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P D Josephy

Publications and source records attributed to P D Josephy.

At least 55 records · Page 3Linked to original sources
PubMed1989

Prostaglandin H synthase-dependent mutagenic activation of benzidine in a Salmonella typhimurium Ames tester strain possessing elevated N-acetyltransferase levels.

Watanabe and colleagues (Biochem. Biophys. Res. Commun. 147: 974-979, 1987) have constructed plasmid-containing derivatives of Salmonella typhimurium Ames tester strain TA1538 with high levels of acetyltransferase activities. In this paper, we describe the mutagenic response of one of these strains, TA1538/1,8-DNP6 (pYG 121), to the bladder carcinogen benzidine and other arylamines. Strain TA1538/1,8-DNP6 (pYG 121) was far more sensitive to benzidine than any previous tester strain, following metabolism of the aromatic amine by hamster hepatic S9, ram seminal vesicle microsomal preparation (RSVM), or purified prostaglandin synthase. Therefore, bacterial acetyltransferase-dependent metabolism of a proximate mutagen is implicated in each of these systems. The mechanism of RSVM-dependent activation of benzidine was examined further. The arachidonic acid-independence and indomethacin insensitivity previously noted with strain TA98 were also observed with the new tester strain. We confirmed that prostaglandin H synthase is the enzyme activity responsible for activation of benzidine by RSVM. Purified prostaglandin H synthase holoenzyme, or apoenzyme reconstituted with heme, supported benzidine activation. However, apoenzyme reconstituted with manganese protoporphyrin IX, which yields enzyme having cyclooxygenase activity but not peroxidase activity, was inactive. Addition of catalase inhibited, and addition of exogenous hydrogen peroxide increased, RSVM-mediated benzidine mutagenicity. We propose that hydrogen peroxide released by the tester strain bacteria (rather than arachidonic acid-derived peroxide) is the oxidizing agent which supports prostaglandin H synthase peroxidase activity in Ames test systems.

Acetyltransferases↗
PubMed1989

Binding of chlorinated benzidines to the rat hepatic aromatic hydrocarbon receptor.

The binding of benzidine, 3,3'-dichlorobenzidine (3,3'-Cl2BZ), and the asymmetrically-substituted chlorinated benzidines 3,5-dichlorobenzidine (3,5-Cl2BZ) and 3,5,3'-trichlorobenzidine (Cl3BZ) to the rat hepatic cytosolic aromatic hydrocarbon (Ah) receptor was measured, in order to assess the mechanism of P-450I induction by 3,3'-Cl2BZ. Cl3BZ is the most mutagenic benzidine derivative in the Ames assay. Binding affinity to the Ah receptor protein was determined by displacement of labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the receptor, measured with the sucrose density gradient centrifugation technique. The rank order of affinities and the apparent inhibitor constants were: Cl3BZ (4 microM) greater than 3,5-Cl2BZ (8.4 microM) greater than 3,3'-Cl2BZ (10 microM). Benzidine did not displace TCDD from the receptor protein. 4-Aminobiphenyl a structural link between the benzidine and biphenyl series competed weakly with TCDD. The 50% inhibition concentration was about 150 microM. The results are consistent with the hypothesis that the induction of P-450 enzymes by 3,3'-Cl2BZ in vivo is mediated by the Ah receptor.

3,3'-Dichlorobenzidine↗
PubMed1989

Activation of aromatic amines by prostaglandin H synthase.

Prostaglandin H synthase catalyzes the first step in the synthesis of prostaglandins from arachidonic acid. The peroxidase activity of this enzyme can support the oxidation of xenobiotics, particularly aromatic amines. This pathway of metabolism may contribute to the activation of carcinogenic aromatic amines in target tissues such as the skin, lung, and bladder. In this review, recent work on this subject is summarized. I emphasize the elucidation of the structures of aromatic amine oxidation products, and their interactions with biological macromolecules. Prostaglandin H synthase supports the activation of benzidine to a mutagenic species in the Ames (Salmonella typhimurium) test, and our studies of the mechanism of this activation are described.

Amines↗
PubMed1989

Prostaglandin hydroperoxidase-dependent activation of heterocyclic aromatic amines.

