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1.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 17(6): 496-500, 2003 Nov.
Article in Chinese | MEDLINE | ID: mdl-14663952

ABSTRACT

OBJECTIVE: To explore the possibility of improving the performance of tissue engineering valve by means of preendothelialization with cultured human umbilical vein endothelial cell(hUVEC) and to develop a new xenogenic bioprosthesis valve material. METHODS: The porcine aortic valves treated by use of glutaraldehyde(GA), epoxychloropropane(EC), L-glutamic acid(L-GA) and cellular extraction(CE) respectively were divided into four groups; group 1(GA), group 2(EC), group 3(EC + L-GA), and group 4(EC + L-GA + CE). The cultured hUVECs were seeded onto the treated porcine aortic valve, then that stuff were examined by means of EC VIII factor staining, living cells counting and microscopy. RESULTS: The cultured hUVEC could adhere to culturing bottle wall an hour later, and propagated to two passages after seven days. The cells increased with serial passage at a 7-day interval. But the hUVEC grew slowly when seeded onto the treated valve material except group 4. The cells in group 4 covered the surface of valve completely seven days later, which could also be seen in group 3 but not completely. There was no cell growing in group 1, and only fewer in group 2. The living cell in groups 3 and 4 were significantly more than in groups 1 and 2 on the 3rd, 7th and 14th days (P < 0.01), meanwhile, the number of cells in group 4 were also significantly more than that in group 3 (P < 0.05). The covering area of cultured cell on the valve material in groups 3 and 4 was significantly larger than that in groups 1 and 2. The covering area of cell in group 4 was over 95%, and higher than that in group 3(60%-70%). The hUVEC of group 4 arranged in pattern of three dimension. So it could resist rising of foreign power from the cardiac cavity of high pressure and flowing volume. There was no cell on the leaflet surface in group 1, and only a few pinch of cells could be seen in group 2. CONCLUSION: The porcine aortic valve can be used to be an ideal xenogeneic valve scaffold; the scaffold of porcine aortic valve should be treated by use of epoxy-chloropropane, L-glutamic acid and cellular extraction, so that a best growing environment to the hUVEC would be given; the cultured hUVECs used to be source of seed living cell had a boundless prospects; the growing velocity of cultured hUVEC was controllable, which facilitated clinical application; and the endothelial cells of xenogeneic valve material which grew compactly onto the scaffold can resist rising of foreign power from the cardiac cavity itself.


Subject(s)
Bioprosthesis , Endothelium, Vascular/cytology , Heart Valve Prosthesis , Tissue Engineering/methods , Animals , Aortic Valve , Cell Adhesion , Cell Count , Cells, Cultured , Endothelium, Vascular/ultrastructure , Glutamic Acid/pharmacology , Glutaral/pharmacology , Humans , Prosthesis Design , Swine , Trichloroepoxypropane/pharmacology , Umbilical Veins/cytology
2.
Carcinogenesis ; 20(8): 1511-20, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10426800

ABSTRACT

Benzene, a ubiquitous environmental pollutant, is known to cause leukemia and aplastic anemia in humans and hematotoxicity and myelotoxicity in rodents. Toxicity is thought to be exerted through oxidative metabolites formed in the liver, primarily via pathways mediated by cytochrome P450 2E1 (CYP2E1). Phenol, hydroquinone and trans-trans-muconaldehyde have all been hypothesized to be involved in benzene-induced toxicity. Recent reports indicate that benzene oxide is produced in vitro and in vivo and may be sufficiently stable to reach the bone marrow. Our goal was to improve existing mathematical models of microsomal benzene metabolism by including time course data for benzene oxide, by obtaining better parameter estimates and by determining if enzymes other than CYP2E1 are involved. Microsomes from male B6C3F1 mice and F344 rats were incubated with [(14)C]benzene (14 microM), [(14)C]phenol (303 microM) and [(14)C]hydroquinone (8 microM). Benzene and phenol were also incubated with mouse microsomes in the presence of trans-dichloroethylene, a CYP2E1 inhibitor, and benzene was incubated with trichloropropene oxide, an epoxide hydrolase inhibitor. These experiments did not indicate significant contributions of enzymes other than CYP2E1. Mathematical model parameters were fitted to rodent data and the model was validated by predicting human data. Model simulations predicted the qualitative behavior of three human time course data sets and explained up to 81% of the total variation in data from incubations of benzene for 16 min with microsomes from nine human individuals. While model predictions did deviate systematically from the data for benzene oxide and trihydroxybenzene, overall model performance in predicting the human data was good. The model should be useful in quantifying human risk due to benzene exposure and explicitly accounts for interindividual variation in CYP2E1 activity.


Subject(s)
Benzene/metabolism , Carcinogens/metabolism , Cytochrome P-450 CYP2E1/metabolism , Microsomes, Liver/metabolism , Models, Biological , Animals , Cytochrome P-450 CYP2E1 Inhibitors , Enzyme Inhibitors/pharmacology , Humans , Male , Mice , Microsomes, Liver/drug effects , Quaternary Ammonium Compounds/pharmacology , Rats , Rats, Inbred F344 , Trichloroepoxypropane/pharmacology
3.
Biochemistry ; 35(49): 15807-13, 1996 Dec 10.
Article in English | MEDLINE | ID: mdl-8961944

