Cooxidation of steroidal and non-steroidal estrogens by purified prostaglandin synthase results in a stimulation of prostaglandin formation.

Estrone (E1), estradiol (E2), the catechol estrogens 2-OHE1 and 2-OHE2, and diethylstilbestrol (DES) were incubated with purified prostaglandin synthase (PHS) in vitro in the presence of arachidonic acid and their PHS-catalyzed cooxidation was determined. 2-OHE1, 2-OHE2, and DES were extensively metabolized by PHS peroxidase activity, E1 and E2 to a lesser extent. The cooxidation of the estrogens is accompanied by an increased prostaglandin formation and an increase in cyclooxygenase activity in vitro; progesterone and nylestriol are without effect. Prostaglandins have been proposed to play a role in events related to early estrogen action in tissues such as the uterus. The cooxidation of estrogens and their metabolites by prostaglandin hydroperoxidase might represent one type of interaction between the hormones and the arachidonic acid cascade that could lead to changes in prostaglandins.

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion.

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.

Co-oxidation of diethylstilbestrol and structural analogs by prostaglandin synthase.

Structural analogs of diethylstilbestrol (DES) with at least one phenolic hydroxyl group are metabolized by prostaglandin H synthase (PHS) from ram seminal vesicle microsomes (RSVM) in vitro in the presence of arachidonic acid (20:4). U.v. spectroscopy revealed the formation of p-quinoid intermediates in incubations of DES, tetrafluoro-DES and dimethylstilbestrol, and tautomerization of the quinones to the respective dien-compounds which were characterized by h.p.l.c. and GC/MS. Indomethacin inhibits the formation of these metabolites which are identical to the major metabolites formed in incubations with horseradish peroxidase/hydrogen peroxide. Covalent binding to protein was observed in incubations of PHS co-substrates. Notably, formation of reactive intermediates which bind to protein is not limited to DES-analogs which form quinone intermediates: radiolabeled hexestrol and E,E-dienestrol yield protein-bound radioactive products upon incubation with RSVM and 20:4, probably via one electron-oxidation to a phenoxy radical. PHS-catalyzed metabolism of structural analogs of DES is accompanied by a concentration-dependent increase in cyclo-oxygenase activity. The measurement of the 20:4-dependent oxygen uptake rates in vitro can serve as a convenient assay for estrogenic compounds which undergo co-oxidation. At high concentrations, however, DES structural analogs can inhibit rather than stimulate PHS. The PHS-catalyzed formation of reactive intermediates from DES structural analogs and their effect on PHS may be of importance for their biological activity in estrogen target tissues with low mono-oxygenase activity.

Biosynthesis of prostaglandins by isolated and cultured airway epithelial cells.

The metabolism of arachidonic acid to prostaglandins and thromboxane by freshly isolated and cultured respiratory tract epithelial cells was examined by HPLC methods. Homogenates prepared from freshly isolated rat and rabbit tracheal epithelial cells did not convert arachidonic acid to prostaglandins. Rat tracheal epithelial cells however, did convert arachidonic acid to uncharacterized metabolites possibly hydroxyfatty acids. In contrast, rat tracheal epithelial cells grown in culture for 9 days acquired the capacity to convert arachidonic acid to PGE2 and related products while cultured rabbit tracheal epithelial cells converted arachidonic acid to TXB2. The conversion of arachidonic acid to PGE2 by rat tracheal cells was studied at various times in culture. The formation of PGE2 appeared to parallel the growth of the cultures. In contrast to freshly isolated rat tracheal cells, enriched rat Clara cell fractions were able to convert 14C-arachidonic acid to prostacyclin (PGI2) ans measured by HPLC analysis of its stable end product 6-keto PGF1 alpha. PGI2 was also the major metabolite of arachidonic acid produced by enriched rat alveolar type II cell fractions. PGF2 alpha, and hydroxyfatty acids were also formed. These results suggest that arachidonic acid metabolism differs in various types of respiratory tract cells and that maintenance of such cells in culture alters the pattern of arachidonic acid metabolism.

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis.

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.

Investigation of potential health effects associated with well water chemical contamination in Londonderry Township, Pennsylvania, U.S.A.

A community health survey was conducted by the Pennsylvania Department of Health in Londonderry Township, Dauphin County, Pennsylvania, in response to concerns about potential health effects associated with residential exposure to chemical contaminants in well water. The data indicate that there were no observable adverse health effects in the exposed group of residents, compared with the control group, which could be ascribed to long-term, low-level exposure to trichloroethylene (TCE) and other volatile organic chemicals. Significantly more individuals in the exposed group than in the control group experienced eye irritation, diarrhea, and sleepiness during the 12-month period prior to the survey. This indicated the possibility of an association of contaminated water with the manifestation of symptoms. It is hypothesized that the increased rate of symptoms observed in the exposed group, when compared to the control group, may have been caused by one or more of the following factors: (1) effect of TCE at a threshold level higher than 28 ppb, (2) effect of a single chemical entity other than TCE, and (3) additive or synergistic effects of several chemicals. It is also possible that there are factors other than water contaminants associated with the recorded symptoms, e.g., stress, that may have had an important influence in the exposed group but not in the control group.

