stilbenes and stilbene-oxide

In this work, a detailed study of mass transfer properties of trans-stilbene oxide (TSO) enantiomers on two Whelk-O1 chiral stationary phases (CSPs) has been performed. The CSPs were prepared by using both fully-porous silica particles of 2.5 μm particle diameter and superficially-porous ones of 2.6 μm particle diameter as base materials. By combining stop-flow and dynamic measurements in normal-phase conditions, the different contributions to mass transfer have been estimated. The study of intraparticle diffusion has revealed that the adsorption of both enantiomers is localized (i.e., characterized by absence of surface diffusion). The determination of thermodynamic binding constants (measured through adsorption isotherms) supports this finding.

Topics: Adsorption; Diffusion; Kinetics; Porosity; Silicon Dioxide; Stereoisomerism; Stilbenes; Temperature

The ultrafast photo-induced ring opening of the oxirane derivative trans-stilbene oxide has been studied through the use of ultrafast UV/UV pump-probe spectroscopy by using photo-ion detection. Single- and multiphoton probe paths and final states were identified through comparisons between UV power studies and synchrotron-based vacuum ultraviolet (VUV) single-photon ionization studies. Three major time-dependent features of the parent ion (sub-450 fs decay, (1.5±0.2) ps, and >100 ps) were observed. These decays are discussed in conjunction with the primary ring-opening mechanism of stilbene oxide, which occurs through C-C dissociation in the oxirane ring. The appearance of fragments relating to the masses of dehydrogenated diphenylmethane (167 amu) and dehydrogenated methylbenzene (90 amu) were also investigated. The appearance of the 167 amu fragment could suggest an alternative ultrafast ring-opening pathway via the dissociation of one of the C-O bonds within the oxirane ring.

Topics: Photons; Stilbenes; Synchrotrons

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (k

Topics: Alcohols; Binding Sites; Biocatalysis; Epoxide Hydrolases; Kinetics; Molecular Dynamics Simulation; Mutagenesis; Recombinant Proteins; Stereoisomerism; Stilbenes; Substrate Specificity

Polysaccharide-based chiral stationary phases (CSPs) are outstandingly suitable to play a key role in chiral HPLC method selection strategies, since they provide high success rates. One reason for this ability is that they adopt a diversity of higher order structures in various eluents, resulting in versatile chiral environments. A potential to extend this versatility further was expected and examined in the present study, based on the recently discovered hysteretic behavior of a widely used chiral selector (CS), amylose tris(3,5-dimethylphenylcarbamate). The hindered transitions of its structure, which are behind the history dependence of its separation ability, were used as a tool to identify distinct states of the chiral selector in order to exploit an extended selectivity space. The identification was carried out using a single diagnostic compound, as opposed to the common approach where testing a library of compounds is required. Eluent mixtures consisting of 2-propanol and either methanol or ethanol were scrutinized in terms of stability and robustness of the observed retentions. The solvent mixtures that were eligible for practical application in these respects were used to construct a screening sequence, including identical compositions combined with different column pretreatment. The gain achievable by using the proposed sequence was then evaluated using 15 enantiomer pairs with focus on resolution, enantiomer elution order and chemoselectivity.

Topics: 2-Propanol; Amylose; Chromatography, High Pressure Liquid; Ethanol; Indans; Methanol; Organic Chemicals; Oxadiazoles; Phenylcarbamates; Solvents; Stereoisomerism; Stilbenes

Seemingly identical chromatographic conditions result in drastically different enantioseparations on amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) columns in mixtures of 2-propanol and methanol. Selectivities of structurally diverse enantiomer pairs depend on the direction from which the composition of the eluent is approached. An alteration in the structure of the chiral stationary phase (CSP) is the only realistic reason behind the dissimilar selectivities in the same eluent. History-dependent retention and recognition mechanisms are indicated by van't Hoff plots and even by a reversal of the enantiomer elution order. The most notable observation is the easy access to markedly different states of the CSP in the same solvent mixture by a short pretreatment with 2-propanol in one case and with methanol in the other, while the transition between the two states is hindered enough to ensure long-term stability for both. Repeatability strongly depends on the composition of the eluent and it is key to utilization and also to rationalization of the phenomenon. From a theoretical point of view, this so-called hysteretic behavior poses another challenge to consider when modeling chiral interactions.

Topics: 2-Propanol; Amylose; Chromatography, High Pressure Liquid; Flavanones; Methanol; Phenylcarbamates; Reproducibility of Results; Rosuvastatin Calcium; Solvents; Stereoisomerism; Stilbenes

The adsorption isotherms of trans-stilbene oxide (TSO) enantiomers have been measured under a variety of normal phase (NP) mobile phases (MPs) on three Whelk-O1 chiral stationary phases (CSPs), prepared respectively on 1.8 μm and 2.5 μm fully porous particles (FPPs) and 2.6 μm superficially porous particles (SPPs). Specific loading of chiral selector (moles per square meter) of the two FPPs was about 20% smaller than that of SPPs (even if they were prepared under exactly the same experimental conditions). Regardless of particle size or format, adsorption was described by means of a Bilangmuir model with ethanol/hexane MPs. On the other hand, in pure hexane, the Tóth isotherm was employed. Interestingly, it was found that selective and nonselective Henry's constants vary in opposite directions by increasing the percentage of strong MP modifier (between 3 and 10%, v/v). Saturation capacity of SPPs (referred only to the porous zone of the particle) was remarkably smaller than those of FPPs. On the other hand, binding constants on both selective and nonselective sites were significantly larger on SPPs. Finally, a correlation between the specific loading of chiral selector and the binding constants of enantiomers was suggested by data, which can be important also to understand the kinetic behavior of these particles in chiral ultrafast applications.