ABSTRACT

Stereoselective epoxidation by cytochrome P450s (P450s) and regioselective hydration by epoxide hydrolase determine the carcinogenic potency of some polycyclic aromatic hydrocarbons (PAHs). In this report, cDNA-expressed human and mouse P450s 1A1 and 1A2 and epoxide hydrolase were used to characterize the stereoselective epoxidation and regioselective hydration at the K-region of benz[a]-anthracene (BA), 7,12-dimethylbenz[a]anthracene (DMBA), chrysene (CR), benzo[a]pyrene (B[a]P), dibenz[a,h]anthracene (DB[a,h]A), and benzo[c]phenanthrene (B[c]Ph) by direct chiral stationary-phase HPLC (CSP-HPLC) analyses. Our results indicated that all P450 isoforms preferentially produced major K-region, S,R-epoxides of BA (95-98%), DMBA (94-97%), B[a]P (91-96%), DB[a,h]A (94-98%), and B[c]Ph (87-92%), and major R,S-epoxide of CR (74-85%) in the presence of 3,3,3-trichloropropylene 1,2-oxide (TCPO), an inhibitor of epoxide hydrolase, suggesting that P450 enzymes exhibited the high stereoselectivity toward one of two stereoheterotopic faces of K-region double bond of the PAHs. Epoxide hydrolase either expressed from recombinant vaccinia virus or contained in human hepatoma G2 cells (HepG2) hydrated the C-O bond of epoxy-ring at the S-carbon of major metabolically-formed K-region epoxide enantiomers of BA, CR, DMBA, B[a]P, and DB[a,h]A to yield 80-98% dihydrodiols enriched in R,R-form and that at the R-carbon of B[c]Ph epoxide to yield 77-92% dihydrodiol enriched in S,S-form, suggesting that epoxide hydrolase was highly regioselective. The various enantiomeric components of dihydrodiol products in the metabolism of PAHs were apparently due to the combined effect of stereoselectivity of the P450s and regioselectivity of epoxide hydrolase. Our results provide a clear understanding of how these enzymes catalyze overall stereoselective metabolism at the K-region of the PAHs.


Subject(s)
Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Epoxide Hydrolases/metabolism , Epoxy Compounds/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Animals , Anthracenes/metabolism , Baculoviridae/genetics , Chrysenes/metabolism , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A2/genetics , Dihydroxydihydrobenzopyrenes/metabolism , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Gene Expression/genetics , Humans , Mice , Models, Chemical , Molecular Conformation , Molecular Structure , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Trichloroepoxypropane/pharmacology , Vaccinia virus/genetics
4.
Chem Res Toxicol ; 9(6): 1007-12, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8870989

ABSTRACT

L-754,394, a furanopyridine derivative, is an experimental anti-HIV agent which has been shown to be an unusually potent and selective inhibitor of cytochrome P450 3A enzymes in a number of mammalian species. In the present studies, L-754,394 was demonstrated to undergo NADPH-dependent metabolic activation in hepatic microsomal preparations from rats, dogs, rhesus monkeys, and humans to electrophilic intermediates which became bound covalently to cellular proteins. The extent of binding was species-dependent, the highest levels being observed with liver microsomes from rhesus monkeys. Inclusion in incubation media of the nucleophilic trapping agents glutathione, cysteine, or methoxyamine led to a modest (15-25%) decrease in the covalent binding, while trichloropropylene oxide, an inhibitor of epoxide hydrolase, had no effect. When L-754,394 was incubated with monkey liver microsomes, the corresponding dihydrofurandiol was identified as a metabolite by liquid chromatography-tandem mass spectrometry. In contrast, when incubations were carried out in the presence of methoxyamine, the O-methyloxime derivative of the ring-opened dihydrodiol tautomer was formed, while inclusion of glutathione or N-acetylcysteine led to the formation of S-linked conjugates of a putative furan epoxide. Collectively, these results are taken to indicate that L-754,394 undergoes cytochrome P450-dependent oxidation of the fused furan ring system, leading to the formation of chemically-reactive intermediates. One or more of these electrophilic species may be responsible for the autocatalytic destruction of cytochrome P450 enzymes which accompanies L-754,394 metabolism in vitro and in vivo.


Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/metabolism , Indans/metabolism , Microsomes, Liver/metabolism , Mixed Function Oxygenases/antagonists & inhibitors , Piperazines/metabolism , Animals , Binding Sites , Biotransformation , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Dogs , Gas Chromatography-Mass Spectrometry , Humans , Hydroxylamines/pharmacology , In Vitro Techniques , Indans/chemistry , Macaca mulatta , Male , Microsomes, Liver/enzymology , Mixed Function Oxygenases/metabolism , NADP/metabolism , Oxidation-Reduction , Piperazines/chemistry , Protein Binding , Rats , Rats, Sprague-Dawley , Species Specificity , Trichloroepoxypropane/pharmacology
5.
Biochim Biophys Acta ; 1290(3): 327-39, 1996 Aug 13.
Article in English | MEDLINE | ID: mdl-8765137

