Ram Seminal Vesicle Microsomes Research Articles

Identification of a Radical Formed in the Reaction Mixtures of Ram Seminal Vesicle Microsomes with Arachidonic Acid Using High Performance Liquid Chromatography-Electron Spin Resonance Spectrometry and High Performance Liquid Chromatography-Electron Spin Resonance-Mass Spectrometry

The reaction of ram seminal vesicle (RSV) microsomes with arachidonic acid (AA) was examined using electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance spectrometry (HPLC-ESR), and high performance liquid chromatography-electron spin resonance-mass spectrometry (HPLC-ESR-MS) combined use of spin trapping technique. A prominent ESR spectrum (αN = 1.58 mT and αHβ = 0.26 mT) was observed in the complete reaction mixture of ram seminal vesicle microsomes with arachidonic acid containing 2.0 mg protein/ml ram seminal vesicle (RSV) microsomal suspension, 0.8 mM arachidonic acid, 0.1 M 4-POBN, and 24 mM tris/HCl buffer (pH 7.4). The ESR spectrum was hardly observed for the complete reaction mixture without the RSV microsomes. The formation of the radical appears to be catalyzed by the microsomal components. In the absence of AA, the intensity of the ESR signal decreased to 16 ± 15% of the complete reaction mixture, suggesting that the radical is derived from AA. For the complete reaction mixture with boiled microsomes, the intensity of the ESR signal decreased to 49 ± 4% of the complete reaction mixture. The intensity of the ESR signal of the complete reaction mixture with indomethacin decreased to 74 ± 20% of the complete reaction mixture, suggesting that cyclooxygenese partly participates in the reaction. A peak was detected on the elution profile of HPLC-ESR analysis of the complete reaction mixture. To determine the structure of the peak, an HPLC-ESR-MS analysis was performed. The HPLC-ESR-MS analysis of the peak showed two prominent ions, m/z 266 and m/z 179, suggesting that the peak is a 4-POBN/pentyl radical adduct. An HPLC-ESR analysis of the authentic 4-POBN/pentyl radical adduct comfirmed the identification.

Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase

Benzidine and N-acetylbenzidine are activated to genotoxic metabolite(s) within the urothelial target tissue, with phase-I and phase-II enzymes being relevant. In principle, both benzidine and N-acetylbenzidine are activated by prostaglandin H synthase (PHS) to reactive intermediates. However, the relative impacts of benzidine and N-acetylbenzidine in this process remain unclear. Two experimental in vitro systems were used in the present comparative investigation: ram seminal vesicle microsomes rich in PHS and porcine urinary bladder epithelial cells (PUBEC) as a model system mimicking the general metabolic situation within the human urothelium. Benzidine, N-acetylbenzidine and N, N′-diacetylbenzidine were incubated with ram seminal vesicle microsomes and arachidonic acid and control incubations were performed with heat-inactivated microsomes. The metabolic disappearance of benzidine, N-acetylbenzidine or N, N′-diacetylbenzidine indicated a rapid turnover by PHS of benzidine and a slower turnover of N-acetylbenzidine. There was almost no PHS-associated metabolism of N, N′-diacetylbenzidine, suggesting that diacetylation of benzidine could represent a pathway of biological inactivation. Under similar conditions, incubations were performed with ram seminal vesicles and benzidine or N-acetylbenzidine upon addition of calf thymus DNA. After re-isolation of the DNA and 32 P -postlabeling, with benzidine 2 distinct adducts were found of unknown nature, and with N-acetylbenzidine a single adduct appeared with co-migrated with the N′-(3′-monophosphodeoxyguanosin-8-yl)- N-acetylbenzidine. PUBEC cells were also incubated with benzidine or N-acetylbenzidine. No DNA adduct was found with benzidine, but a total of five adducts was produced from N-acetylbenzidine. The major adduct again co-migrated with N′-(3′-monophosphodeoxyguanosin-8-yl)- N-acetylbenzidine. When benzidine was incubated with PUBEC cells N-acetylbenzidine and, with some delay, N, N′-diacetylbenzidine were formed. Application of Lineweaver–Burk plots for the formation of N-acetylbenzidine from benzidine revealed a K m of 56.4 μM and a V max of 7.05 nmol/h per 10 6 PUBEC cells. The investigations generally support a key role of N-acetylbenzidine at the target site of the urothelium.