Topics: Adsorption; Chemistry Techniques, Analytical; Kinetics; Particle Size; Porosity; Stereoisomerism; Stilbenes

Retention factors for two enantiomers of trans-stilbene oxide, k

Topics: Chemistry Techniques, Analytical; Chromatography, Supercritical Fluid; Methanol; Pressure; Solvents; Stereoisomerism; Stilbenes; Temperature

A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/β-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.

Topics: Binding Sites; Catalysis; Enzyme Activation; Enzyme Stability; Epoxide Hydrolases; Kinetics; Models, Chemical; Molecular Docking Simulation; Protein Binding; Protein Conformation; Stilbenes; Substrate Specificity

Reduced plate heights (hr) of <2 were observed for the first time during the chiral separation of enantiomers, on sub-2μm particles with supercritical fluid chromatography (SFC). The enantiomers of trans-stilbene oxide, were separated on a 4.6×50mm, 1.8μm R,R-Whelk-O1 column, with hr as low as 1.93. The plumbing of a commercial SFC instrument was modified to create a low dispersion version. Without the modification performance was considerably worse. vanDeemter like plots of reduced plate height vs. flow rate, for trans-stilbene oxide, indicate that the optimum flow varied with% modifier. On a 4.6×250mm, 5μm R,R- Whelk-O1 column, the optimum flow was >4mL/min for 5% methanol in CO2, decreasing to <2mL/min for 40% methanol (more than a factor of 2). For a 4.6×50mm column packed with 1.8μm particles the optimum appeared to be near, or >5mL/min with 2.5%, 5%, and 10% methanol, decreasing to between 3 and 3.5mL/min at 40% methanol. This is the first time such shifts have been characterized. Since the solutes were the same in all cases, the differences are likely due to changes in solute diffusion coefficients caused by changes in modifier concentration, and pressure. Pump pressure requirements sometimes exceeded 500bar. It is shown that a 5mL/min flow rate is inadequate for use with 1.8μm particles in a 4.6mm ID column format. Instead, it is suggested to decrease the ID of the column to 3mm, where the optimum flow rates are on the order of 2mL/min with decreased tubing variance. Nevertheless, a number of sub-1min chromatograms are presented.

Topics: Carbon Dioxide; Chromatography, Supercritical Fluid; Equipment Design; Kinetics; Methanol; Pressure; Stereoisomerism; Stilbenes

In an earlier part of this study (performance evaluation) it was observed, for home-made capillary electrochromatography (CEC) columns, that smaller particle diameters do not always generate higher efficiencies. This phenomenon was further examined in this study, evaluating Van Deemter curves. Naphthalene and trans-stilbene oxide were analyzed on four 3 µm and four 5 µm chlorinated polysaccharide-based chiral stationary phases (CSPs) applying voltages ranging from 5 to 30 kV. Neither the 3 nor the 5 µm packings generated systematically the highest efficiencies. The varying column efficiencies were optimized by evaluating nine packing procedures for both 3 and 5 µm CSPs. Again it was observed that smaller particle-size packings were not necessarily beneficial for the efficiency of the CEC analysis. This observation was statistically evaluated. A variability study evaluated different precision estimates related to column packing and replicate measurement conditions. The best columns with the highest efficiencies (for chiral separations) and good precision, that is, the lowest RSD values, were generated by the packing procedure in which an MeOH-slurry and a water rinsing step of 8 h were applied.

Topics: Capillary Electrochromatography; Particle Size; Polysaccharides; Stilbenes

In the chromatographic separation of enantiomers the order of elution is determined by the strength of diasteromeric interactions between the components of the mixture and a chiral stationary phase. For analytical purposes, it is ideal to have the minor component elute first, whereas in the preparative mode a faster elution of the major component is desirable. Here we describe a stationary phase constructed from a polyacetylene that bears 2,2'-bisphenol-derived side chains in which chirality can be switched in the solid state prior to use. Both the macromolecular helicity of the polymer backbone and the axial chirality of the side chains can be switched in the solid state by interaction with a chiral alcohol, but importantly are maintained after removal of the chiral alcohol because of a memory effect. The chiral stationary phase thus prepared was used to separate the enantiomers of trans-stilbene oxide with the enantiomer elution order determined by the preseparation treatment.