ABSTRACT

Mouse liver microsomes oxidized linoleic acid to form 9,10- or 12,13-epoxyoctadecenoate. These monoepoxides were subsequently hydrolyzed to their corresponding diols in the absence of the microsomal epoxide hydrolase inhibitor, 1,2-epoxy-3,3,3-trichloropropane. Furthermore, both 9,10- and 12,13-epoxyoctadecenoates were oxidized to diepoxyoctadecanoate at apparently identical rates by mouse liver microsomal P-450 epoxidation. Both epoxyoctadecanoates and diepoxyoctadecanoates were converted to tetrahydrofuran-diols by microsomes. Tetrahydroxides of linoleate were produced as minor metabolites. Arachidonic acid was metabolized to epoxyeicosatrienoates, dihydroxyeicosatrienoates, and monohydroxyeicosatetraenoates by the microsomes. Microsomes prepared from clofibrate (but not phenobarbital) -treated mice exhibited much higher production rates for epoxyeicosatrienoates and vic-dihydroxyeicosatrienoates. This indicated an induction of P-450 epoxygenase(s) and microsomal epoxide hydrolase in mice by clofibrate and not by phenobarbital. Incubation of synthetic epoxyeicosatrienoates with microsomes led to the production of diepoxyeicosadienoates. Among chemically generated diepoxyeicosadienoate isomers, three of them possessing adjacent diepoxides were hydrolyzed to their diol epoxides which cyclized to the corresponding tetrahydrofuran-diols by microsomes as well as soluble epoxide hydrolase at a much higher rate. Larger cyclic products from non-adjacent diepoxides were not observed. The results of our in vitro experiments suggest that linoleic and arachidonic acid can be metabolized to their tetrahydrofuran-diols by two consecutive microsomal cytochrome P-450 epoxidations followed by microsomal or soluble epoxide hydrolase catalyzed hydrolysis of the epoxides. Incubation experiments with the S-9 fractions indicate that the soluble epoxide hydrolase is more important in this conversion. This manuscript is the first report of techniques for the separation and identification of regio and geometrical isomer of an interesting class of oxylipins and their metabolism by liver microsomes and S-9 fractions to THF-diols.


Subject(s)
Arachidonic Acid/metabolism , Epoxy Compounds/metabolism , Linoleic Acids/metabolism , Microsomes, Liver/metabolism , Animals , Clofibrate/pharmacology , Cytochrome P-450 Enzyme System/drug effects , Enzyme Induction , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxy Compounds/chemistry , Linoleic Acid , Male , Mice , Oxidation-Reduction , Phenobarbital/pharmacology , Trichloroepoxypropane/pharmacology
6.
J Toxicol Environ Health ; 48(3): 273-94, 1996 Jun 28.
Article in English | MEDLINE | ID: mdl-8656450

ABSTRACT

The purpose of this study was to investigate the toxicity of styrene and styrene oxide in the lung in comparison to the toxicity in the liver. Pneumotoxicity caused by styrene or styrene oxide was measured by elevations in the release of gamma-glutamyltranspeptidase (GGT) and lactate dehydrogenase (LDH) into bronchoalveolar lavage fluid (BALF), while hepatotoxicity was measured by increases in serum sorbitol dehydrogenase (SDH) in non-Swiss Albino (Hsd:NSA) mice. Intraperitoneal administration of styrene at doses of 500-1000 mg/kg caused consistent dose-dependent increases in both sets of biomarkers with the hepatic effect appearing earlier than the pulmonary effect. Pyridine, phenobarbital, and beta-naphthoflavone, inducers of CYP2E1, CYP2B, and CYP1A, respectively, increased the toxicity of styrene. Pyridine and phenobarbital treatments increased mortality due to styrene. Styrene oxide exists in two enantiomeric forms: (R)- and (S)-styrene oxide, and the differential toxicities of the two enantiomers and racemic styrene oxide were compared. In all studies, (R)-styrene oxide caused greater toxicity than the (S) enantiomer, especially in the liver. Trichloropropene oxide, an epoxide hydrolase inhibitor, was used to inhibit styrene oxide detoxification and increased its hepatotoxicity, while buthionine sulfoxamine, a glutathione depletor, did not. These results demonstrated the greater role of epoxide hydrolase in styrene oxide detoxification.


Subject(s)
Carcinogens/toxicity , Chemical and Drug Induced Liver Injury , Epoxy Compounds/toxicity , Lung Diseases/chemically induced , Styrenes/toxicity , Animals , Benzoflavones/pharmacology , Bronchoalveolar Lavage Fluid/chemistry , Buthionine Sulfoximine , Carcinogens/administration & dosage , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/metabolism , Epoxy Compounds/administration & dosage , Injections, Intraperitoneal , L-Iditol 2-Dehydrogenase/blood , L-Iditol 2-Dehydrogenase/metabolism , L-Lactate Dehydrogenase/metabolism , Liver/enzymology , Liver/pathology , Liver Diseases/pathology , Male , Methionine Sulfoximine/analogs & derivatives , Methionine Sulfoximine/pharmacology , Mice , Phenobarbital/pharmacology , Pyridines/pharmacology , Styrene , Styrenes/administration & dosage , Trichloroepoxypropane/pharmacology , beta-Naphthoflavone , gamma-Glutamyltransferase/metabolism
7.
Biochem Mol Med ; 56(2): 131-43, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8825076

ABSTRACT

Previous animal research has suggested that the phenytoin arene oxide metabolite is teratogenic in acute studies and that the fetal effects were increased after injecting an inhibitor of microsomal epoxide hydrolase (mEH) (Martz et al., Pharmacol Exp Ther 203:231-239, 1977, Barcellona et al., Teratog Carcinog Mutagen 7:159-168, 1987). We have studied the effects of chronic oral phenytoin exposure in utero and the mEH inhibitor trichloropropene oxide (TCPO) on the prenatal growth and development of an inbred mouse strain with a low incidence of spontaneous oral clefting (C57BL/6J). Chronic daily gastric gavage of phenytoin produced a plasma level (mean 10.7 micrograms/ml on gestation Day 8) within the range recommended to prevent epilepsy in humans; this did not produce an increase in oral clefting or ventricular septal defects in the exposed C57BL/6J pups. It did produce a significant delay in prenatal growth and development, including phalangeal ossification. However, except for percentage resorptions/implantation, there was no synergism between phenytoin and TCPO in contrast to the finding reported by Martz et al. in Swiss mice. This issue was also assessed in a test of the fetal effect of phenytoin injected with TCPO, as had been done by Martz et al. There were no oral clefts or ventricular septal defects or a difference (P > 0.05) in prenatal growth and development in these C57BL/6J pups compared to the chronic gastric phenytoin plus TCPO group. This suggests either that differences in the genotypes of Swiss and C57BL/6J mice may be a contributing factor or that other teratogenic mechanisms were involved.