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase — 32P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the 32P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin- N 6-yl)aristolactam I, 7-(deoxyguanosin- N 2-yl)aristolactam I and 7-(deoxyadenosin- N 6-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin- N 2-yl)aristolactam II and 7-(deoxyadenosin- N 6-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (α-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers.

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 microM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 microM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(2) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 microM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11 (E) CLA isomer being the most effective and the 9(Z),11 (Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The Ki values obtained by Dixon replots range from 18.7 microM for the most effective isomer, 9(Z),11 (E) CLA, to 105.3 microM for the least effective isomer, 9(2),11(2) CLA. The Ki value for flurbiprofen with ram seminal vesicle microsomes was 0.33 microM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.

Prostaglandin H synthase-2 inhibitors interfere with prostaglandin H synthase-1 inhibition by nonsteroidal anti-inflammatory drugs

Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with 100 nM of the prostaglandin H synthase-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) prior to exposure to the prostaglandin H synthase inhibitors aspirin, indomethacin, ibuprofen or naproxen. Activity of the enzyme was measured by following the conversion of arachidonic acid to prostaglandin E 2 and prostaglandin F2α. Although prostaglandin H synthase-1 activity was not altered by these concentrations of the prostaglandin H synthase-2 inhibitors, it was found that exposure to these agents prior to aspirin or indomethacin (irreversible prostaglandin H synthase inhibitors) significantly attenuated the inhibition obtained by the latter inhibitors. On the other hand, the same concentrations of the prostaglandin H synthase-2 inhibitors did not interfere with prostaglandin H synthase-1 inhibition that was induced by naproxen or ibuprofen (competitive prostaglandin H synthase inhibitors). Attenuation of the indomethacin inhibition of prostaglandin H synthase-1 by prostaglandin H synthase-2 inhibitors was observed only when the microsomes were pre-exposed to DuP-697 or NS-398 in the absence, but not in the presence, of arachidonic acid. The effect of DuP-697 was found to be irreversible, however, washing away the agent reversed the action of NS-398. Similar phenomena have been reported by us in bovine aortic endothelial cells and in human dermal fibroblasts. Attenuation of the inhibition by aspirin and indomethacin, without altering the enzyme's basal activity or the inhibition induced by ibuprofen or naproxen may suggest the possibility that the prostaglandin H synthase-2 specific inhibitors DuP-697 and NS-398 affect prostaglandin H synthase-1 by interaction with a site different from the enzyme's catalytic site.

PGHS-2 inhibitors, NS-398 and DuP-697, attenuate the inhibition of PGHS-1 by aspirin and indomethacin without altering its activity

Since the discovery of the inducible form of prostaglandin (PG) H synthase (PGHS), PGHS-2, considerable effort has been made to design selective inhibitors of this isozyme. N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) have been shown to interact reversibly with PGHS-1, while irreversibly inhibiting PGHS-2 in a time-dependent manner. In the present study we have tested the effects of DuP-697 and NS-398 on the activity of PGHS-1 and further explored the interactions between these agents and the inhibition of PGHS-1 by aspirin, indomethacin and ibuprofen. Three independent experimental systems, namely bovine aortic endothelial cells (BAEC), human fibroblasts and ram seminal vesicle microsomes were used to investigate the effects of DuP-697 and NS-398 on PGHS-1. The results show that DuP-697 and NS-398, at concentrations ranges which do not inhibit PGHS-1 activity, significantly attenuated the inhibition of PGHS-1 that was caused by aspirin and indomethacin. The same concentrations of DuP-697 and NS-398 did not affect the inhibition of PGHS-1 that was induced by the competitive reversible inhibitors ibuprofen and naproxen. Similar effects of DuP-697 and NS-393 were obtained with ram seminal vesicle microsomes. These results suggest that PGHS-2 inhibitors DuP-697 and NS-398 possibly interact with PGHS-1 at a site different from the enzyme’s catalytic site, thus causing attenuation of PGHS-1 inhibition by aspirin and indomethacin without altering PGHS-1 basal activity or the ibuprofen-induced inhibition.