Topics: Macromolecular Substances; Models, Molecular; Molecular Structure; Phenols; Polyynes; Stereoisomerism; Stilbenes

A chemometric approach is used for studying the combined effect of temperature, pressure and co-solvent fraction in analytical and preparative supercritical fluid chromatography (SFC). More specifically, by utilizing design of experiments coupled with careful measurements of the experimental conditions the interaction between pressure, temperature and co-solvent fraction was studied with respect to productivity, selectivity and retention in chiral SFC. A tris-(3,5-dimethylphenyl) carbamoyl cellulose stationary phase with carbon dioxide/methanol as mobile phase and the two racemic analytes trans-stilbene oxide (TSO) and 1,1′-bi-2-naphthol (BINOL) were investigated. It was found for the investigated model system that the co-solvent fraction and pressure were the parameters that most affected the retention factors and that the co-solvent fraction and column temperature were most important for controlling the selectivity. The productivity in the preparative mode of SFC was most influenced by the co-solvent fraction and temperature. Both high co-solvent fraction and temperature gave maximum productivity in the studied design space.

Topics: Carbon Dioxide; Chromatography, Supercritical Fluid; Methanol; Naphthols; Pressure; Solvents; Stilbenes; Temperature

The chromatographic enantioseparation of trans-stilbene oxide (TSO) was studied experimentally and theoretically, where the preparative column was packed with 20 μm Chiralcel OD stationary phase and hexanes/2-propanol were used as mobile phase. The bed porosity, axial dispersion coefficient, mass transfer coefficient and the column efficiency were determined according to the pulse response experiments. The adsorption equilibrium isotherms of single and racemic mixture of trans-stilbene oxide were measured by the frontal analysis, the linear-Langmuir isotherm model was used to fit the experimental data, and the relative parameters were estimated for the competitive adsorption equilibrium of TSO enantiomers. Elution profiles were measured for the separation of TSO enantiomers on Chiralcel OD preparative column, and the experimental data were compared with the simulated results predicted by the mathematical model that accounted for axial dispersion and linear driving force mass transfer model. The adsorption equilibrium and kinetic information obtained in this work are useful for the scale up and optimization of both batch and continuous chromatographic enantioseparation systems.

Topics: Adsorption; Cellulose; Chromatography, Liquid; Models, Chemical; Phenylcarbamates; Stereoisomerism; Stilbenes

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems.

Topics: Chromatography; Stereoisomerism; Stilbenes

Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds.

Topics: Catalysis; Catalytic Domain; Epoxide Hydrolases; Epoxy Compounds; Hydrogen Bonding; Models, Molecular; Molecular Dynamics Simulation; Quantum Theory; Stereoisomerism; Stilbenes

Preparative supercritical fluid chromatography (SFC) has become the preferred method for the rapid purification of drug candidates during the pharmaceutical discovery process. This paper will discuss the evaluation of injection techniques for preparative SFC. A thorough evaluation of mixed stream vs. modifier stream™ injection was performed. It was shown that for the majority of the compounds evaluated, modifier stream injection gave better resolution relative to mixed stream injection. The improvement in resolution with modifier stream injection increased as injection volume increased. In addition, a study evaluating the effect of dissolution solvent on chromatographic performance for the preparative resolution of enantiomers using SFC showed that dissolution solvent had minimal impact on preparative resolution for the preparative SFC separation of trans stilbene oxide.

Topics: Chromatography, Supercritical Fluid; Models, Chemical; Pharmaceutical Preparations; Stereoisomerism; Stilbenes

Acid/base mobile phase modifiers affect enantioseparations in ways that are not yet understood for the lack of systematic studies, which makes the scale-up of preparative separations difficult to predict. Shifts of the selectivity of certain pairs of enantiomers upon exposure of the column to these modifiers is amply documented. Furthermore, once the modifier has been removed from the mobile phase, the improved selectivity remains, this phenomenon has been named the memory effect. We selected four enantiomeric pairs for a systematic study of this memory effect. The selectivity of 4-chlorophenylalanine ethyl ester (4CPEE) improves after a solution of ethanesulfonic acid (ESA) is percolated through the column. The selectivity of propranolol HCl and Tröger's base increases after a solution of diiospropylethylamine is percolated through the column. The selectivity of these three pairs of enantiomers is inversely affected by percolation of the opposite acid/base solution. Each of these four compounds reached an equilibrium concentration that maintained the separation of the enantiomeric pairs. In contrast, the selectivity of trans-stilbene oxide (TSO) is not affected by either acid/base modifier. Preparative separations can be used to detect changes in the active surface of the chiral polymer stationary phase by measuring the change in selectivity and resolution when modifiers are used. Preparative method development was carried out on analytical columns and scale-up to 1cm ID columns were performed in this study.