Heterocyclic aromatic amines, derived from the pyrolysis of amino acids and proteins, are potent mutagens in the Ames Salmonella assay with rodent liver activation. Additionally, heterocyclic aromatic amines are multipotent carcinogens. We report evidence that these compounds are substrates for the hydroperoxidase activity of prostaglandin H synthase, as measured by alterations in UV/visible spectra, and are bioactivated to macromolecule-reactive species by this enzyme. Indirect electron paramagnetic resonance studies indicate that this activation may occur via a one-electron mechanism. 2-Amino-3-methylimidazo[4,5f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) are direct-acting mutagens in TA98. The mutagenicity of IQ and MeIQ, but not Trp-P-2, were enhanced by activation with ram seminal vesicle microsomes (a rich source of prostaglandin H synthase). Subsequent experiments utilized the newly constructed tester strain TA1538/1,8-DNP6 (pYG 121), which has enhanced arylamine N-acetyltransferase activity. In this strain IQ, MeIQ and 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) were mutagenic with ram seminal vesicle microsome activation. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was a weak direct-acting mutagen, and was not activated by the ram seminal vesicles (RSV) system. The responses of IQ and MeIQ were markedly enhanced in TA1538/1.8-DNP6 (pYG 121), relative to TA98. These data are consistent with the involvement of prostaglandin H synthase-catalyzed activation in heterocyclic aromatic amine-induced extrahepatic neoplasia.

Amines↗
PubMed1989

Re-evaluation of the effect of ellagic acid on dimethylnitrosamine mutagenicity.

Dimethylnitrosamine (DMN) is activated to mutagenic species in the Ames test (Salmonella typhimurium strain TA100) by hamster hepatic S9 preparation. This S9 activity is induced by administration of ethanol to the animals. The organic solvents dimethylsulphoxide (DMSO) and N-methyl-2-pyrrolidinone (MP) inhibit this mutagenicity, apparently because they inhibit DMN demethylase activity (assayed as formaldehyde production). Ellagic acid, dissolved in DMSO or MP, had no inhibitory effect on DMN mutagenicity, beyond the effect of the solvent vehicle.

Animals↗
PubMed1988

Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material.

Ram seminal vesicle (RSV) microsomal preparations activate benzidine and other arylamines to mutagenic species in a modified Ames assay. We have examined the mechanism of this activation process in more detail. The mutagenic effect was neither arachidonic acid-dependent nor indomethacin inhibitable. The mutagenic species was stable for at least 30 min in experiments in which addition of bacteria was delayed. Acetylbenzidine was a much more potent mutagen than benzidine in this system. Substitution of the acetylase-deficient tester strain TA98/1,8-DNP6 for strain TA98 markedly reduced the mutagenicity of acetylbenzidine and completely eliminated the mutagenicity of benzidine. Benzidine analogues 3,3'-dimethoxybenzidine (o-dianisidine), o-tolidine and 3,3',-5,5'-tetramethylbenzidine were not mutagenic in the RSV activation system. RSV-dependent activation of all radiolabeled congeners examined resulted in covalent binding to calfthymus DNA. The rank order of binding was: 3,3'-dichlorobenzidine greater than benzidine greater than o-dianisidine greater than acetylbenzidine greater than tetramethylbenzidine. This binding required active enzyme and arachidonic acid or hydrogen peroxide. The reactive species was short-lived: delayed addition of DNA reduced the level of binding nearly to zero. Binding was inhibitable by indomethacin, but this inhibition was incomplete in the cases of dichlorobenzidine and acetylbenizidine. We conclude that the extracellular generation of peroxidase-catalyzed oxidation products does not explain the RSV microsome-dependent mutagenicity observed with these compounds.

Animals↗
PubMed1987

Peroxidative metabolism of benzidine derivatives by Salmonella typhimurium.

Benzidine and several derivatives are activated to mutagenic species in an H2O2-dependent Ames test system. Optical and electron paramagnetic resonance (EPR) spectroscopy are employed in studies of the H2O2-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, and provide direct evidence for peroxidase activity in Salmonella typhimurium. The acetylase-proficient Ames tester strain TA98 and its acetylase-deficient derivative TA98/1,8-DNP6 are equally responsive to H2O2-dependent mutagenicity; enzymatic acetylation appears not to be involved in activation of benzidine, in this system. The H2O2-dependent mutagenicity of benzidine and oxidation of TMB are observed when the assays are carried out in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethane sulfonic acid (MES) buffer, at the same pH. This difference is interpreted in terms of the effects of these buffers on the intracellular pH of the bacteria. The H2O2-dependent mutagenicity of several benzidine congeners is also described.

Benzidines↗
PubMed1987

Benzidine activation in the Ames test: roles of hepatic N-acetyltransferase and other cytosolic and microsomal factors.