The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical.

The oxidation of aminopyrine to an aminopyrine cation radical was investigated using a solubilized microsomal preparation or prostaglandin H synthase purified from ram seminal vesicles. Aminopyrine was oxidized to an aminopyrine cation radical in the presence of arachidonic acid, hydrogen peroxide, t-butyl hydroperoxide or 15-hydroperoxyarachidonic acid. Highly purified prostaglandin H synthase, which processes both cyclo-oxygenase and hydroperoxidase activity, oxidized aminopyrine to the free radical. Purified prostaglandin H synthase reconstituted with Mn2+ protoporphyrin IX, which processes only cyclo-oxygenase activity, did not catalyze the formation of the aminopyrine free radical. Aminopyrine stimulated the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11-13-eicosatetraenoic acid. Approximately 1 molecule of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was reduced for every 2 molecules of aminopyrine free radical formed, giving a stoichiometry of 1:2. The decay of the aminopyrine radical obeyed second-order kinetics. These results support the proposed mechanism in which aminopyrine is oxidized by prostaglandin H synthase hydroperoxidase to the aminopyrine free radical, which then disproportionates to the iminium cation. The iminium cation is further hydrolyzed to the demethylated amine and formaldehyde. Glutathione reduced the aminopyrine radical to aminopyrine with the concomitant oxidation of GSH to its thiyl radical as detected by ESR of the glutathione thiyl radical adduct.

An electron spin resonance study of o-semiquinones formed during the enzymatic and autoxidation of catechol estrogens.

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone-free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated either enzymatically (using horseradish peroxidase-H2O2 or tyrosinase-O2) or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). In the peroxidase system, radicals are produced by one-electron oxidation of the catechol estrogen and their decay is by a second-order pathway, consistent with their disproportionation to quinone and catechol products. With tyrosinase-O2, radical generation occurs indirectly. Initial hydroxylation of phenolic estrogen (at either the 2- or 4-position) gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. A competing mechanism for radical production involves autoxidation of the catechol. Results obtained from the estrogen systems have been compared with those from the model compound 5,6,7,8-tetrahydro-2-naphthol.

Effect of aspirin and indomethacin on the formation of benzo(a)pyrene-induced pulmonary adenomas and DNA adducts in A/HeJ mice.

Previous results in various in vitro systems suggest that prostaglandin endoperoxide synthetase (PES) could serve as either an alternative or an additional enzyme to the cytochrome P-450-dependent monooxygenases for the formation of mutagenic, cell-transforming, and DNA-binding metabolites of carcinogens. To test this hypothesis in vivo, we examined the effect of PES inhibitors on benzo(a)pyrene (BP)-induced pulmonary adenoma and BP metabolite:DNA adduct formation in A/HeJ mice. Animals were treated with a dosage regimen of aspirin which inhibited PES but had no effect on the cytochrome P-450-dependent oxidation of 7,8-dihydrodihydroxybenzo(a)pyrene. Aspirin did not significantly alter the number of pulmonary adenomas per mouse at either dose of BP (6.0 and 3.0 mg per mouse, administered twice, 2 weeks apart). In addition, aspirin treatment did not depress the in vivo formation of BP metabolite:DNA adducts in lung or liver at either dose of BP (6.0 and 0.06 mg/mouse). The lower dose of BP was used in the adduct study to assess the effect of aspirin at a more environmental exposure level of BP. Treatment with indomethacin, another PES inhibitor, also did not reduce the pulmonary BP metabolite:DNA adduct levels. The failure of PES inhibitors to reduce the number of pulmonary adenomas and BP metabolite:DNA adduct levels suggests that cooxidation of BP during prostaglandin biosynthesis may not play a significant role in BP-induced pulmonary adenomas.

A free radical mediated cooxidation of tetramethylhydrazine by prostaglandin hydroperoxidase.

Prostaglandin endoperoxide synthase catalyzed the oxidation of tetramethylhydrazine to the tetramethylhydrazine radical cation, as detected by electron spin resonance spectroscopy. Oxidation of tetramethylhydrazine by prostaglandin endoperoxide synthase also resulted in the N-demethylation of tetramethylhydrazine leading to the formation of formaldehyde. Both the radical and formaldehyde formation were dependent on arachidonic acid, inhibited by indomethacin, and supported by 15-hydroperoxyarachidonic acid, indicating that the metabolism was peroxidative. We propose that tetramethylhydrazine undergoes two sequential one-electron oxidations yielding an iminium cation, which is then hydrolyzed to yield formaldehyde. Metabolism of hydrazines by prostaglandin endoperoxide synthase may be of importance in tissue containing low mixed-function oxidase activity.