Topics: Adsorption; Amylose; Chromatography, High Pressure Liquid; Phenylcarbamates; Polymers; Stereoisomerism; Stilbenes

Acid/base mobile phase modifiers affect enantioseparations in ways that are not fully understood yet, for the lack of systematic studies. This makes chiral analysis of some pharmaceuticals difficult to reproduce. Once a column has been exposed to a modifier, the selectivity of certain pairs of enantiomers may change, for the better or the worse. We study the behavior of five enantiomeric pairs, three which are highly sensitive to the addition of certain modifiers and two that have little sensitivity to these modifiers. Their use permits the determination of the extent of the memory effect response on individual columns. The selectivity of 4-chlorophenylalanine methyl and ethyl ester, and of ketoprofen improve as a solution of ethanesulfonic acid is percolated through the column. As a result, these pairs are most useful for the determination of the extent of acid memory effect on a column. The selectivity of propranolol HCl and, to a lesser degree, Tröger's base increases as a solution of diisopropylethylamine is percolated through the column. The separation of each one of these five pairs is inversely affected by the percolation of the opposite acid/base solution. We used trans-stilbene oxide (TSO) as a 'standard' to determine the column stability because no memory effect is observed for it (its retention, enantioselectivity, and resolution remain constant). Understanding whether a column is under the influence of the memory effect is critical to both the analysis of pharmaceutical ingredients and to the development of preparative purification techniques for racemic mixtures. Thus, columns that were unreliable for method development and method transfer, due to the memory effect and a lack of proper solvent exposure records, can now be used.

Topics: Alkanesulfonates; Amylose; Chromatography, Liquid; Fenclonine; Ketoprofen; Models, Chemical; Phenylcarbamates; Propranolol; Stereoisomerism; Stilbenes

Picosecond time-resolved transmission Raman data were acquired for 1 mm thick powder samples of trans-stilbene, and a Monte Carlo model was developed that can successfully model the laser and Raman pulse profiles. Photon migration broadened the incident (approximately 1 ps) probe pulse by two orders of magnitude. As expected from previous studies of Raman photon migration in backscattering mode, the transmitted Raman pulse was broader than the transmitted laser pulse and took longer to propagate through the sample. The late-arriving photons followed tortuous flight paths in excess of 50 mm on traversing the 1 mm sample. The Monte Carlo code was also used to study the spatial resolution (lateral and depth) of steady-state transmission Raman spectroscopy in the diffusion regime by examining the distribution of Raman generation positions as a function of incident beam size, sample thickness, and transport length. It was predicted that the lateral resolution should worsen linearly with sample thickness (typically the resolution was about 50% of the sample thickness), and this is an inevitable consequence of operating in the diffusion regime. The lateral resolution was better at the sample surface (essentially determined by the probe beam diameter or the collection aperture) than for buried objects, but transmission sampling was shown to be biased towards the mid-point of thick samples. Time-resolved transmission experiments should improve the lateral resolution by preferentially detecting snake photons, subject to constraints of signal-to-noise ratio.

Topics: Computer Simulation; Lasers; Models, Theoretical; Monte Carlo Method; Photons; Scattering, Radiation; Spectrum Analysis, Raman; Stilbenes; Time

Chromatographic behavior of nonracemic mixtures, viz., mandelic acid and stilbene oxide as analytes has been studied in detailed by enantiomer self-disproportionation on achiral ordered mesoporous material M41S and regular silica gel as stationary phases. Enantiomer self-disproportionation gave enhanced separation of analytes. The extent and magnitude of enantiomer self-disproportionation is dependent on the optical purity of the starting non-racemic molecules, presence of intermolecular hydrogen bonding/pi-pi interactions and the nature of eluents used. The present study and previous literature data suggest that percentage ee of a nonracemic mixture needs to be determined before any chromatographic purification is taken up as enantiomer self-disproportionation phenomenon could occur during purification. The data show that enantiomer self-disproportionation of nonracemic mixtures can be harnessed for its enantioenrichment on inexpensive achiral stationary phases.

Topics: Chromatography, Gel; Hydrogen Bonding; Mandelic Acids; Porosity; Silica Gel; Silicon Dioxide; Solvents; Stereoisomerism; Stilbenes; Surface Properties; Temperature

A facile strategy based on click chemistry for preparation of the structurally well-defined native beta-cyclodextrin (beta-CD) based chiral stationary phase (CSP) was proposed. The beta-CD CSP was evaluated by enatioseparation of benzoin, trans-stilbene oxide, Troger's base, bendroflumethiazide, ketoprofen, chlorthalidone, three flavanone compounds and two beta-adrenergic blocking agents under reversed phase high performance liquid chromatography. The chromatographic results demonstrate the chiral separation ability of click beta-CD CSP and illustrate the usefulness of click chemistry in the preparation of beta-CD based CSP.

Topics: Benzoin; beta-Cyclodextrins; Chromatography, High Pressure Liquid; Silicon Dioxide; Stereoisomerism; Stilbenes

This study presents a simple method for measuring long-range heteronuclear coupling constants between protons and proton-bearing carbons. The approach involves recording two conventional 1D-TOCSY experiments in which the offset of the selective proton pulse is set on the low- and high-frequency 13C satellites of an isolated proton signal, Hi. Long-range heteronuclear coupling values between the 13Ci bonded to Hi and the protons Hj,k...n that belong to the same spin system were easily determined from the relative displacement of the relayed Hj,k...n signals in the satellite-selective TOCSY spectra. The sense of the displacement indicated the sign of the coupling constants.