Benzidine (BZ) is a known animal and human carcinogen, and is mutagenic in the Ames test using strain TA 98. Several workers have shown that hepatic S9 fraction from hamster is much more effective than is rat S9, as an activation system for BZ in the Ames test. We show that rat microsomal fraction inhibits hamster S9 activation of BZ. Hamster microsomal fraction, supplemented with glucose-6-phosphate dehydrogenase (G6PdeH), gives a BZ dose-dependent mutagenic response, in the absence of cytosolic fraction. Rat microsomal fraction, in contrast, gives relatively little activation, under comparable conditions. Activation was enhanced when hamster or rat cytosol was added back to a mixture of hamster microsomes and G6PdeH. When strain TA 98 was replaced by strain TA 98/1,8-DNP6, very little activation of BZ was observed. Partially purified mouse liver acetyltransferase effectively activated BZ to mutagenic products in the presence of acetyl coenzyme A (CoASAc)/hamster microsomes/G6PdeH. Hamster and rat liver cytosol contain a CoASAc-dependent as well as a CoASAc-independent cytosolic activating factor of BZ. Hamster but not rat microsomal activation of BZ is enhanced in the presence of CoASAc. The biochemical mechanisms of BZ activation in the Ames test are discussed in light of these results.

Acetyl Coenzyme A↗
PubMed1987

Synthesis and mutagenicity of 3-halogenated and 3,3',5,5'-tetrahalogenated benzidines.

3-Fluorobenzidine (FBz), 3-chlorobenzidine (ClBz), 3-bromobenzidine (BrBz), 3,3',5,5'-tetrafluorobenzidine (F4Bz), and 3,3',5,5'-tetrachlorobenzidine (Cl4Bz) were synthesized and tested for their ability to revert Salmonella typhimurium. F4Bz was the only compound to show direct-acting mutagenicity and was equally potent in strain TA98 and the acetylase-deficient strain TA98/1,8-DNP6. In the presence of hamster liver S9, all compounds except Cl4Bz were mutagenic. The relative mutagenicities in TA98 were FBz greater than ClBz greater than BrBz greater than F4Bz greater than Bz greater than Cl4Bz = 0. In TA98/1,8-DNP6 the trend was F4Bz approximately BrBz approximately ClBz greater than FBz greater than Bz greater than Cl4Bz = 0.

Animals↗
PubMed1987

Unambiguous synthesis of asymmetrically substituted chlorinated benzidines, and a study of their mutagenicity in the Ames test: potent activity of 3,5,3'-trichlorobenzidine.

3,5-Dichlorobenzidine and 3,5,3'-trichlorobenzidine were prepared by the reaction of 2,6-dichloronitrosobenzene with aniline and 2-chloroaniline, respectively, to give the appropriate substituted azobenzene; reduction and rearrangement gave the desired benzidine derivatives. The products were purified and characterized by mass spectrometry and [1H] nuclear magnetic resonance spectroscopy. The mutagenic activities of the chlorinated benzidines were determined, using the Ames tester strain TA98. 3,5,3'-Trichlorobenzidine is the most potent benzidine derivative yet reported. The activities of five chlorinated benzidines are compared, and rules for the effect of substitution on activity are discussed.

Animals↗
PubMed1987

Studies on the mechanism of action of diallyl sulfide, an inhibitor of the genotoxic effects of cyclophosphamide.

Diallyl sulfide, a component of garlic oil, has been previously shown to inhibit induction of nuclear aberrations in the colons of mice treated with 1,2-dimethylhydrazine, a colon carcinogen. The ability of this agent to block cyclophosphamide-induced nuclear aberrations in urinary bladder and hair follicles was tested. [14C]Cyclophosphamide was injected and urine was collected to determine the disposition of metabolites of cyclophosphamide in mice treated with diallyl sulfide. This agent was capable of blocking nuclear aberration induction by cyclophosphamide in bladder and hair follicles. The effect was not mediated through inhibition of mitosis as determined by [3H]thymidine autoradiography in hair follicles. Diallyl sulfide pretreatment decreased the amount of radioactivity excreted in the urine in the first 24 h following cyclophosphamide treatment and blocked the appearance of acrolein, a cytotoxic metabolite of cyclophosphamide, in the urine over this time period. These results suggest that diallyl sulfide acts by conjugating the toxic metabolites of cyclophosphamide, thereby limiting their systemic circulation and diverting their route of excretion from the urine.