Subject(s)
Anticonvulsants/toxicity , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Phenytoin/toxicity , Teratogens/toxicity , Trichloroepoxypropane/pharmacology , Animals , Anticonvulsants/blood , Body Weight/drug effects , Drug Interactions , Female , Heart Defects, Congenital/chemically induced , Mice , Mice, Inbred C57BL/embryology , Microsomes/enzymology , Phenytoin/blood , Pregnancy
8.
Biochem Pharmacol ; 50(9): 1361-6, 1995 Oct 26.
Article in English | MEDLINE | ID: mdl-7503784

ABSTRACT

Idiosyncratic hypersensitivity reactions with carbamazepine have been postulated to be due to a deficiency of microsomal epoxide hydrolase (HYL1), although this is based on indirect evidence. Using 3H-cis stilbene oxide (0.5 Ci/mmol) as a substrate, we have developed a radiometric HPLC assay sensitive enough to measure the kinetic parameters of HYL1 in lymphocytes. The intra-assay coefficient of variation was 8%. Enzyme activity has been measured in lymphocytes from six carbamazepine hypersensitive patients, six patients on carbamazepine without any adverse effects, and twelve drug-naive healthy volunteers. No significant difference was observed in three kinetic parameters of the enzyme among these three groups. The values for Km, Vmax, and intrinsic clearance ranged from 6.1-89.9 microM, 3.0-23.2 pmoles diol formed/min/mg protein, and 0.147-0.493 microliter/min/mg protein. There was no difference in enzyme activity between patients currently on carbamazepine and healthy volunteers, indicating a lack of induction of lymphocyte HYL1 by carbamazepine. Co-incubation of lymphocytes with 1,1,1-trichloropropene oxide, an inhibitor of hepatic HYL1, resulted in an 82% inhibition of activity, similar to that observed with the hepatic enzyme. The healthy volunteers were genotyped as being either GSTM1 positive (n = 6) or GSTM1 negative (n = 6). This did not affect the kinetic parameters of lymphocyte microsomal epoxide hydrolase. Our results suggest that there is normal HYL1 activity in lymphocytes of hypersensitive patients using cis-stilbene oxide as a substrate.


Subject(s)
Anticonvulsants/adverse effects , Carbamazepine/adverse effects , Drug Hypersensitivity/enzymology , Epoxide Hydrolases/blood , Lymphocytes/enzymology , Adolescent , Adult , Aged , Chromatography, High Pressure Liquid , Drug Hypersensitivity/blood , Drug Hypersensitivity/etiology , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Female , Glutathione Transferase/metabolism , Humans , Hydrolysis , Isoenzymes/metabolism , Kinetics , Male , Microsomes/enzymology , Middle Aged , Radiometry , Stilbenes/metabolism , Trichloroepoxypropane/pharmacology , Tritium
9.
Xenobiotica ; 24(10): 989-1001, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7900414

ABSTRACT

1. Benzo[a]pyrene (BaP) metabolism was studied in microsomes of the pyloric caeca (main digestive tissue and site of P450) of the echinoderm sea star (starfish) Asterias rubens. 2. NADPH-dependent metabolism of BaP produced phenols (36% of total metabolism), quinones (19%), dihydrodiols (25%) and putative protein adducts (20%). 3. NADH-dependent rates of BaP metabolism were approximately twice those found for NADPH-dependent metabolism, and metabolite formation was shifted towards dihydrodiols and quinones. 4. Cumene hydroperoxide (CHP)-dependent rates of BaP metabolism were also higher than NADPH-dependent rates by a factor of six for quinone and putative protein adduct production, and by a factor of four for phenol and dihydrodiol production. 5. Microsomal rates of BaP metabolism in BaP-exposed sea stars appeared to be elevated more in the case of NADPH-dependent than for CHP-dependent metabolism (respectively, increases of 130 and 41%), indicating the induction of forms of P450 preferentially catalysing NADPH-dependent metabolism. 6. 1,1,1-Trichloropropene-2,3-oxide (TCPO) inhibited dihydrodiol formation from both NADPH- and CHP-dependent BaP metabolism, indicating the involvement of epoxide hydratase in BaP metabolism. 7. Incubations of pyloric caeca microsomes with BaP and a superoxide anion radical-generating system (xanthine/xanthine oxidase) produced putative protein adducts but no free metabolites.