Topics: Carbon; Hydrogen; Isoleucine; Magnetic Resonance Spectroscopy; Menthol; Organic Chemicals; Protons; Stilbenes

Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.

Topics: Alkenes; Alkylation; Amino Acid Sequence; Binding Sites; Cysteine; Epoxy Compounds; Glutathione Transferase; Humans; Kinetics; Mutagenesis, Site-Directed; Protein Engineering; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stereoisomerism; Stilbenes

The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.

Topics: Alkylation; Amino Acid Sequence; Binding Sites; Catalysis; Epoxide Hydrolases; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Solanum tuberosum; Stereoisomerism; Stilbenes; Substrate Specificity; Tyrosine

trans-Stilbene oxide (TSO) induces drug metabolizing enzymes in rat and mouse liver. TSO is considered a phenobarbital-like compound because it induces Cyp2B mRNA expression in liver. Phenobarbital increases Cyp2B expression in liver via activation of the constitutive androstane receptor (CAR). The purpose of this study was to determine whether TSO induces gene expression in mouse liver via CAR activation. TSO increased CAR nuclear localization in mouse liver, activated the human Cyp2B6 promoter in liver in vivo, and activated a reporter plasmid that contains five nuclear receptor 1 (NR1) binding sites in HepG2 cells. TSO administration increased expression of Cyp2b10, NAD(P)H:quinone oxidoreductase (Nqo1), epoxide hydrolase, heme oxygenase-1, UDP-glucuronosyl-transferase (Ugt) 1a6 and 2b5, and multidrug resistance-associated proteins (Mrp) 2 and 3 mRNA in livers from male mice. Cyp2b10 and epoxide hydrolase induction by TSO was decreased in livers from CAR-null mice, compared with wild-type mice, suggesting CAR involvement. In contrast, TSO administration induced Nqo1 and Mrp3 mRNA expression equally in livers from wild-type and CAR-null mice, suggesting that TSO induces expression of some genes through a mechanism independent of CAR. TSO increased nuclear staining of the transcription factor Nrf2 in liver, and activated an antioxidant/electrophile response element luciferase reporter construct that was transfected into HepG2 cells. In summary, in mice, TSO increases Cyp2b10 and epoxide hydrolase expression in mice via CAR, and potentially induces Nqo1 and Mrp3 expression via Nrf2. Moreover, our data demonstrate that a single compound can activate both CAR and Nrf2 transcription factors in liver.

Topics: Animals; Base Sequence; Cell Nucleus; Constitutive Androstane Receptor; Gene Expression Regulation; Liver; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Multidrug Resistance-Associated Protein 2; NF-E2-Related Factor 2; Oligonucleotide Probes; Receptors, Cytoplasmic and Nuclear; Stilbenes; Transcription Factors

trans-Stilbene oxide (TSO) is a synthetic proestrogen that induces phase I and II drug-metabolizing enzymes in rat liver. The purpose of this study was to determine whether TSO also induces transporter expression in rat liver and whether gene induction in rat liver after TSO occurs in a constitutive androstane receptor (CAR)-dependent manner. Total RNA was isolated from male rat livers after treatment with TSO for up to 4 days (200 mg/kg, i.p., twice daily), and the mRNA levels for each gene were quantified. CYP2B1/2, CYP3A1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1, multiple drug resistance protein (Mdr) 1a and 1b, as well as multidrug resistance-associated protein (Mrp) 2, 3, and 4 mRNA were increased in livers after TSO treatment. To determine whether TSO activates gene expression in a CAR-dependent manner, male and female Wistar-Kyoto (WKY) rats were treated with TSO for 3 days. TSO induced CYP2B1/2, UGT2B1, and Mdr1b in males more than in females, suggesting that TSO could increase their expression via CAR. Conversely, TSO induced CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 similarly in both genders, indicating that induction of these genes occurs independently of CAR. TSO treatment also increased the activity of a CAR binding element luciferase reporter construct in HepG2 cells transfected with rat CAR and in mouse liver. Additionally, TSO increased antioxidant response element/electrophile response element luciferase reporter construct activity in HepG2 cells. In conclusion, in WKY rat liver, TSO increases CYP2B1/2, UGT2B1, and Mdr1b mRNA expression in a gender-dependent manner and CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 in a gender-independent manner.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Cell Line, Tumor; Constitutive Androstane Receptor; Cytochrome P-450 CYP2B1; Cytochrome P-450 CYP3A; Epoxide Hydrolases; Female; Genes, Reporter; Glucuronosyltransferase; Humans; Liver; Luciferases; Male; Mice; Mice, Inbred C57BL; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Rats; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Response Elements; RNA, Messenger; Sex Factors; Stilbenes; Transcription Factors; Transfection; Up-Regulation

[reaction: see text] A stereoselective/stereospecific synthesis of polysubstituted tetrahydronaphthols based on the Michael addition of ortho-lithiated stilbene oxides to alpha,beta-unsaturated Fischer carbene complexes followed by an unusual cyclization of the corresponding intermediate in a 6-endo-tet mode is described.