Peroxygenase Metabolism of N-Acetylbenzidine by Prostaglandin H Synthase: FORMATION OF AN N-HYDROXYLAMINE

Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product, 4'-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mM ascorbic acid was present. Similar results were observed whether 0.2 mM arachidonic acid or 0.5 mM H2O2 was used as the substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by electrospray ionization/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4'-nitro-4-acetylaminobiphenyl, but not for N'HA. A 97% enrichment was observed for one atom of 18O, and a 17 +/- 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N'HA by PHS and suggest a stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products.

N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism.

N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-post-labeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared with that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-post-labeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and were shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product that eluted at the same time from the HPLC (32P-dpG-ABZ). Additional experiments demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced approximately 40-fold more dGp-ABZ than horseradish peroxidase (1943 +/- 339 versus 49 +/- 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e. arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different.

Free radical oxidation of (E)-retinoic acid by prostaglandin H synthase.

Cooxidative metabolism of all-trans (E)-retinoic acid (RA) by prostaglandin H synthase was investigated employing ram seminal vesicle microsomes (RSVM) or purified, RSVM-derived enzyme. RA was shown to undergo hydroperoxide [H2O2 or 5-phenyl-4-penten-1-yl hydroperoxide (PPHP)]- or arachidonic acid-dependent cooxidation by microsomal prostaglandin H (PGH) synthase as evidenced by UV spectroscopic analysis of reaction mixtures. Cooxidation of RA by microsomal or purified PGH synthase, using PPHP as substrate, was characterized by uptake of dioxygen which was first order with respect to enzyme concentration. Dioxygen uptake was inhibited by the peroxidase reducing substrate 2-methoxyphenol. In addition, O2 uptake was inhibited by the spin trap nitrosobenzene. ESR spin trapping studies, using alpha-phenyl-N-tert-butylnitrone (PBN) as the spin trap, demonstrated the formation of RA-PBN adducts, characterized by hyperfine coupling constants of alpha H = 3.2 G and alpha N = 15.8 G. Reverse phase HPLC analysis of reaction mixtures demonstrated the formation of 4-hydroxy-RA, 5,6-epoxy-RA, 4-oxo-RA, (13Z)-retinoic acid, and other geometric isomers which were identified on the basis of cochromatography with synthetic standards, UV spectroscopy, and/or mass spectrometry. Mechanisms are proposed for the hydroperoxide-dependent, PGH synthase-catalyzed oxidation of RA that are consistent with these results.

An extract of Desmodium adscendens activates cyclooxygenase and increases prostaglandin synthesis by ram seminal vesicle microsomes

AbstractMicrosomes from ram seminal vesicles were used as a source of cyclooxygenase enzyme to evaluate the effect of an n‐butanol fraction of Desmodium adscendens (nBF) on prostaglandin (PG) synthesis. 1‐14C‐arachidonic acid (14C‐AA) conversions into prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2a) and the endoperoxide intermediate, prostaglandin H2 (PGH2) were analysed radiometrically and compared among reaction mixtures with different concentrations of the cyclooxygenase enzyme, with and without GSH and with or without preincubation with nBF, tyramine and hordenine, phenolic compounds present in D. adscendens. The percentage of 14C‐AA converted in the various reaction mixtures was used to indicate activation of cyclooxygenase enzyme (Cox). nBF activated Cox at sub‐optimal enzyme concentrations and increased PG synthesis at all enzyme concentrations used, shifting the increased production in favour of PGE2 under certain conditions. Tyramine was more effective than nBF in activating Cox. The results indicate that D. adscendens contains compounds, other than phenolics, which modulate PG synthesis and could lead to production of relaxant prostanoids.

