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We describe the enzymological regulation of the formation of prostaglandin (PG) D2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2 (prostacyclin), and thromboxane (Tx) A2 from arachidonic acid. We discuss the three major steps in prostanoid formation: (a) arachidonate mobilization from monophosphatidylinositol involving phospholipase C, diglyceride lipase, and monoglyceride lipase and from phosphatidylcholine involving phospholipase A2; (b) formation of prostaglandin endoperoxides (PGG2 and PGH2) catalyzed by the cyclooxygenase and peroxidase activities of PGH synthase; and (c) synthesis of PGD2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2, and TxA2 from PGH2. We also include information on the roles of aspirin and other nonsteroidal anti-inflammatory drugs, dexamethasone and other anti-inflammatory steroids, platelet-derived growth factor (PDGF), and interleukin-1 in prostaglandin metabolism.

The mutagenic response of L5178Y mouse lymphoma cells to the model aromatic amine carcinogens, benzidine (BNZ) and 2-aminofluorene (2-AF) in the presence of the mammalian peroxidase prostaglandin H synthase (PHS) was examined. Standard incubation conditions for mouse lymphoma cells and the PHS system were developed. The cells were exposed to BNZ and 2-AF with purified PHS in the presence or absence of a peroxide, 5-phenyl-4-pentenyl hydroperoxide (PPHP) which is required for PHS-dependent amine oxidation. Incubations were carried out in a medium consisting of Hanks' balanced salt solution with calcium and magnesium and 0.1% pluronic F-68. BNZ by itself or in the presence of PPHP induced a weak mutagenic response in mouse lymphoma cells, but the addition of PHS or PHS and its co-factor PPHP increased the mutagenic response approximately 5-fold over that observed in the absence of PHS. A maximal mutagenic response for BNZ was observed after incubation with the complete activating system, PHS and PPHP. These data are in agreement with the fact that BNZ is an excellent substrate for PHS. When 2-AF was incubated with mouse lymphoma cells, only a minimal mutagenic response was observed. Incubation of 2-AF with either PPHP or PPHP and PHS (complete peroxidase system produced a significant enhancement in mutagenic response. Thus, the mutagenic response of the mouse lymphoma cells to 2-AF was dependent on the peroxide, PPHP but not the enzyme PHS. These data suggest that 2-AF, which is a poor PHS substrate, is oxidized by a different catalyst than PHS. This work demonstrates that BNZ and 2-AF are converted by peroxide-dependent mechanisms to mutagens that can be detected in mammalian cells.

The reaction of malondialdehyde (MDA) with cytosine nucleosides was investigated, and the structures of 1:1 and 3:1 MDA-cytosine adducts were identified. Ultraviolet and NMR spectroscopy indicates the structure of the 1:1 adduct is N4-(3-oxo-1-trans-propenyl)cytosine (M1C) and the structure of the 3:1 adduct is 6-(5*,7*-diformyl-2*H-3*,6*-dihydro-2*,6*-methano-1*,3*-oxazoci n-3*yl)-2- oxopyrimidine (M3C). Both adducts are analogous to previously identified MDA-adenine adducts. The time courses of adduct formation in the reaction of MDA with deoxycytidine at different pH's and using different sources of MDA were determined. M3C-deoxyribose is the major product at acidic pH whereas M1C-deoxyribose appears to be the sole adduct formed at neutral pH. These results suggest that adducts formed between nucleic acid bases and oligomers of MDA may not play a major role in MDA mutagenesis.

Prostaglandin-H synthase is unique among enzymes of the plant and animal kingdom in its ability to biosynthesize and metabolize hydroperoxides. Its cyclooxygenase activity oxygenates polyunsaturated fatty acids to hydroperoxy endoperoxides, and its peroxidase activity reduces the hydroperoxy group to hydroxy groups. Higher oxidation states of the peroxidase oxidize reducing substrates to electron-deficient derivatives that react with macromolecular nucleophiles. In the case of aromatic amines, the electron-deficient derivatives are mutagenic to bacterial and mammalian cells. beta-Dicarbonyl compounds and retinoic acid are oxidized to carbon-centered radicals that react with O2 to form peroxyl free radicals. Peroxyl radicals are the most stable oxy radicals and are able to diffuse some distance from the site of their generation. Peroxyl radicals are also formed during lipid peroxidation and in the reaction of polyunsaturated fatty acid hydroperoxides with metal complexes and metalloproteins. Peroxyl radicals epoxidize isolated doubled bonds of compounds such as 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol); 3,4-dihydroxy-3,4-dihydrobenzo(a)anthracene; and aflatoxin B1. The epoxide products represent the ultimate carcinogenic forms of the respective compounds. Techniques for quantitating the extent of peroxidase dependent or peroxyl radical-dependent metabolism in vivo make use of differences in the structure or stereochemistry of reactive intermediates formed by peroxidases relative to cytochromes P-450. Differences in the relative amounts of hydrolysis products and DNA adducts derived from anti- and syn-dihydrodiolepoxides following application of BP-7,8-diol to mouse skin in vivo indicate peroxyl radicals play a significant role in metabolism of BP-7,8-diol in uninduced animals.