Topics: Cyclization; Hydrocarbons; Lithium; Macromolecular Substances; Methane; Molecular Structure; Naphthols; Stereoisomerism; Stilbenes

Topics: Chromatography, Liquid; Reproducibility of Results; Statistics as Topic; Stilbenes

1. By sequencing genomic DNA from 72 established cell lines derived from Japanese individuals, we detected 25 single nucleotide alterations in the microsomal epoxide hydrolase (EPHX1) gene. Of them, five were exonic alterations resulting in amino acid alterations (77C>G, T26S; 128G>C, R43T; 337T>C, Y113H; 416A>G, H139R; 823A>G, T275A). The T26S, R43T, Y113H and H139R substitutions were found at relatively high frequencies and seemed to be polymorphic, and T26S and T275A were novel. 2. To examine the effects of these amino acid alterations on EPHX1 function, EPHX1 cDNA constructs of wild-type and five variants were transfected into COS-1 cells, and their hydrolytic activities for cis-stilbene oxide were determined in vitro. Although all of the transfectants expressed EPHX1 mRNA and protein at similar levels, the variant H139R protein was expressed at a significantly higher level (128% of the wild-type). K(m) values were not significantly different between the wild-type and variants. 3. Increase (140%) in the enzymatic activity (V(max)) of the variant H139R was accompanied by the increased EPHX1 protein level without any significant change in the intrinsic EPHX1 activity. On the other hand, the variant R43T showed increased values for V(max) and clearance (V(max)/K(m)) (around 130%) both on a microsomal protein basis and on a EPHX1 protein basis. 4. These results suggest that R43T as well as H139R increase epoxide hydrolase activity.

Topics: Amino Acid Substitution; Animals; Blotting, Northern; Blotting, Western; Cell Line; Chlorocebus aethiops; COS Cells; DNA; DNA Primers; Epoxide Hydrolases; Exons; Hydrolysis; Introns; Kinetics; Microsomes, Liver; Plasmids; Polymorphism, Single Nucleotide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stilbenes

Treatment with the microsomal enzyme inducer trans-stilbene oxide (TSO) can decrease biliary excretion of acetaminophen-glucuronide (AA-GLUC) and increase efflux of AA-GLUC into blood. The hepatic canalicular multidrug resistance protein (Mrp) 2 and sinusoidal protein Mrp3 transport AA-GLUC conjugates into bile and blood, respectively. Thus, TSO-induced alterations in the vectorial excretion of AA-GLUC may occur via increased hepatic Mrp3 levels. The goal of this study was to determine whether TSO, diallyl sulfide (DAS), and oltipraz (OLT) treatments can up-regulate Mrp3 protein expression, and whether treatment with DAS and OLT can correspondingly increase hepatovascular efflux of AA metabolites. Rats were administered phenobarbital, TSO, DAS, OLT, or vehicle for 4 days. Interestingly, all of the chemicals increased the plasma concentration and urinary excretion of AA-GLUC and decreased its biliary excretion. In control animals, approximately 77% and 23% of AA-GLUC was excreted into bile or urine, respectively, whereas with inducer-pretreated animals, <32% of AA-GLUC was excreted into bile and >68% was excreted into urine. Correspondingly, all of the compounds increased hepatic Mrp3 mRNA levels by 13- to 37-fold and protein levels by 2- to 6-fold, respectively. In conclusion, these studies correlate increased Mrp3 protein levels in liver with increased hepatovascular excretion of AA-GLUC and suggest that induction of Mrp3 affects the route of drug excretion.

Topics: Acetaminophen; Allyl Compounds; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Transporters; Bile; Blotting, Western; Branched DNA Signal Amplification Assay; Drug Resistance, Multiple; Kidney; Liver; Male; Mitochondrial Proteins; Phenobarbital; Pyrazines; Rats; Rats, Sprague-Dawley; Ribosomal Proteins; RNA, Messenger; Saccharomyces cerevisiae Proteins; Stilbenes; Sulfides; Thiones; Thiophenes; Time Factors

In order to investigate the fundamental data for the resistance of gamma radiation sterilization of polyvinylchloride (PVC), the formulations of the antioxidants such as commercial Irganox series and inorganic, processing aids, stabilizer aids, trans-stilbene oxide (StO) and so on have been carried out. The control and irradiated PVC samples with 1.5, 2.5 and 4.0 Mrad were characterized by mechanical tester, colorimetry, and extractant in water. Irganox 1010 was more effective than Irganox 1076 for color changes whereas Irganox 1076 was more effective than Irganox 1010 for the change of extractant. It was also observed the significant diminution of color changes for inorganic antioxidants as CaO and ZnO. Oxidized paraffin wax as lubricant, styrene-methylmetacrylate copolymers as processing aids, and nontoxic debenzoylmethane as secondary stabilizer did not show good stabilization for the irradiation. The new proposed radiation stabilizer in this study, StO, showed the significant improvement of gamma radiation resistance for the plasticized PVC. The possible mechanism could be explained that an epoxy functional group stabilizes effectively the processes of dehydrogenation, the formation of hydroperoxides, and the formation of oxygen containing groups, and the synergetic effects of an epoxy compound are more notable for the prevention of radiation oxidation in the presence of an aromatic group.