Prostaglandin-H synthase mediated metabolism and mutagenic activation of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ).

Prostaglandin-H synthase (PHS), a mammalian peroxidase of interest for the extrahepatic formation of reactive intermediates of carcinogens, catalyzes in vitro the metabolic activation of the mutagen and carcinogen 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ). Incubation of 14C-labeled IQ with ram seminal vesicle microsomes (RSVM), a rich source of PHS, resulted in protein binding and generated products mutagenic in S. typhimurium YG1024. The mutagenic activity produced in IQ/PHS incubations was stable and extractable with ethyl acetate. Upon fractionation of such extracts by HPLC and subsequent analysis, two metabolites were identified as 2,2'-azo-bis-3-methylimidazo[4,5-f]quinoline (azo-IQ) and 3-methyl-2-nitro-imidazo[4,5-f]quinoline (nitro-IQ) confirmed by comparison of HPLC retention times, UV/VIS-, 1H-NMR-spectroscopy, and mass spectrometry of synthesized standards. Azo-IQ was obtained by chemical oxidation of IQ with meta-sodium periodate. It was the major metabolite in PHS incubations, but has not been detected in monooxygenase incubations. Azo-IQ, without metabolic activation, was much less mutagenic in S. typhimurium YG1024 (308 rev/nmol) than nitro-IQ and 3-methyl-2-nitroso-imidazo[4,5-f]quinoline (nitroso-IQ), two other S9-independent mutagens which have been synthesized by chemical oxidation of IQ with sodium nitrite. Nitro-IQ was formed only in trace amounts but due to its potent mutagenicity in S. typhimurium YG1024 (2 x 10(6) rev/nmol) it accounted for most of the mutagenic activity of the incubations. These data show that PHS-mediated in vitro metabolism of IQ results in its metabolic activation; thus PHS may contribute to the genotoxicity of IQ in extrahepatic tissues.

Protection of conjugated linoleic acids against 2-amino-3- methylimidazo[4,5-f]quinoline-induced colon carcinogenesis in the F344 rat: a study of inhibitory mechanisms.

Grilled ground beef contains a number of heterocyclic amine carcinogens, such as 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), as well as anticarcinogenic conjugated linoleic acids (CLA). In the present study, CLA was administered to male F344 rats by gavage on alternating days in weeks 1-4, while IQ was given by gavage every other day in weeks 3 and 4 (100 mg/kg body wt). Rats were killed 6 h after the final carcinogen dose 16 in order to score colonic aberrant crypt foci (ACF). In the ACF study, CLA had no effect on the size of the foci, but inhibited significantly (P < 0.05) the number of ACF/colon, from 4.3 +/- 2.4 in controls to 1.1 +/- 1.3 in CLA-treated rats (mean +/- SD, n = 10). Rats given CLA also had significantly lower IQ-DNA adducts in the colon as determined by 32P-postlabeling analysis; relative adduct labeling levels (RAL x 10(7) for the major adduct were 9.13 +/- 2.6 in controls versus 5.42 +/- 1.8 in CLA-treated animals (P < 0.05). Mechanism studies indicated that CLA and other fatty acids interact with certain heterocyclic amines in a manner consistent with substrate-ligand binding. However, no such interaction occurred with IQ, and CLA failed to inhibit significantly the mutagenicity of N-hydroxy-IQ in the Salmonella assay. Liver microsomes from CLA-treated rats exhibited lower activities for dealkylation of 7-ethoxyresorufin and methoxyresorufin and activated IQ to DNA binding species less effectively than microsomes from control animals. Direct addition of CLA to the in vitro incubation inhibited IQ-DNA binding and was associated with increased recovery of unmetabolized parent compound. In the Salmonella assay, CLA inhibited the mutagenic activity of IQ in the presence of S9 or ram seminal vesicle microsomes. Collectively, these results support a mechanism involving inhibition of carcinogen activation by CLA, as opposed to direct interaction with the procarcinogen, scavenging of electrophiles or selective induction of phase I detoxification pathways.

Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo[4,5- f]quinoline (nitro-IQ) from IQ

The mutagenic effects of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) following activation by ram seminal vesicle microsomes (RSVM, a source of prostaglandin H synthase, PHS) were studied in Salmonella typhimurium tester strains possessing elevated levels of acetyl-CoA:arylamine N-acetyltransferase (NAT). The metabolites formed by RSVM were extracted and fractionated by high pressure liquid chromatography (HPLC). One isolable product accounted for most of the direct-acting mutagenicity observed in the extracts. The metabolite was identified as 2-nitro-3-methylimidazo[4,5- f]quinoline (nitro-IQ). Since nitro-IQ is a potent direct-acting mutagen, its role in IQ genotoxicity warrants further study.

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho -anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N -acetyltransferase (YG1029). p -Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o -anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p -anisidine was greater than with o -anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p -anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N -acetyltransferase enzymes play an important role in their metabolism to mutagenic species.

Mutagenic activation of benzidine requires prior bacterial acetylation and subsequent conversion by prostaglandin H synthase to 4-nitro-4'-(acetylamino)biphenyl.

We have used the Ames test in combination with prostaglandin H synthase (PHS) to study the bioactivation of benzidine as well as other aromatic amines. Previous investigations established that the formation of benzidine mutagens by PHS is dramatically enhanced in Salmonella typhimurium strains with high levels of acetyl CoA-dependent arylamine N-acetyltransferase/arylhydroxylamine O-acetyltransferase activity despite the fact that acetylation of aromatic amines decreases their susceptibility to oxidation by peroxidases. In this study, we used a new strain (YG1012) that has very high acetylation capability to investigate the metabolism and mutagenicity of benzidine and N-acetylbenzidine catalyzed by PHS (from ram seminal vesicle microsomes) and horseradish peroxidase (HRP). YG1012 bacteria rapidly acetylated benzidine to N-acetylbenzidine and N,N'-diacetylbenzidine. Preincubation of the bacteria with benzidine before addition of PHS increased the mutagenicity. Under conditions identical to those used to assess mutagenicity, PHS metabolized benzidine rapidly, but the substrate was not totally consumed, with about 40% of the original concentration remaining intact. These data suggest that conversion to N-acetylbenzidine may be the initial step in the bioactivation of benzidine in the PHS-mediated Ames assay. N-Acetylbenzidine is a cosubstrate for PHS peroxidase activity as measured by 5-phenyl-4-pentenyl hydroperoxide reduction, spectral changes, and formation of protein adducts. N-Acetylbenzidine was converted to mutagens by PHS but not HRP, with enhanced mutagenicity observed in bacteria with high acetylation activity. We used reverse- phase HPLC to characterize the metabolites of N-acetylbenzidine formed by PHS and HRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.

Modification of benzo(a)pyrene induced chromosomal damage in mouse bone marrow by Vitamin A

Benzo(a)pyrene (BP) is one of the major atmospheric pollutants emitted in coal furnaces, automobile exhausts etc. It exerts its mutagenic/carcinogenic effects only after metabolic activation (Kliesch et al 1982). The metabolic conversion of BP and majority of polycyclic aromatic hydrocarbons is catalysed in vivo by the microsomal mixed function oxydase system. It was reported that during the process of oxygenation of BP leading to the formation of the ultimate mutagenic product, arachidonic acid (AA) was converted to prostaglandins (PGs) of 2 series (Sivarajah et al 1978). Further, the oxygenated product of BP namely 7,8-dihydrodiol was mutagenic to Salmonella typhimurium TA98 on incubation with ram seminal vesicle microsomes in the presence of AA (Marnett et al 1978).