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.

Dihydrodiolepoxides (BPDE) are reactive derivatives that appear to mediate the genotoxic effects of benzo[a]pyrene (BP) and several other polycyclic hydrocarbons. Their formation requires sequential epoxidation, hydration, and epoxidation steps. The first epoxidation step is catalyzed by cytochrome P-450 isoenzymes whereas the second is catalyzed by cytochromes P-450 and assorted species of oxygen radicals (in particular peroxyl radicals). The stereochemistry of epoxidation of the procarcinogen 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is a useful tool with which to dissect the contributions of cytochrome P-450-dependent and independent pathways. [32P]-Postlabeling techniques were developed to analyze the diastereomeric composition of BPDE-deoxynucleoside-bis-phosphate adducts recovered from DNA of target tissues for BP tumorigenesis. Application of these techniques revealed that cytochrome P-450 is the major contributor to BPDE production in mouse skin in vivo. Following administration of BP or (+)-BP-7,8-diol to female CD-1 mice, only minor amounts of adducts derived from peroxyl radical epoxidation were detected. In contrast, pretreatment of the animals with the tumor promoter tetradecanoyl phorbol acetate (TPA) led to the formation of sizeable amounts of non-cytochrome P-450-dependent adducts following coincident administration of (+)-BP-7,8-diol with a second dose of TPA. The results demonstrate that although oxygen radicals appear to play a minor role in tumor initiation by BP, they may play a major role in tumor promotion by phorbol esters.

The effects of 13-cis-retinoic acid on iron/ascorbate-dependent lipid peroxidation were investigated with rat liver microsomes. 13-cis-retinoic acid effectively inhibited malondialdehyde generation and molecular oxygen consumption associated with lipid peroxidation. Under the conditions employed, inhibition was complete at concentrations as low as 25 microM and the IC50 was 10 microM. Evidence for concomitant retinoid oxidation by microsomal unsaturated fatty acid-derived peroxyl radicals was demonstrated by detection of several retinoid-derived metabolites, including 5,8-oxy-13-cis-retinoic acid, generated during lipid peroxidation. The data indicate that 13-cis-retinoic acid inhibits lipid peroxidation by scavenging lipid peroxyl radicals with its conjugated polyene system. Its antioxidant properties may contribute to the pharmacological activities of this and related retinoids.

Malondialdehyde (MDA) forms oligomeric adducts with DNA bases. It has been proposed that the 2:1 MDA-guanosine (M2G) and 3:1 MDA-adenosine (M3A) adducts result from sequential addition of MDA molecules to the nucleoside base. Reaction of 1:1 MDA-guanosine (M1G) and 1:1 MDA-adenosine (M1A) adducts with MDA does not produce the multimeric adducts. This suggests they arise by reaction of the nucleoside bases with oligomers of MDA. If so, the proposed structure for M3A is inconsistent with the addition of an MDA trimer to adenosine. Therefore, we investigated the structure of this molecule. Two-dimensional double quantum filtered COSY and hetero-COSY NMR experiments were performed, and a series of insensitive nuclei assignment by polarization transfer (INAPT) NMR spectra were also recorded. The results of these experiments revealed the presence of a propano group and two alpha,beta-unsaturated aldehydes. The UV spectrum of M3A displayed a maximum at 326 nm, similar to that of N6-[3-oxo-1(E)-propenyl]adenosine (M1A). The adducts were reduced with sodium borohydride for comparison of the UV and NMR spectra. On the basis of our results, a new structure for M3A is proposed which is tentatively named 6(5*,7*-diformyl-2*H-3*,6*-dihydro-2*,6*-methano-1*,3*-oxazocin -3*-yl)-9-beta-D-ribofuranosylpurine.