Subject(s)
Benzene Derivatives/pharmacology , Benzo(a)pyrene/metabolism , Microsomes/metabolism , NADP/pharmacology , NAD/pharmacology , Starfish/metabolism , Animals , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Kinetics , Phenols/metabolism , Proteins/metabolism , Pylorus/ultrastructure , Quinones/metabolism , Trichloroepoxypropane/pharmacology , Xanthine , Xanthine Oxidase/metabolism , Xanthines/metabolism
10.
Food Chem Toxicol ; 30(6): 467-73, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1500033

ABSTRACT

The effect of modulating epoxide metabolism by inhibiting microsomal and cytosolic epoxide hydrolases and depleting glutathione, on the cytotoxicity of trans-anethole has been examined in freshly isolated rat hepatocytes in suspension. Hepatocytes derived from female Sprague-Dawley CD rats by collagenase perfusion were incubated in suspension and sampled at intervals over a 6-hr period. Cytotoxicity was assessed by the leakage of lactate dehydrogenase into the culture medium and in the cells after lysis. Glutathione was determined by fluorimetry. Anethole showed a dose-dependent cytotoxicity at concentrations ranging from 5 x 10(-4) to 5 x 10(-3) M, with concentrations of 10(-3) M and above causing greater than 63% leakage of lactate dehydrogenase in 6 hr. Microsomal epoxide hydrolase was inhibited by trichloropropene oxide (10(-4) M) and cyclohexene oxide (10(-3) M), and cytosolic epoxide hydrolase by 4-fluorochalcone oxide (5 x 10(-4) M). Cellular glutathione was depleted by diethyl maleate (5 x 10(-4) M), and its synthesis inhibited by 2.5 x 10(-3) M-L-buthionine (S,R)-sulphoximine. Suspensions treated with a sub-cytotoxic concentration of anethole (5 x 10(-4) M) showed a rapid increase in cytotoxicity when 4-fluorochalcone oxide was present (complete loss of viability within 2 hr), while pretreatment of hepatocytes with diethyl maleate in combination with buthionine sulphoximine, to deplete glutathione, slowly increased the cytotoxic response at later times (after 4 hr of incubation). The association of the effects of 4-fluorochalcone oxide with the inhibition of cytosolic epoxide hydrolase is strengthened by the inability of chalcone oxide, a close structural analogue of 4-fluorochalcone oxide, which has no effect on epoxide hydrolase or glutathione conjugation, to influence the effects of anethole on hepatocytes. These data are discussed in terms of the role of anethole epoxide in the cytotoxicity of trans-anethole.


Subject(s)
Anisoles/toxicity , Epoxy Compounds/metabolism , Flavoring Agents/toxicity , Liver/drug effects , Allylbenzene Derivatives , Animals , Antimetabolites/pharmacology , Buthionine Sulfoximine , Cell Survival/drug effects , Cells, Cultured , Chalcone/analogs & derivatives , Chalcone/pharmacology , Chalcones , Cyclohexanes/pharmacology , Cyclohexenes , Dose-Response Relationship, Drug , Epoxide Hydrolases/antagonists & inhibitors , Female , Flavoring Agents/metabolism , Glutathione/analysis , L-Lactate Dehydrogenase/metabolism , Liver/cytology , Maleates/pharmacology , Methionine Sulfoximine/analogs & derivatives , Methionine Sulfoximine/pharmacology , Rats , Rats, Inbred Strains , Trichloroepoxypropane/pharmacology
11.
Drug Metab Dispos ; 20(3): 440-5, 1992.
Article in English | MEDLINE | ID: mdl-1521503

ABSTRACT

2',3'-Allylic epoxide derivatives of allylbenzene and its analogs estragole, eugenol, and safrole were synthesized, and their enzymatic conversion to dihydrodiols by cytosolic and microsomal epoxide hydrolases was examined. All four epoxides were good substrates for both epoxide hydrolases, with Michaelis constants in the low micromolar range. Two putatively selective inhibitors of cytosolic and microsomal epoxide hydrolases, trichloropropylene oxide and nordihydroguaiaretic acid, were used to inhibit the hydrolysis of these allylic epoxides. Minimal selectivity toward either hydrolase was seen with either inhibitor, suggesting that the "selectivity" of these inhibitors is highly substrate-dependent. The susceptibilities of these epoxides to rapid hydrolysis by both epoxide hydrolases may explain their low genotoxic potencies in vivo.


Subject(s)
Epoxide Hydrolases/metabolism , Epoxy Compounds/metabolism , Lignans , Allylbenzene Derivatives , Anisoles/chemistry , Anisoles/metabolism , Catalysis , Cytosol/metabolism , Epoxide Hydrolases/antagonists & inhibitors , Epoxy Compounds/chemical synthesis , Eugenol/chemistry , Eugenol/metabolism , Guaiacol/analogs & derivatives , Guaiacol/pharmacology , Hydrolysis , Microsomes/metabolism , Safrole/chemistry , Safrole/metabolism , Trichloroepoxypropane/pharmacology
12.
Carcinogenesis ; 12(11): 2053-8, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1718618

ABSTRACT

The formation of RNA and DNA adducts by the environmental pollutant 2-nitrofluorene (2-NF) has been investigated in rat liver in vivo. The adduct pattern was studied after trifluoroacetic acid hydrolysis of DNA or RNA, followed by analysis of the adducts by HPLC. This was also done by enzymatic hydrolysis of DNA, followed by 32P-postlabeling. Both after oral and i.v. administration of [3H]2-NF, one major adduct was found. This adduct did not co-migrate with one of the known adducts of 2-(acetyl)-aminofluorene, N-deoxyguanosin-8-yl-2-aminofluorene (dG-C8-AF), which could have been formed after nitroreduction of 2-NF. 32P-Postlabeling revealed that two minor adducts were also formed, one of which was dG-C8-AF. The observation that the major adduct was also formed after i.v. administration of 2-NF to bile duct-catheterized rats makes a role for the intestinal microflora in the formation of this adduct very unlikely. In vitro experiments with inhibitors of the enzyme epoxide hydrolase indicated that epoxidation of 2-NF may play a role in the microsomal bioactivation of this compound.