Topics: Antioxidants; Biocompatible Materials; Color; Cross-Linking Reagents; Excipients; Gamma Rays; Materials Testing; Plasticizers; Polyvinyl Chloride; Radiation Dosage; Sensitivity and Specificity; Sterilization; Stilbenes; Tensile Strength

A new series of trans-stilbene benzenesulfonamide derivatives were designed and synthesized as potential antitumor agents. These new compounds were evaluated in the National Cancer Institute's 60 human tumor cell line in vitro screen. Compounds 9-13 were cytotoxic against several cell lines. Notably, two compounds, 9 and 12, demonstrated selective cytotoxic activity against BT-549 breast cancer (GI(50)=0.205 microM) and HT-29 colon cancer (GI(50)=0.554 microM), respectively.

Topics: Antineoplastic Agents; Benzene; Cell Division; Drug Screening Assays, Antitumor; Humans; Models, Molecular; Molecular Conformation; Molecular Structure; Neoplasms; Stilbenes; Structure-Activity Relationship; Sulfonamides; Tumor Cells, Cultured

About 50% and 15% of Caucasians lack the glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes and the corresponding enzyme activity, respectively. Both of these polymorphisms have been shown to affect the genotoxicity of some epoxides in cultured human lymphocytes. Especially GSTT1 appears to be important in whole-blood cultures, probably because GSTT1 activity is high in erythrocytes. The in vitro genotoxicity of trans-stilbene oxide (TSO), a model substrate for GSTM1, has been shown to depend on individual GSTM1 activity. The potential role of GSTM1 genotype, and the possible interference of GSTT1 genotype, has not previously been examined in this context. We have studied TSO-induced sister chromatid exchanges (SCEs) in 72 h whole-blood lymphocyte cultures from 24 healthy human donors, representing different combinations of GSTM1 and GSTT1 positive and null genotypes. TSO clearly increased SCEs in cultures of all donors. The mean number of SCEs per cell induced by 75 and 150 microM TSO was, respectively, 1.5- and 1.3-times higher in cultures of GSTM1 null than GSTM1 positive donors. In another experiment, GSTM1 null individuals showed, in comparison with GSTM1 positive subjects, a 1.8-fold SCE induction by 50 microM TSO. GSTT1 genotype did not have an unequivocal effect. Our findings suggest that the lack of the GSTM1 gene, resulting in reduced detoxification capacity, increases individual sensitivity to the genotoxic effects of TSO.

Topics: Adult; Cells, Cultured; Dose-Response Relationship, Drug; Female; Genotype; Glutathione Transferase; Homozygote; Humans; Lymphocytes; Male; Middle Aged; Polymorphism, Genetic; Sister Chromatid Exchange; Stilbenes; Time Factors

The present study provides the first evidence that stilbene oxide and styrene oxide are reductively metabolized to the corresponding olefins in rats. When cis- or trans-stilbene oxide was given orally to rats, both cis- and trans-stilbene were isolated from the urine and feces. Styrene was also isolated from the urine and feces of rats given styrene oxide. These metabolites were identified unequivocally by UV and mass spectral comparison with authentic samples, and on the basis of their TLC and HPLC behavior. However, these olefins were not detected in the urine or feces of antibiotics-treated rats dosed with cis- or trans-stilbene oxide. Cecal contents of the untreated rats exhibited olefin oxide reductase activities toward cis- and trans-stilbene oxides under anaerobic conditions. The results suggest that intestinal bacteria play an important role in the reduction of olefin oxides to the corresponding olefins in the animal body.

Topics: Alkenes; Animals; Biotransformation; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Epoxy Compounds; Feces; Male; Mass Spectrometry; Oxidation-Reduction; Rats; Rats, Wistar; Spectrophotometry, Ultraviolet; Stilbenes

1. This study provides the first evidence that stilbene oxide and styrene oxide are reductively metabolized to the corresponding alkenes by intestinal bacteria in animals. 2. When trans- or cis-stilbene oxide were incubated with the caecal contents of rat under anaerobic conditions, both trans- and cis-stilbene were isolated from the incubation mixture. Styrene oxide was also reduced to styrene by rat caecal contents. 3. Caecal contents of mouse, hamster and guinea pig also exhibited alkene oxide reductase activities toward cis- and trans-stilbene oxides, and styrene oxide. In contrast, liver microsomes or cytosol exhibited no epoxide reductase activities toward these substrates. 4. Seven pure strains of intestinal bacteria exhibited alkene oxide reductase activities of varying degrees under anaerobic conditions, with the highest activity being observed in Clostridium sporogenes. 5. Cell-free extracts of either the intestinal bacteria in rat caecal contents or C. sporogenes exhibited reductase activity when supplemented with both NAD(P)H and FMN under anaerobic conditions. Reductase activity was also observed on addition of the photochemically reduced form of FMN instead of both NAD(P)H and FMN.