The peroxidase activity of prostaglandin (PGH) synthase catalyzes the reduction of PGG2 and other natural and synthetic hydroperoxides by reducing substrates. Sulfides serve as reductants by incorporating the oxo ligand from the ferryl-oxo complex which represents the higher oxidation state of the peroxidase (Compound I). A series of alkylaryl sulfides and substituted dihydrobenzo[b]thiophenes were synthesized to determine the electronic and steric requirements of PGH synthase for sulfide reducing substrates. Kinetic parameters were determined for most of the molecules by determining their ability to support reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of PGH synthase purified from ram seminal vesicle microsomes. Electron-donating groups on the aryl moiety para to the sulfide enhanced reducing substrate activity (p = -0.8). As expected from previous results, the major oxidation product of p-methylthioanisole was the corresponding sulfoxide. The presence of a para-amino group increased binding to the enzyme and changed the reduction mechanism from oxygen transfer to electron transfer. The major oxidation product of p-(dimethylamino)thioanisole was identified as p-(methylamino)thioanisole; an equivalent amount of formaldehyde was produced. Increasing the size of the alkyl group attached to sulfur decreased the ability of the sulfide to act as a peroxidase reductant. The maximal turnover for reduction by p-methoxyphenylalkyl sulfides decreased 10-fold on substitution of isopropyl for ethyl. Chiral derivatives of benzo[b]thiophenes demonstrated differences in the ability of the two enantiomers to support reduction. Introduction of a carboxylic acid moiety anywhere in the molecule decreased the maximal turnover for reduction. Esterification of the carboxylate doubled the extent of reduction relative to the free acid. The results are used to develop models for the interaction of sulfides with Compound I of PGH synthase.

The incorporation of tritiated deoxythymidine ([3H]dThd) into colonic DNA of male Sprague-Dawley rats treated intrarectally (i.r.) with bile salts and other substances has been investigated. Instillation of sodium deoxycholate caused an increase in the incorporation of [3H]dThd which was maximal 12 h after treatment. The level of incorporation showed a steep linear dose response from 0.5 mM to 15 mM bile salt. Higher concentrations of deoxycholate up to 300 mM only slightly increased the extent of incorporation when compared to the lower concentration. Several other substances also increased the extent of [3H]dThd incorporation; these include: chenodeoxycholate, lithocholate, sodium dodecyl sulfate, dioctyl sulfosuccinate, corn oil, beef fat, and trioctanoin. Substances which had no effect on [3H]dThd incorporation include cholesterol, dehydrocholate, sodium acetate, dextrose, and mineral oil. Many of the agents which increase colonic [3H]dThd incorporation are also known to enhance colonic tumorigenesis. These findings indicate similarities between the short-term effects, in their respective target tissues, of colon tumor promoters, and classical mouse skin tumor promoters.

The role of prostaglandin H (PGH) synthase and peroxyl radicals as well as cytochrome P-450 in the metabolism of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was examined in fresh skin keratinocytes isolated from hairless mice. Labeled (+)-BP-7,8-diol was oxidized after incubation with the keratinocytes to syn- and anti-diolepoxides in greater than a 4:1 ratio as estimated by h.p.l.c. analysis of the stable hydrolysis products. Formation of diolepoxides was dependent on cell number and the concentration of BP-7,8-diol. Incubation in the presence of the PGH synthase substrate, 20:4 or the inhibitor, indomethacin did not alter the total formation or the ratio of diolepoxides. However, the addition of butylated hydroxyanisole (1 micron) an inhibitor of peroxyl radical dependent-metabolism significantly inhibited diolepoxide formation. The time course for the formation of the anti-diolepoxide and lipid peroxidation, measured as malondialdehyde was determined. The results suggest an excellent correlation between peroxyl radical and diolepoxide formation. Pretreatment of mice with the cytochrome P-450 inducer, beta-naphthoflavone greatly altered the metabolism of (+)-BP-7,8-diol by keratinocytes. The major metabolite was the syn-diolepoxide with significant formation of two unknown metabolites. Pretreatment of mice with BP-7,8-diol did not induce aryl hydrocarbon hydroxylase activity but did increase the yield of syn-diolepoxide formed from labeled (+)-BP-7,8-diol by 1.5-fold. Our results suggest that peroxyl radical-mediated metabolism is primarily responsible for the oxidation of (+)-BP-7,8-diol in control animals while the cytochrome P-450 system is primarily responsible for oxidation in animals pretreated with inducers.

The malondialdehyde (MDA) content of cervical mucus from 23 healthy adult females was measured using an ion-pairing HPLC method capable of detecting 10 pmol MDA. Ten of the women were wearing copper IUDs, four were wearing plastic IUDs, and nine controls were not wearing an IUD. Cervical mucus was sampled during the follicular, periovulatory, luteal, and menstrual phases. The study was designed to determine if there is a relationship between MDA formation and the use of a copper IUD. A total of 79 samples were analyzed. Only 16 of the samples had sufficient MDA for reliable quantitation with the level ranging from 0.1 nmol/g to 2.32 nmol/g. In 19 of the samples, trace levels (less than 0.06 nmol/g) were detected but could not be reliably quantitated. In the remaining 44 samples, no MDA was detectable. There was no correlation between the presence of copper- or non-copper-containing IUDs and the level of MDA. These results are contrary to a previously published report that used a less specific method for MDA analysis.