Subject(s)
DNA Damage , DNA/drug effects , Fluorenes/metabolism , Liver/metabolism , 2-Acetylaminofluorene/analogs & derivatives , 2-Acetylaminofluorene/metabolism , Administration, Oral , Animals , Carcinogens , Chromatography, High Pressure Liquid , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Epoxide Hydrolases/physiology , Epoxy Compounds/pharmacology , Injections, Intravenous , Male , Masoprocol/pharmacology , RNA/drug effects , Rats , Rats, Inbred Strains , Trichloroepoxypropane/pharmacology , Trifluoroacetic Acid/pharmacology
13.
Proc Natl Acad Sci U S A ; 87(8): 3037-41, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2326264

ABSTRACT

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators.


Subject(s)
8,11,14-Eicosatrienoic Acid/metabolism , Fatty Acids, Unsaturated/metabolism , Glutathione/metabolism , Hippocampus/metabolism , 8,11,14-Eicosatrienoic Acid/analogs & derivatives , 8,11,14-Eicosatrienoic Acid/chemical synthesis , Animals , Carbon Radioisotopes , Epoxide Hydrolases/antagonists & inhibitors , Evoked Potentials , Hippocampus/physiology , Indicators and Reagents , Male , Rats , Rats, Inbred Strains , Trichloroepoxypropane/pharmacology
14.
J Environ Pathol Toxicol Oncol ; 10(1-2): 79-88, 1990.
Article in English | MEDLINE | ID: mdl-2121958

ABSTRACT

The function of nuclear envelope in the activation of aflatoxin B1 (AFB1) was investigated. Pretreatment of rats by phenobarbital or polychlorobiphenyl induced markedly the nuclei-dependent binding of AFB1 to DNA as well as the drug-metabolizing enzymes, such as aminopyrine-N-demethylase and epoxide hydratase. The high sensitivity of AFB1 activation to SKF-525A, a low inducibility of 3-methylcholanthrene for AFB1 activation and the removal of nuclear AFB1 activation by Triton X-100 indicate that the AFB1 binding to DNA is mediated by P-450 system localized in the nuclear envelope. In the intact nuclei, trichloropropane oxide, a potent inhibitor of epoxide hydratase, caused no significant increment in AFB1 binding to DNA. While in the reconstructed system composed of microsomes and stripped nuclei prepared by Triton X-100 treatment, this inhibitor caused a marked elevation of AFB1 binding. This evidence supports an assumption that AFB1-2,3-oxide produced by the microsomal oxygenase may be hydrolyzed by the microsomal epoxide hydratase, whereas the nuclear envelope-mediated AFB1-2,3-oxide is insensitive to the nuclear epoxide hydratase.


Subject(s)
Aflatoxins/metabolism , DNA/metabolism , Nuclear Envelope/metabolism , Aflatoxin B1 , Animals , Biotransformation , Glutathione/metabolism , In Vitro Techniques , Male , Microsomes, Liver/metabolism , Octoxynol , Polyethylene Glycols/pharmacology , Rats , Rats, Inbred Strains , Trichloroepoxypropane/pharmacology
15.
Cancer Res ; 49(21): 5816-22, 1989 Nov 01.
Article in English | MEDLINE | ID: mdl-2507138

ABSTRACT

The immunosuppressive actions of benzo(a)pyrene have been proposed to be mediated by reactive metabolites rather than the parent compound. Reactive metabolites which suppress splenic humoral immune responses are thought to be generated within the spleen rather than in distant tissues. Although the spleen has been shown to be capable of metabolizing benzo(a)pyrene, the relative amounts and types of metabolites generated have not been determined. In this study, high-pressure liquid chromatography was used to separate benzo(a)pyrene metabolites generated by splenic microsomes. The major metabolites generated by the splenic microsomal preparations of untreated female B6C3F1 mice were found to be the 9,10- and 7,8-dihydrodiols and 9-, 7-, and 3-hydroxy benzo(a)pyrene. The 1,3-, 3,6-, and 6,12-diones and 4,5-dihydrodiol constituted only a small fraction of the metabolites generated. The generation of all metabolites were inhibited by alpha-naphthoflavone and antiserum to NADPH-cytochrome P-450 reductase, whereas SKF 525-A had only a minimal effect. Dihydrodiol production was completely inhibited by the epoxide hydrolase inhibitor, trichloropropylene oxide. Benzo(a)pyrene pretreatment of mice produced a dramatic increase in the amount of metabolites formed; however, the pattern of metabolites remained similar to that generated by splenic microsomes of untreated mice. The role of prostaglandin synthetase in generating these metabolites was also examined. The addition of arachidonic acid in place of NADPH resulted in the formation of only quinones. Dihydrodiols and phenols were undetectable. The results of this study indicate that splenocytes may be capable of generating the 7,8-dihydrodiol, the precursor to the highly reactive 7,8-dihydrodiol-9,10-epoxide. Furthermore, the addition of the 7,8-dihydrodiol-9,10-epoxide to splenocyte cultures resulted in a decreased in vitro antibody forming cell response to sheep red blood cells. Thus, benzo(a)pyrene-induced immunosuppression may be mediated by the dihydrodiol-epoxide generated within the spleen. Since benzo(a)pyrene exposure was found to increase its own metabolism, immunosuppression produced by the administration of benzo(a)pyrene over several days may be the result of an increased production of immunosuppressive metabolites. The pattern of metabolites generated and the effects of the two types of cytochrome P-450 inhibitors suggests that the major isozyme of cytochrome P-450 that mediates the metabolism of benzo(a)pyrene within the spleen of untreated mice may be similar to the isozyme induced in the liver upon pretreatment with polycyclic aromatic hydrocarbons.