Allyl Compounds↗
PubMed1986

Mutagenicity of thionitrites in the Ames test. The biological activity of thiyl free radicals.

Thiols, such as glutathione and cysteine, are mutagenic in the Ames test, using Salmonella typhimurium strain TA 100 and rat kidney S-9 preparation [Glatt et al. Science 220, (1983)]. Formation of thiyl free radicals, RS., has been implicated in this effect. We have prepared thionitrite (nitrosylmercaptan) derivatives of glutathione and other thiols. These unstable derivatives decompose by homolysis, yielding RS. radicals. Glutathione thionitrite is mutagenic to strains TA 100 and TA 102, in the absence of activation by mammalian S-9 preparations. We suggest that this mutagenicity is evidence for the role of thiyl free radicals as biological reactive intermediates. Since alkyl nitrites readily convert thiols to thionitrites, our findings have implications for the toxicology of nitrosating drugs, such as amyl nitrite.

Mutagenicity Tests↗
PubMed1986

Comparative metabolism and mutagenicity of azo and hydrazone dyes in the Ames test.

Enteric bacterial and hepatic azoreductase enzymes are capable of reducing azo dyes to yield the constituent aromatic amines. Azo dyes based on benzidine and benzidine congeners have received particular attention because of their widespread use and the known carcinogenicity of benzidine to humans. Azo dyes based on beta-diketone coupling components exist preferentially as the tautomeric hydrazones. A series of hydrazone dyes based on benzidine and benzidine congeners was prepared and characterized by NMR and UV-visible spectroscopy. These dyes were tested for mutagenicity using a modified Ames assay and, unlike the true azo dyes, showed no significant mutagenic activity. The hydrazone dyes were resistant to enzymatic reduction by FMN-supplemented hamster-liver post-mitochondrial supernatant (S-9); under identical conditions, azo dyes such as trypan blue were rapidly reduced.

Animals↗
PubMed1986

Synthesis and mutagenicity of 3,3'-dihalogenated benzidines.

3,3'-Difluorobenzidine (F2Bz), and 3,3'-dibromobenzidine (Br2Bz) were synthesized and compared with 3,3'-dichlorobenzidine (Cl2Bz) for ability to revert Salmonella typhimurium. The relative mutagenicities in all systems are Cl2Bz approximately equal to Br2Bz greater than F2Bz greater than Bz. F2Bz, Cl2Bz, and Br2Bz are direct-acting mutagens towards S. typhimurium strain TA98. The acetylase-deficient derivative TA98/1,8-DNP6 displays no resistance to induction of mutagenesis by these compounds, in the absence of mammalian activation. With addition of hamster hepatic S-9 activation the mutagenicity of these compounds increases greatly. TA98/1,8-DNP6 shows some resistance to this mutagenicity. Multiple mechanisms must exist for the genotoxicity of 3,3'-dihalogenated benzidines.

3,3'-Dichlorobenzidine↗
PubMed1986

Benzidine: mechanisms of oxidative activation and mutagenesis.

Benzidine oxidative activation may proceed by peroxidase-catalyzed one-electron oxidation via free radical intermediates, or by N-acetylation followed by monooxygenase-catalyzed N-hydroxylation. The peroxidase route has been examined by using horseradish peroxidase or prostaglandin H synthase in vitro. In the presence of nucleophiles such as phenols, thiols, or nucleic acids, isolable adducts are formed. The structures of these adducts have been elucidated by spectroscopic methods. The Ames test provides a useful system for studying benzidine bioactivation to mutagenic intermediates. An endogenous bacterial acetylase plays an important auxiliary role in the hepatic S9-dependent activation of benzidine. Bacterial peroxidases may also support benzidine oxidation in the Ames test.

Acetylation↗
PubMed1985

An electron spin resonance investigation of the iron-catalyzed reaction of metronidazole with cysteine.

Metronidazole is used in the treatment of protozoal and bacterial infections, and has been used as a radiation sensitizer in experimental research and clinical trials. This drug is rapidly decomposed by cysteine in the presence of ferric or ferrous iron. Electron spin resonance spectroscopy demonstrates the formation of two complexes composed of iron, cysteine, and NO. The nitric oxide is probably formed by the reduction of inorganic nitrite formed by the cleavage of the metronidazole nitro group from the imidazole ring. No such reaction occurred with the 2-nitroimidazole drug, misonidazole.

Chemical Phenomena↗