The peroxidase activity of prostaglandin H (PGH) synthase catalyzes reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol with a turnover number of approximately 8000 mol of 5-phenyl-4-pentenyl hydroperoxide/mol of enzyme/min. The kinetics and products of reaction establish PGH synthase as a classical heme peroxidase with catalytic efficiency similar to horseradish peroxidase. This suggests that the protein of PGH synthase evolved to facilitate peroxide heterolysis by the heme prosthetic group. Comparison of an extensive series of phenols, aromatic amines, beta-dicarbonyls, naturally occurring compounds, and nonsteroidal anti-inflammatory drugs indicates that considerable differences exist in their ability to act as reducing substrates. No correlation is observed between the ability of compounds to support peroxidatic hydroperoxide reduction and to inhibit cyclooxygenase. In addition, the resolved enantiomers of MK-410 and etodolac exhibit dramatic enantiospecific differences in their ability to inhibit cyclooxygenase but are equally potent as peroxidase-reducing substrates. This suggests that there are significant differences in the orientation of compounds at cyclooxygenase inhibitory sites and the peroxidase oxidation site(s). Comparison of 5-phenyl-4-pentenyl hydroperoxide reduction by PGH synthase and horseradish peroxidase reveals considerable differences in reducing substrate specificity. Both the cyclooxygenase and peroxidase activities of PGH synthase inactivate in the presence of low micromolar amounts of hydroperoxides and arachidonic acid. PGH synthase was most sensitive to arachidonic acid, which exhibited an I50 of 0.6 microM in the absence of all protective agents. Inactivation by hydroperoxides requires peroxidase turnover and can be prevented by reducing substrates. The I50 values for inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid are 4.0 and 92 microM, respectively, in the absence and presence of 500 microM phenol, a moderately good reducing substrate. The ability of compounds to protect against hydroperoxide-induced inactivation correlates directly with their ability to act as reducing substrates. Hydroquinone, an excellent reducing substrate, protected against hydroperoxide-induced inactivation when present in less than 3-fold molar excess over hydroperoxide. The presence of a highly efficient hydroperoxide-reducing activity appears absolutely essential for protection of the cyclooxygenase capacity of PGH synthase. The peroxidase activity is, therefore, a twin-edged sword, responsible for and protective against hydroperoxide-dependent inactivation of PGH synthase.(ABSTRACT TRUNCATED AT 400 WORDS)

Human liver microsomes oxidized 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [B(a)P-7,8-diol] to products that yield DNA adduct formation and umu gene expression in the tester system Salmonella typhimurium TA1535/pSK1002. The umu response is correlated to levels of microsomal cytochrome P-450NF (P-450NF) and nifedipine oxidation in different human liver samples used for activation, and both the (+)- and (-)-enantiomers of B(a)P-7,8-diol gave similar results in these and other assays. The microsomal umu response was inhibited by antibodies raised against P-450NF. 7,8-Benzoflavone stimulated the B(a)P-7,8-diol-dependent umu response observed with purified P-450NF and human liver and lung microsomes. Thus, P-450NF appears to be the major enzyme involved in the activation of B(a)P-7,8-diol in human liver and possibly lung. Similar results were obtained for the activation of trans-9,10-dihydroxy-9,10-dihydrobenzo(b)fluoranthene and trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz(a)anthracene, compounds that are known to form highly tumorigenic diol-epoxides. The major product of the oxidation of (+)-B(a)P-7,8-diol was the cis-syn isomer of benzo(a)pyrene-7,8,9,10-tetraol[7 beta, 8 alpha, 9 beta, 10 beta-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]. Studies on the nature of the human liver enzymes involved in the formation of B(a)P-7,8-diol [from benzo(a)pyrene] indicate that neither P-450NF, P-450PA, P-450j, P-450DB, nor P-450MP is involved. The correlation of 7,8-diol formation with phenacetin O-deethylation in a set of liver samples and the partial inhibition of the reaction by 7,8-benzoflavone and anti-rat P-450 beta NF-B suggest that the enzyme involved may be P1-450, the human ortholog of rat P-450 beta NF-B, which catalyzes both the formation of B(a)P-7,8-diol and its subsequent oxidation in tissues of polycyclic hydrocarbon-treated rats. The differential effects of inhibitors indicate that benzo(a)pyrene 3-hydroxylation, 4,5-epoxidation, and 9,10-epoxidation are catalyzed by an enzyme(s) distinct from that which forms the 7,8-epoxide. The roles of the human P-450 enzymes differ from the rodent orthologs in the paradigm for bioactivation of polycyclic hydrocarbons; further, flavones appear to have opposing effects on diol formation and further epoxidation in both human liver and lung.