Prostaglandin synthetase and cytochrome P-450-dependent metabolism of (+/-)benzo(a)pyrene 7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells.

The metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases was studied using enriched fractions of Clara cells and alveolar type II cells from rat lung. Arachidonic acid-fortified fractions enriched in Clara cells and alveolar type II cells metabolized BP-7,8-diol to the 7,10/8,9-tetrol of benzo(a)pyrene and the 7/8,9,10-tetrol of benzo(a)pyrene. These tetrols are formed upon solvolysis of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene (BP diol-epoxide I). Arachidonic acid-dependent metabolism of BP-7,8-diol to BP diol-epoxide I in enriched Clara cells and alveolar type II cells was completely inhibited by indomethacin, a classical inhibitor of prostaglandin synthetase. Enriched Clara cells and alveolar type II cells also metabolized BP-7,8-diol to BP diol-epoxide I in the presence of NADPH. Amounts of BP diol-epoxide I-derived tetrols formed from BP-7,8-diol by the prostaglandin synthetase-dependent and the cytochrome P-450-dependent pathways varied significantly between the two pulmonary cell fractions examined. In fractions enriched in Clara cells, cytochrome P-450-dependent BP-7,8-diol oxidation was higher than was prostaglandin synthetase-dependent BP-7,8-diol oxidation; while in fractions of alveolar type II cells, prostaglandin synthetase-dependent BP-7,8-diol oxidation to BP diol-epoxide I predominated. Pretreatment of rats with beta-naphthoflavone resulted in a 2- to 3-fold increase in BP diol-epoxide I formation by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases in both enriched Clara cells and alveolar type II cells. These increases in BP-7,8-diol oxidation to BP diol-epoxide I appear to be due to induction of the two enzymatic pathways in both pulmonary cell types. No qualitative changes in the pattern of BP-7,8-diol metabolism by either enzymatic pathway in enriched Clara cells or alveolar type II cells were observed following beta-naphthoflavone treatment. The results suggest that pulmonary prostaglandin synthetase may serve as either an additional or an alternative bioactivating enzyme to the cytochrome P-450-dependent monooxygenases for the formation of reactive chemical carcinogens in the lung.

Activation of some aromatic amines to mutagenic products by prostaglandin endoperoxide synthetase.

Cooxidation of xenobiotics may occur during prostaglandin biosynthesis. The ability of prostaglandin endoperoxide synthetase to cooxidize several aromatic amines and other chemicals to mutagenic products was tested with the standard Salmonella tester strains. The microsomal fraction of ram seminal vesicles, a rich source of prostaglandin endoperoxide synthetase, in the presence of the prostaglandin endoperoxide synthetase substrate arachidonic acid metabolized benzidine, 2-aminofluorene, 2-naphthylamine, and 2,5-diaminoanisole to mutagenic products. 1-Napthylamine, 2-aminoanthracene, 2-acetylaminofluorene, and 2,4-diaminoanisole were negative or weakly mutagenic. N-Nitrosodimethylamine, N-nitrosomorpholine, the pesticide Aminocarb, and di(2-ethylhexyl)phthalate were not activated to mutagenic products by the ram seminal vesicle microsomal fraction.

Disposition, elimination and metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate after subchronic dermal application in male cats.

The metabolism, distribution, and excretion of the insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) were studied in the male cat. Each cat was given a daily dermal dose of 0.5 mg/kg [14C]EPN for 10 consecutive days. Fifteen days after the last dose, the cats had excreted 62% of the cumulative dose in the urine and 10% in the feces. No 14CO2 was detected in the expired air. O-Ethyl phenylphosphonic acid (EPPA) was identified as the major urinary and fecal metabolite. Phenylphosphonic acid (PPA) was the second highest metabolite. Only traces of the intact EPN were recovered in the urine and feces. The disposition studies performed 1, 5, 10 and 15 days after the administration of the last dose showed that EPN was the major compound identified in the brain, spinal cord, sciatic nerve, adipose tissue, plasma and kidney. Most of the radioactivity in the liver was identified as EPPA followed by PPA. The time course of plasma EPN, determined after the 10th daily dose was biphasic. The slower process had a half-life of 17.0 days. After tissue distribution was completed, tissue elimination was adequately represented as a single first-order process.

The formation of sulfur trioxide radical anion during the prostaglandin hydroperoxidase-catalyzed oxidation of bisulfite (hydrated sulfur dioxide).