Evaluation of nitrofurantoin on the two stages of urinary bladder carcinogenesis in the rat

The possibility that nitrofurantoin is a complete carcinogen or is an initiator or promoter of urinary bladder carcinogenesis was evaluated in male weanling F344 rats. No increase in tumor incidence was observed in rats fed nitrofurantoin at a level of 0.18% of the diet for 2 years compared to a control group. Also, no evidence of bladder initiating activity by nitrofurantoin was observed using sodium saccharin (5% of the diet) as a promoter, and no promoting activity was observed when nitrofurantoin was fed after initiation by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (0.2% of the diet for 4 weeks). In a second experiment, nitrofurantoin (at a dose of 0.187% of the diet) was administered for 6 weeks to rats with a rapidly proliferating bladder epithelium following freeze ulceration, and then the rats were treated with 5% sodium saccharin in the diet for 98 weeks. In additional rats, labelling index following [ 3H]thymidine injection, determined after 12 weeks of feeding nitrofurantoin, was not increased above control levels in the urinary bladder, stomach, duodenum, or liver. Metabolism of nitrofurantoin by prostaglandin H synthase (PHS) was examined using solubilized ram seminal vesicle microsomes. The rate of nitrofurantoin metabolism by PHS was much less than that observed with benzidine, and the proportion of total metabolite bound to protein was also much less than that with benzidine. These results are consistent with previous reports describing the lack of effect of nitrofurantoin on urinary bladder carcinogenesis.

Alkylaryl sulfides as Peroxidase Reducing Substrates for Prostaglandin H Synthase: Probes for the Reactivity and Environment of the Ferryl-Oxo Complex

Abstract 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.

Metabolism of 20-hydroxyeicosatetraenoic acid by cyclooxygenase: Formation and identification of novel endothelium-dependent vasoconstrictor metabolites

Abstract We recently demonstrated that 20-hydroxyeicosatetraenoic acid (20-HETE) constricts rat aortic rings. The contractile response was partially dependent on the presence of endothelium and was abolished by pretreatment of the rings with either indomethacin or the endoperoxide/thromboxane receptor antagonist, SQ29548. Addition of GSH or SnCl2 to the organ bath diminished the contractile response of 20-HETE, whereas preincubation of the rings with a thromboxane synthase inhibitor did not affect the 20-HETE induced contractions. Short time incubation (2 min) of 20-HETE with ram seminal vesicle microsomes in the presence of p-hydroxymercurybenzoate yielded metabolites which migrated similarly on thin layer chromatography to the known arachidonate endoperoxides prostaglandin (PG) G2 and PGH2 and possess vasoconstrictory properties. The vasoconstriction was dose-dependent with a half-life of approximately 6.3 +/- 0.6 min. Addition of SQ29548 to the aortic ring bath 1 min after metabolite elicited vasoconstriction produced immediate relaxation. Furthermore, pretreatment of the rings with SQ29548 totally abolished the contraction. SnCl2 reduction of the metabolites produced in incubation of rat seminal vesicles with 20-HETE and p-hydroxymercurybenzoate resulted in a single radioactive peak which was further identified by gas chromatography/mass spectrometry as 20-hydroxy-PGF2 alpha. The inhibitory effect of SQ29548, the appearance of labile metabolites with a half-life of approximately 6 min and the production of 20-hydroxy-PGF2 alpha by SnCl2 reduction clearly indicate that the vasoconstrictor metabolites of 20-HETE are the labile endoperoxides of 20-HETE, 20-hydroxy-PGG2, and 20-hydroxy-PGH2.