The effect of intrarectal instillation of hydroperoxy and hydroxy fatty acids on colonic DNA synthesis and ornithine decarboxylase activity in male Sprague-Dawley rats was examined. A mixture of hydroperoxy-arachidonic acid isomers was prepared by methylene blue-sensitized photooxygenation. Pure 13-hydroperoxy-9,11-octadecadienoic acid was prepared by the action of soybean lipoxygenase on linoleic acid. Sodium borohydride reduction yielded the respective hydroxy fatty acids. Twelve hr after instillation of solutions of either hydroperoxy or hydroxy fatty acids, at concentrations up to 10 mM, DNA synthesis was increased in a dose-dependent fashion up to 240% above control values. The induction of ornithine decarboxylase occurred over a similar concentration range 3 hr after instillation of oxidized linoleic acid. In this case, the hydroxy acids (49-fold increase at 10 mM), were more stimulatory than the hydroperoxy derivatives (23-fold at 10 mM). Highly purified linoleic and arachidonic acids did not stimulate either activity at concentrations up to 50 mM. These data indicate that autoxidation products of unsaturated fatty acids, likely components of high-fat diets, can evoke proliferative responses in colonic mucosa. These responses may be relevant to the promotional effect of high dietary fat on colon carcinogenesis.

Arachidonic acid is oxidized by a NADPH-dependent oxygenase of rat liver microsomes to a number of oxygen-containing products, which can be resolved by HPLC. Several of these products have been purified and characterized. They exhibit an absorbance in the UV region of the spectrum that has a maximum at approximately 235 nm, indicative of the presence of a conjugated diene function. Mass spectral analysis of the trimethylsilyl ether derivatives of the methyl esters of the hydrogenated and nonhydrogenated metabolites shows that they are the 9-, 11-, 12-, and 15-monohydroxy derivatives of arachidonic acid, the hydroxyicosatetraenoic acids (HETEs). Their UV absorbance and chromatographic properties suggest that these products possess cis,transdiene geometry characteristic of HETEs isolated from other mammalian sources. THe isolation of these isomeric HETEs suggests that cytochrome P-450 may play a role in the oxidative metabolism of arachidonic acid to physiologically and pharmacologically important hydroxylated unsaturated fatty acids.

Salmonella tester strain TA102 carries the hisG428 ochre mutation on the multicopy plasmid pAQ1. DNA sequence analysis of 45 spontaneous revertants of hisG428 on the chromosome in the presence of pKM101 (strain TA103) indicates that hisG428 revertants fall into three major categories: (i) small, in-frame deletions (3 or 6 base pairs) that remove part or all of the ochre triplet; (ii) base substitution mutations at the ochre site; (iii) extragenic ochre suppressors. Deletion revertants are identified in a simple phenotypic screen by their resistance to the inhibitory histidine analog thiazolealanine, which feedback inhibits the wild-type hisG enzyme but not the enzyme resulting from the deletions. The effect of various genetic backgrounds on the generation of spontaneous deletion revertants was examined. The error-prone repair system encoded in the pKM101 plasmid markedly increased the frequency of total spontaneous reversion events in all genetic backgrounds except recA but did not affect the frequency of spontaneous deletion revertants in any background except polA. The presence of a polA mutation increased the frequency of spontaneous deletion revertants by 2-fold in the absence of pKM101 and by 20-fold with pKM101. The presence of a uvrB mutation or a recA mutation suppressed the generation of spontaneous deletion revertants to approximately 1/2.5. When hisG428 was in multiple copies on pAQ1, the frequency of spontaneous deletion revertants increased by 40-fold, which is the approximate copy number of pAQ1. Mutagenic agents that induce single-strand breaks in DNA (e.g., x-rays, bleomycin, and nalidixic acid) induced deletion revertants in TA102. These agents induced deletion revertants only in hisG428 on pAQ1 and only in the presence of pKM101. Deletion revertants were not induced by frameshift mutagens (i.e., ICR-191 and 9-aminoacridine). These results indicate that different pathways exist for the generation of spontaneous and mutagen-induced deletion revertants of hisG428.

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.

Monoclonal antibodies against prostaglandin H (PGH) synthase have been used to precipitate cyclooxygenase and peroxidase activities from detergent-solubilized preparations of ram seminal vesicle microsomes. Approximately 85% of the solubilized cyclooxygenase activity was precipitated using an excess of anti-PGH synthase antibody; under similar conditions, immunoprecipitation of 60% of the diphenylisobenzofuran peroxidase, 75% of the phenylbutazone peroxidase, and 50% of the epinephrine peroxidase activities occurred. In contrast, less than 10% of the cyclooxygenase or peroxidase activities could be precipitated with nonimmune, control antibody preparations. These data indicate that the hydroperoxidase activity of PGH synthase is the major peroxidase activity catalyzing the co-oxidation of xenobiotics in ram seminal vesicle microsomes.