The mechanism of prostaglandin synthase-dependent (bi)sulfite (hydrated sulfur dioxide) oxidation was investigated using an enzyme preparation derived from ram seminal vesicles. The horseradish peroxidase-catalyzed oxidation of (bi)sulfite was used as a model system. Incubation of (bi)sulfite with prostaglandin synthase and arachidonic acid, 15-hydroperoxyarachidonic acid, or H2O2 results in the formation of the reactive sulfur trioxide anion radical (SO3(-)). The horseradish peroxidase/H2O2 system also oxidizes (bi)sulfite to SO3(-). This free radical reacts with oxygen resulting in oxygen consumption by these incubations. The free radical was detected with the indirect electron spin resonance technique of spin trapping. The SO3(-) radical adduct formed by the reaction of SO3(-) with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, gives a nitroxide free radical with a nearly unique electron spin resonance spectrum (aH = 16.0 G and aN = 14.7 G). Using the spin-trapping technique, the SO3(-) could be detected even in incubations of guinea pig lung microsomes. When arachidonic acid-derived prostaglandin G2 was the source of hydroperoxide, formation of SO3(-) could be inhibited by indomethacin. When 15-hydroperoxyarachidonic acid or hydrogen peroxide was used to drive the enzymatic oxidation of (bi)sulfite, indomethacin had no effect. This hydroperoxidase activity was not nearly as heat-labile as the cyclo-oxygenase reaction which forms prostaglandin G2. Finally, the peroxidatic oxidation of (bi)sulfite may occur in vivo in competition with the mitochondrial sulfite oxidase, which oxidizes (bi)sulfite to sulfate without the formation of free radicals.

Metabolism of N-alkyl compounds during the biosynthesis of prostaglandins. N-Dealkylation during prostaglandin biosynthesis.

The microsomal fraction of ram seminal vesicles (RSV), when fortified with arachidonic acid, catalyzed the dealkylation of various N-methyl compounds. These included an analogous series of monomethyl- and dimethyl-substituted anilines as well as the drugs aminopyrine and benzphetamine. In contrast, S-alkyl and O-alkyl compounds were poor substrates for dealkylation by RSV microsomes fortified with fatty acid. RSV microsomal N-dealkylation was completely dependent on enzyme and arachidonic acid and could be inhibited by the prostaglandin synthetase inhibitors indomethacin, phenylbutazone, and flufenamic acid as well as by anaerobic conditions. Butylated hydroxyanisole also inhibited the reaction, whereas SKF-525A and metyrapone, which are inhibitors of cytochrome P-450-dependent N-dealkylation, did not. In addition to arachidonic acid, N-dealkylation was elicited by 15-hydroperoxyarachidonic acid, tert-butyl-hydroperoxide, and hydrogen peroxide; these latter reactions were not inhibited by either prostaglandin synthetase inhibitors or anaerobic conditions but did require the presence of microsomal protein. The time course of RSV N-dealkylation, which paralleled O2 consumption by this tissue (an indicator of prostaglandin biosynthesis) implied arachidonic acid-dependent irreversible self-inactivation of catalytic activity. Apparently, oxidizing agents are formed during the interaction of hydroperoxide intermediates of prostaglandin biosynthesis with prostaglandin synthetase, with the oxidizing agents then causing both substrate N-dealkylation and destruction of the enzyme. The metabolism of N-alkyl compounds during the biosynthesis of prostaglandins may provide an additional xenobiotic oxidation pathway to cytochrome P-450-dependent monooxygenases.

A free radical mechanism of prostaglandin synthase-dependent aminopyrine demethylation.

The mechanism of prostaglandin synthase-dependent N-dealkylation has been investigated using an enzyme preparation derived from ram seminal vesicles. Incubation of an N-alkyl substrate, aminopyrine, with enzyme and arachidonic acid, 15-hydroperoxyarachidonic acid, or tert-butyl hydroperoxide resulted in the formation of the transient aminopyrine free radical species. Formation of this radical species, which was detected by electron paramagnetic resonance spectroscopy and/or absorbance at 580 nm, was maximal approximately 30 s following initiation of the reaction and declined thereafter. Free radical formation corresponded closely with formaldehyde formation in this system, in terms of dependence upon substrate and cofactor concentration, as well as in terms of time course. Both aminopyrine free radical and formaldehyde formation were inhibited by indomethacin and flufenamic acid, inhibitors of prostaglandin synthase. The results suggest that the aminopyrine free radical is an intermediate in the prostaglandin synthase-dependent aminopyrine N-demethylase pathway. The aminopyrine free radical electron paramagnetic resonance spectrum revealed that this species is a one-electron oxidized cation radical of the parent compound. A reaction mechanism has been proposed in which aminopyrine undergoes two sequential one-electron oxidations to an iminium cation, which is then hydrolyzed to the demethylated amine and formaldehyde. Accordingly, the oxygen atom of the aldehyde product is derived from neither molecular nor hydroperoxide oxygen, but from water.