Subject(s)
Benzo(a)pyrene/metabolism , Microsomes/metabolism , Spleen/metabolism , Animals , Biotransformation , Chromatography, High Pressure Liquid , Female , Immune Sera , Kinetics , Mice , Mice, Inbred Strains , Microsomes/drug effects , NADPH-Ferrihemoprotein Reductase/antagonists & inhibitors , NADPH-Ferrihemoprotein Reductase/metabolism , Proadifen/pharmacology , Trichloroepoxypropane/pharmacology , Tritium
16.
Toxicol Appl Pharmacol ; 99(3): 466-73, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2749733

ABSTRACT

The polycyclic aromatic hydrocarbon naphthalene is bioactivated by cytochromes P450 to an electrophilic epoxide intermediate, which subsequently is metabolized to naphthoquinones (NQ) and possibly to a free radical intermediate. These reactive intermediates may bind covalently to lenticular tissues, cause oxidant stress and/or lipid peroxidation, thereby initiating cataracts. To evaluate this hypothesis, male C57BL/6 or DBA/2 mice were treated with naphthalene or one of several naphthoquinone and naphthol metabolites, in the presence or absence of modulators of chemical bioactivation and detoxification. In C57BL/6 mice, cataracts were caused by naphthalene (500-2000 mg/kg ip) in a dose-dependent fashion. The incidence of naphthalene-induced cataracts was decreased by pretreatment with the P450 inhibitors SKF 525A and metyrapone, the antioxidants caffeic acid and vitamin E, the glutathione (GSH) precursor N-acetylcysteine, and the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (p less than 0.05). Naphthalene cataractogenicity was enhanced by pretreatment with the cytochrome P450 inducer phenobarbital and the GSH depletor diethyl maleate (DEM) (p less than 0.05), and was unaffected by pretreatment with the prostaglandin synthetase inhibitors aspirin or naproxen, or the epoxide hydrolase inhibitor trichloropropene oxide. Cataracts were initiated by 1,2-NQ and 1,4-NQ (5-250 mg/kg ip) in a dose-dependent fashion, with a molar potency about 10-fold higher than that for naphthalene. NQ cataractogenicity was enhanced by pretreatment with DEM (p less than 0.05). 1-Naphthol (56 to 562 mg/kg ip) demonstrated a cataractogenic potency intermediary to that for naphthalene and NQ. DBA/2 mice treated with naphthalene (2000 mg/kg ip), 1,4-NQ (65-250 mg/kg ip), 1,2-NQ (30-250 mg/kg ip), or DEM followed by 1,4-NQ (125 mg/kg ip) did not develop cataracts. These results suggest that naphthalene cataractogenesis in C57BL/6 mice requires P450-catalyzed bioactivation to a reactive intermediate, which may be the NQ and/or a free radical derivative, either of which is dependent upon GSH for detoxification.


Subject(s)
Cataract/chemically induced , Naphthalenes/toxicity , Animals , Cytochrome P-450 Enzyme System/physiology , Glutathione/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Naphthalenes/metabolism , Naphthoquinones/metabolism , Trichloroepoxypropane/pharmacology
17.
Mutagenesis ; 4(1): 45-50, 1989 Jan.
Article in English | MEDLINE | ID: mdl-2654551

ABSTRACT

The mutagenicity of rutacridone and rutacridone epoxide was investigated using Salmonella typhimurium without as well as with different metabolic activation systems. Rutacridone epoxide was found to be a direct acting mutagen in S. typhimurium strains TA98, TA100 and TA1538; addition of rat liver preparations resulted in a marked decrease of mutagenicity. In contrast, rutacridone required metabolic conversion to exhibit mutagenic activity. Neither of the compounds had any effect on tester strain TA1978. S9 mixes as well as microsomal and cytosolic preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated rats were used to study the activation and deactivation capacities of the enzyme mixtures. In addition, the influence of enzyme inhibitors on the activation and deactivation of the furoacridones were tested. Evidence is presented that rutacridone is metabolized by rat liver enzymes to the corresponding epoxide as the ultimate mutagen.


Subject(s)
Alkaloids/toxicity , Mutagenicity Tests , Cytosol/enzymology , Microsomes, Liver/enzymology , Mutagenicity Tests/methods , Mutation , Salmonella typhimurium/drug effects , Trichloroepoxypropane/pharmacology
18.
Carcinogenesis ; 9(12): 2291-5, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3191575