The in vivo metabolism of malondialdehyde (MDA) by male and female Swiss mice was investigated. Distribution of an i.p. dose of MDA is rapid and uniform throughout the body. Conversion of 14C-labeled MDA to CO2 is complete 4 hours after an i.p. dose of 5 mumol to 200 mumol with no signs of short term toxicity. The yields of CO2 from [1-14C]-beta-alanine, [3-14C]-beta-alanine, [1-14C]-sodium acetate, and [2-14C]-sodium acetate were also determined. Comparison of the yields of CO2 from this series of compounds suggests the intermediacy of malonic semialdehyde in the metabolism of MDA. High doses (600 mumol) of beta-alanine or acetate given prior to 14C-MDA reduced the yield of 14CO2. Ethanol and disulfiram were both inhibitors of MDA metabolism, indicating the involvement of aldehyde dehydrogenase in the oxidation of MDA. These data demonstrate the ability of animal tissues to rapidly remove exogenously administered MDA. They also have implications with respect to the possible pathological consequences of in vivo MDA generation.

Pulse radiolysis and electron spin resonance experiments have been performed on the antithrombotic and antimetastatic agent, nafazatrom. Results show that nafazatrom is an extremely reactive scavenger of free radicals. The rate of its reaction with Br-2 is higher than rates found for biologically important antioxidants, tocopherol and ascorbate. The radical formed by oxidation of nafazatrom is indicated by ESR to have a structure similar to phenoxyl radical. This radical is found to decay at a rate approaching diffusion controlled rates. The ease of oxidation of nafazatrom makes it ideally suited to act as an antioxidant. This property may be an important determinant of its pharmacological activities.

Prostaglandin H (PGH) synthase reacts with organic hydroperoxides and fatty acid hydroperoxides on a millisecond time scale to generate an intermediate that is spectrally similar to compound I of horseradish peroxidase. Compound I of PGH synthase is converted to compound II within 170 ms. Compound II decays to resting enzyme in a few seconds. Thus, the peroxidase reaction of PGH synthase appears to involve a cycle of native enzyme, compound I, and compound II, typical of heme-containing peroxidases. The Soret absorption maximum of compound I appears to occur at 412 nm but a small amount of compound II may be present. Soret maxima occur at 420, 433, and 419 for compound II, the ferrous enzyme, and the oxyferrous enzyme (compound III), respectively. Rapid scan analysis of the reaction of PGH synthase with arachidonic acid reveals the absorbance of compound II but no evidence for ferrous or oxyferrous enzyme.

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.

The microsomal fraction of sheep vesicular glands has been found to oxygenate 1,3-diphenylisobenzofuran, luminol, and the carcinogenic hydrocarbon benzopyrene when incubated with arachidonic acid. The oxygenations demonstrate an absolute dependence on enzyme and fatty acid and can be completely inhibited by indomethacin and 2,3-dimercaproptopanol, inhibitors of prostaglandin synthetase. The oxygenations can also be stimulated by the hydroperoxy endoperoxide, prostaglandin G2, and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. These latter reactions are not inhibited by indomethacin or dimercaptopropanol but do require the microsomal enzyme system. The involvement of superoxide anion in the transformations could not be demonstrated. The oxygenations occurring in the presence of arachidonic acid appear to arise via the interaction of a microsomal enzyme system with hydroperoxide intermediates of prostaglandin biosynthesis. The ability of various sulfur reagents (reduced glutathione, alpha-lipoic acid, methional) to inhibit co-oxygenation is probably related to their ability to stimulate the conversion of the intermediate to prostaglandins.

Strains of Salmonella typhimurium that carry a nonsense mutation at the site of reversion detect a variety of naturally occurring and synthetic carbonyl compounds as direct-acting mutagens. TA104 is reverted efficiently by formaldehyde, alpha, beta-unsaturated aldehydes (enals), and dicarbonyl compounds, such as diacetyl and glutaraldehyde. This strain is much more sensitive to carbonyl mutagenesis than is TA100, a strain previously reported to detect aldehydes as mutagens, or any other characterized strains of Salmonella. Long-chain enals are very toxic to TA104, but addition of a reduced glutathione chase following an incubation period decreases this toxicity, thus enabling the detection of 4-hydroxy-pentenal, a homolog of the lipid peroxidation product, 4-hydroxy-nonenal, as a mutagen. This is the first report of the mutagenicity of a hydroxy-enal, a class of enals produced by lipid peroxidation. Testing conducted with strains that carry the nonsense mutation in different repair backgrounds indicates that the presence of pKM101 and the deletion of the uvrB gene facilitate the detection of enals and dicarbonyls, but not malondialdehyde, as mutagens. Since carbonyl compounds are widely distributed in foods, are generated during cellular metabolism, and are present in body fluids, they may make a significant contribution to the risk of human cancer.

