Gleason. and T. J. Slaga. Inhibition of polycyclic aromatic 26. Zenser, T. V., Mattammal, M. B., and Davis. B. B. Metabolism of A/-(4-<5- hydrocarbon-induced skin tumors by cyclooxygenase inhibitors. Winter Prosta nitro-2-furyl)-2-thlazolyl]formamide by prostaglandin endoperoxide synthe glandin Meeting, Snowbird. Utah, April 1980. tase. Cancer Res., 40: 114-118, 1980.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. Prostaglandin Endoperoxide Synthetase-dependent Cooxidation of (±)- trans-7,8-Dihydroxy-7,8-dihydrobenzo(a )pyrene in C3H/10T½ Clone 8 Cells

Jeff A. Boyd, J. Carl Barrett and Thomas E. Eling

Cancer Res 1982;42:2628-2632.

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