ABSTRACT

The conversion of cyclopento[c,d]pyrene (CPP) to forms which are mutagenic to Salmonella typhimurium strain TA98 has been demonstrated in systems which generate peroxyl radicals. The systems examined included prostaglandin H synthase (PHS) and arachidonic acid, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) and hematin, and the autoxidation of the sulfite ion. In all cases concentration-dependent activation of CPP was observed at hydrocarbon concentrations between 10 and 100 microM. Neither CPP nor the peroxyl radical systems alone were mutagenic or toxic to the tester strain. The use of hydroxygen peroxide with PHS, a peroxidative system which does not yield peroxyl radicals, does not activate CPP. The involvement of a CPP epoxide was examined using 1,1,1-trichloropropene-2,3-oxide. Addition of this epoxide hydrolase inhibitor to incubations of CPP with the PHS/arachidonic acid system resulted in a 210% increase in induced revertants relative to the system in the absence of the inhibitor. The addition of pure rat liver microsomal epoxide hydrolase to incubations of CPP with the 15-HPETE/hematin system resulted in a concentration-dependent loss of mutagenicity, further supporting the intermediacy of an epoxide. The site of metabolism of CPP is the cyclopenteno double bond based on the formation of products which display distinct pyrene-type fluorescence spectra. The involvement of the cyclopenteno double bond also is shown by the inability of the 15-HPETE/hematin system to activate 3,4-dihydrocyclopenteno[c,d]pyrene as a mutagen. CPP is the first environmentally-relevant carcinogenic hydrocarbon found to be activated directly by peroxyl radical systems without prior biotransformation to a diol derivative by the cytochrome P-450 system. These findings expand the range of potentially toxic substrates to be considered for activation by peroxyl radical pathways.


Subject(s)
Carcinogens/metabolism , Mutagens/metabolism , Pyrenes/metabolism , Animals , Biotransformation , Epoxy Compounds/metabolism , Free Radicals , In Vitro Techniques , Leukotrienes/pharmacology , Lipid Peroxides/pharmacology , Rats , Spectrometry, Fluorescence , Trichloroepoxypropane/pharmacology
19.
Br J Clin Pharmacol ; 26(5): 569-76, 1988 Nov.
Article in English | MEDLINE | ID: mdl-3061424

ABSTRACT

1. All trials of drug therapy for intermittent claudication published in English during the period 1965-1985 were reviewed. A total of 75 trials had studied 33 different pharmacological agents. Treadmill exercise, the most reproducible method of evaluating symptoms in this condition, was used in 49% of trials. 2. Oxpentifylline was the drug that had been most frequently studied. In seven placebo-controlled trials the average response to oxpentifylline, compared with placebo and weighted for sample-size, was 65% improvement in claudication distance. There was, however, a significant negative relation between sample-size and response (rs = -0.79, P less than 0.05), suggesting that this estimate was likely to have been biased by non-publication of negative results. 3. One third of all trials were uncontrolled; 84% of these reported benefit from drug treatment, compared with 32% of placebo-controlled trials (P less than 0.001). Sample-sizes varied from seven to 227 patients; 31% of trials reported data from less than 20 patients and these were likely to have had insufficient statistical power. 4. Overall, 57 of the 75 trials (76%) had at least one of the following deficiencies: an uncontrolled design; not double-blind; failure to use treadmill exercise; less than 20 patients included in the analysis. Thus, a priori three-quarters of all trials were unlikely to have made a satisfactory assessment of drug efficacy. 5. The information available does not establish convincingly that any drug consistently improves exercise performance in intermittent claudication. In view of the deficiencies in previous trials, we propose guidelines for future studies with regard to trial design, sample-size and methods of exercise testing.


Subject(s)
Hydrocarbons, Chlorinated/pharmacology , Intermittent Claudication/drug therapy , Pentoxifylline/therapeutic use , Theobromine/analogs & derivatives , Trichloroepoxypropane/pharmacology , Clinical Trials as Topic , Exercise Test , Female , Humans , Male , Meta-Analysis as Topic
20.
Mutagenesis ; 3(3): 233-7, 1988 May.
Article in English | MEDLINE | ID: mdl-3045485

ABSTRACT

1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are environmental pollutants and are S9-mediated mutagens in the Chinese hamster ovary (CHO) cell/hypoxanthine-guanine phosphoribosyl transferase assay. In this study, we examined the pathways leading to the mutagenic activation of these compounds in CHO cells. The microsomal metabolites of 1- and 3-nitro-BaP, the 1- and 3-nitro-BaP trans-7,8-dihydroxy-7,8-dihydrodiols (trans-7,8-dihydrodiols) and the 1- and 3-nitro-BaP trans-9,10-dihydrodiols, were isolated and tested for mutagenicity. At the concentrations assayed, both trans-9,10-dihydrodiols were non-mutagenic with and without S9 activation. In contrast, the trans-7,8-dihydrodiols of 1- and 3-nitro-BaP were direct-acting mutagens and these responses were similar in magnitude to the S9-mediated mutagenicities of the parent nitro-BaPs. S9 increased the mutagenic responses of the trans-7,8-dihydrodiols approximately 20-fold. Inhibition of epoxide hydrolase decreased the S9-mediated mutagenicity of 1-nitro-BaP by half, but doubled the S9-mediated mutagenicity of 3-nitro-BaP. These results suggest that in CHO cells: (i) the major route of mutagenic activation of 1- and 3-nitro-BaP involves S9-generated derivatives of the trans-7,8-dihydrodiols, e.g. bay-region diol epoxides; (ii) reactive nitroarene oxides may contribute to mutation induction by 3-nitro-BaP; and (iii) metabolic routes involving trans-9,10-dihydrodiol formation result in detoxification.


Subject(s)
Benzopyrenes/pharmacokinetics , Microsomes/metabolism , Mutagens/pharmacokinetics , Animals , Benzopyrenes/toxicity , Biotransformation , Cells, Cultured , Cricetinae , Epoxide Hydrolases/antagonists & inhibitors , Liver , Mutagenicity Tests , Subcellular Fractions , Trichloroepoxypropane/pharmacology
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