A Tween 20-solubilized preparation of prostaglandin endoperoxide synthase has been shown to metabolize 7,8-dihydrobenzo[a]pyrene (H2BP) to a form highly mutagenic to Salmonella typhimurium strain TA98. The arachidonic acid-dependent metabolism of H2BP by microsomal and purified prostaglandin endoperoxide synthase has been studied and the products identified. A spectral investigation of the metabolism indicated the bay-region double bond as the primary site of metabolism. Radiolabeled H2BP was synthesized and incubated with the enzyme preparations and the metabolites were separated by reverse phase high performance liquid chromatography and quantitated by liquid scintillation counting. Radioactive products were characterized by co-chromatography with chemically synthesized standards, UV-visible spectra, and mass spectrometry of acetate derivatives. The major polar products were determined to be trans- and cis-9,10-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8,9,10-tetrahydrobenzo[a]pyrene-9-one in a ratio of 1:1.2:0.4. The inclusion of 5 mM 3,3,3-trichloropropene-1,2-oxide, an epoxide hydrolase inhibitor, produced the same products but in a ratio of 1:2.3:1.2. Incubations with purified prostaglandin endoperoxide synthase yielded the three products in a ratio of 1:2.8:0.7. The major nonpolar product was identified as benzo[a]pyrene. The polar products of metabolism, the effects of 3,3,3-trichloropropene-1,2-oxide on their distribution, and the detection of a mutagenic intermediate support the conclusion that H2BP is co-oxygenated during prostaglandin biosynthesis to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

A method for the analysis of malondialdehyde (MDA) by ion-pairing HPLC is described. The method is direct, no derivitization is required, and sample preparation is minimal. After removal of particulates, the samples are injected directly onto an octadecylsilane column which is eluted with 14% (v/v) acetonitrile in 50 mM myristyltrimethylammonium bromide. 1 mM phosphate, pH 6.8. Detection is accomplished by monitoring absorbance at 254 nm or for greater sensitivity at 267 nm. The lower limit for reliable quantitation is 5 pmol MDA and the dynamic range extends to at least 4 nmol MDA. The method has been applied to the quantitation of MDA production during microsomal lipid peroxidation and to an assessment of the stability of MDA in microsomal and urine samples.

Lipid peroxidation triggered by ascorbate or reduced nicotinamide adenine dinucleotide in rat liver microsomes can initiate the epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. The stereochemistry of epoxidation is indicative of a peroxide-dependent free radical process. Since the epoxides formed may be the most carcinogenic derivatives of benzo[a]pyrene yet identified, lipid peroxidation can effect the metabolic activation of proximate carcinogens to ultimate carcinogens.

Reaction of 10-hydroperoxyoctadec-8-enoic acid (10-OOH-18:1) (50 microM) with hematin (0.5 microM) in sodium phosphate buffer containing Tween 20 (200 microM) generates 10-oxooctadec-8-enoic acid, 10-oxodec-8-enoic acid (10-oxo-10:1), and 10-hydroxyoctadec-8-enoic acid in relative yields of 79, 4, and 17%, respectively. The product profile and relative distribution are unaffected by 1 mM butylated hydroxyanisole. Approximately 5% of the hydroperoxide isomerizes from the 10- to the 8-position. 10-Oxo-10:1 most likely arises via beta-scission of an intermediate alkoxyl radical to the aldehyde and the n-octyl radical. To test this, 10-hydroperoxyoctadeca-8,12-dienoic acid was reacted with hematin under identical conditions. 10-Oxooctadeca-8,12-dienoic acid, 10-oxodec-8-enoic acid, and 10-hydroxyoctadeca-8,12-dienoic acid are formed in relative yields of 50, 45, and 5%, respectively. The product ratios are constant with time and hydroperoxide to catalyst ratio and unaffected by inclusion of phenolic antioxidants. The higher yield of 10-oxo-10:1 from 10-OOH-18:2 compared to 10-OOH-18:1 is due to the higher rate of beta-scission of the intermediate alkoxyl radical from the former to the resonance-stabilized octenyl radical. Two products of reaction of the 2-octenyl radical with O2, octenal and octenol, were detected in 10% yield relative to 10-oxo-10:1. Inclusion of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) led to epoxidation by both 10-OOH-18:1 and 10-OOH-18:2. Studies with isotopically labeled hydroperoxide or O2 indicated approximately 65% of the epoxide oxygen was derived from O2 and 35% from hydroperoxide oxygen, consistent with the involvement of peroxyl free radicals as the oxidizing agents. The available evidence indicates that hematin reduces the fatty acid hydroperoxides homolytically to alkoxyl radicals that are oxidized to ketones, reduced to alcohols, or undergo beta-scission to aldehydes. Carbon radicals generated during these reactions couple to O2, generating peroxyl free radicals that epoxidize BP-7,8-diol. The smaller percentage of epoxidation that results from hydroperoxide oxygen may arise from oxidation of the hydroperoxide group to peroxyl radicals or from heterolytic cleavage of the hydroperoxide to alcohol and an iron-oxo complex.

A new generation of heteroatom analogs of arachidonic acid are documented as powerful and selective inhibitors of the cytochrome P-450-dependent arachidonic acid oxygenase reaction (IC50, 5-10 microM) with little effect on either cyclooxygenase or soybean lipoxidase at 100 microM. The imidazole derivatives, ketoconazole and clotrimazole, are potent and selective inhibitors of the arachidonic acid epoxygenase and lipoxidase-like activities of phenobarbital-induced rat liver microsomal fractions (IC50, 2.0 and 0.3 microM, respectively). In contrast, the w/w-1 oxygenase activity of ciprofibrate-induced microsomal fractions was relatively resistant to inhibition by these compounds (IC50, 50 and 25 microM for ketoconazole and clotrimazole, respectively). Nordihydroguaiaretic acid (NDGA), eicosatetraynoic acid (ETYA), and indomethacin, extensively utilized inhibitors of the cyclooxygenase and lipoxygenase branches of the arachidonate cascade, also inhibit cytochrome P-450-dependent arachidonic acid metabolism. In decreasing order of potency, they were NDGA, ETYA, and indomethacin (IC50, 15, 40, and 70 microM, respectively).

The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.

Treatment of prostaglandin (PG)H synthase purified from ram seminal vesicle microsomes with trypsin cleaves the 70-kDa subunits into 33- and 38-kDa fragments (Chen, Y.-N. P., Bienkowski, M. J., and Marnett, L. J. (1987) J. Biol. Chem. 262, 16892-16899). In contrast to a minimal decrease in cyclooxygenase activity, peroxidase activity declines rapidly following trypsin treatment. The time course for loss of guaiacol peroxidase activity corresponds closely to the time course for protein cleavage. The ability of trypsin-treated enzyme to support catalytic reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of reducing substrates is significantly reduced. The products of metabolism of 10-hydroperoxy-8,12-octadecadienoic acid indicate that trypsin-treated enzyme catalyzes homolytic scission of the hydroperoxide bond in contrast to the heterolytic scission catalyzed by intact enzyme. Spectrophotometric titrations of hematin addition to trypsin-treated PGH synthase indicate approximately a 50% reduction in heme binding. These observations suggest that trypsin treatment of PGH synthase decreases the ability of the protein to bind prosthetic heme at a site that controls peroxidase activity. Comparison of the N-terminal sequence of the 38-kDa fragment of trypsin-treated PGH synthase to the amino acid sequence of the intact protein indicates that cleavage occurs between Arg253 and Gly254. Based on literature precedents and the results of the present investigations, we propose that the heme prosthetic group that controls the peroxidase activity of PGH synthase binds to the His residue of the sequence His250-Tyr251-Pro252-Arg253 located immediately adjacent to the trypsin cleavage site.

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.

Malondialdehyde and a series of acrolein derivatives substituted in the beta-position with good leaving groups react with guanine and guanine nucleosides to form two different types of adducts. The reaction with guanosine is typical. One adduct exhibits ultraviolet absorbance maxima at 253, 319, and 348 nm and is fluorescent. Its NMR spectrum exhibits three new aromatic proton resonances derived from malondialdehyde. The mass spectrum exhibits an M + 1 at 320. The spectroscopic properties are consistent with the structure, 3-beta-D-erythro-pentofuranosyl-pyrimido[1,2-alpha]purin-10(3H)-one (PyP-ribose). The second guanosine adduct is an equal mixture of diastereomers that exhibit ultraviolet maxima at 217 and 244 nm and mirror image circular dichroism spectra. The NMR spectrum and mass spectrum (M + 1 = 392) indicate the addition of two molecules of MDA to one molecule of guanosine. Two-dimensional NMR (COSY) analysis reveals the presence of propano and enal functionalities. The spectroscopic and chemical properties suggest an oxadiazabicyclo[3.3.1]nonene structure that is confirmed by X-ray crystallography. Comparison of the deoxyguanosine adducts of malondialdehyde to those of the structurally related carbonyl compounds, methyl glyoxal and acrolein, provides a structural basis to explain the unique ability of malondialdehyde to induce frameshift mutations in bacterial mutagenesis systems.

Enzyme preparations from sheep seminal vesicles display an enhanced ability to synthesize prostaglandins, particularly prostaglandin F from polyunsaturated fatty acids if alpha-lipoic acid is present in the incubation mixture prior to the addition of fatty acid. The stimulation by lipoate is reversible, time dependent, and involves modifications of V and Km for oxygenase activity. Product studies, structure vs. activity studies, and purification data indicate that lipoate exerts it effect by a mechanism distinct from a glutathione-like metabolism of the endoperoxide linkage in prostaglandin G and prostaglandin H. In addition, product studies suggest that lipoate is not a cofactor for the endoperoxide isomerase component of prostaglandin synthetase. Purification of the endoperoxide synthesizing activity by ion-exchange chromatography and isoelectric focusing yields preparations which are more responsive to lipoate than microsomal preparations.