Topics: Animals; Bacteria; Clostridium; Cricetinae; Epoxy Compounds; Guinea Pigs; Intestinal Mucosa; Intestines; Male; Mice; Rats; Rats, Wistar; Stilbenes

A liver microsome-mediated activation of the proestrogens trans-stilbene and trans-stilbene oxide was found in this study. trans-Stilbene and trans-stilbene oxide were negative in estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7 and growth assay in rat pituitary tumor cell line MtT/E-2. However, these compounds exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. In contrast, cis-stilbene and cis-stilbene oxide did not show estrogenic activity after such incubation. When trans-stilbene was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, two metabolites were detected by HPLC. They were identified unequivocally as trans-4-hydroxystilbene and trans-4, 4'-dihydroxystilbene by mass and UV spectral comparison with authentic samples. The oxidase activity of the liver microsomes toward trans-stilbene was inhibited by SKF 525-A and alpha -naphthoflavone. Minor activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. trans-4-Hydroxystilbene and trans-4,4'-dihydroxystilbene exhibited significant estrogenic activities. These results suggest that the estrogenic activities of trans-stilbene and trans-stilbene oxide were due to formation of hydroxylated metabolites.

Topics: Animals; Biotransformation; Estrogens; Humans; Male; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Stilbenes; Tumor Cells, Cultured

Oxidative biotransformation, coupled with genetic variability in enzyme expression, has been the focus of hypotheses interrelating environmental and genetic factors in the etiology of central nervous system disease processes. Chemical modulation of cerebral cytochrome P450 (P450) monooxygenase expression character may be an important determinant of in situ metabolism, neuroendocrine homeostasis, and/or central nervous system toxicity resulting from exposure to neuroactive drugs and xenobiotic substances. To examine the capacity of the rat brain to undergo phenobarbital (PB)-mediated induction, we developed reverse transcription-polymerase chain reaction methods and evaluated the effects of several PB-like inducers on P450 and microsomal epoxide hydrolase gene expression. Animals treated i.p. with four daily doses of PB demonstrated markedly induced levels of CYP2B1, CYP2B2, and CYP3A1 mRNA in the striatum and cerebellum. In contrast, 1 or 2 days of PB treatment resulted in unchanged or even slightly decreased levels of CYP2B1 and CYP2B2 in the brain, although the latter treatments produced marked induction of the corresponding genes in the liver. Only slight increases in epoxide hydrolase RNA levels resulted in brains of PB-treated animals. Substantial activation of cerebral CYP2B1, CYP2B2, and CYP3A1 mRNA levels also resulted when animals were treated with the neuroactive drugs diphenylhydantoin and amitryptiline, and with the potential PB-like xenobiotic inducers trans-stilbene oxide and diallyl sulfide, whereas dichlorodiphenyltrichloroethane was less efficacious. Although the time course of the induction response is delayed in brain relative to that required for the liver, these results clearly establish that brain P450s are markedly PB inducible.

Topics: Allyl Compounds; Amitriptyline; Animals; Aryl Hydrocarbon Hydroxylases; Blotting, Northern; Brain; Cytochrome P-450 CYP2B1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; DDT; Enzyme Induction; Epoxide Hydrolases; Liver; Male; Mixed Function Oxygenases; Phenobarbital; Phenytoin; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Steroid Hydroxylases; Stilbenes; Sulfides

Recent epidemiological studies proposed that the glutathione S-transferase (GST) M1-null genotype may contribute to diseases associated with oxidative stress. The genetic polymorphism exhibited by the GSTM1 may be an important factor in risk toward oxidant chemicals. In this study, we investigated the effect of GSTM1-null genotype in lymphocyte and oxidative stress-dependent inhibition of platelet aggregation. To determine whether GSTM1 deficiency is a genetic determinant of cell toxicity toward oxidant chemicals, lymphocytes were incubated in vitro with low levels of benzo(a)pyrene (BaP), cumene hydroperoxide (CumOOH), or trans-stilbene oxide that do not decrease cell viability, and were assessed for oxidative damage and for the lymphocyte-dependent inhibition of platelet response. Malondialdehyde and carbonyl levels, and the oxidation of cisparinaric acid, were used as biomarkers of oxidative stress in lymphocytes. Following stimulation by BaP or CumOOH, when peroxidation-dependent changes in these parameters were compared between the GSTM1-null genotype and the positive genotype, no significant differences were found between the two genotypes. On the other hand, preincubation of the lymphocytes with BaP or CumOOH attenuated their inhibitory action on ADP-induced platelet aggregation. However, our results indicate that lymphocytes of individuals with the GSTM1-null genotype have greater inhibitory activity on platelet function after exposure to BaP, but not CumOOH, although they are not more susceptible to in vitro oxidative stress.

Topics: Adult; Benzene Derivatives; Benzo(a)pyrene; Blood Platelets; Female; Glutathione Transferase; Humans; Leukocytes, Mononuclear; Lymphocytes; Male; Malondialdehyde; Oxidants; Oxidative Stress; Stilbenes

Glutathione S-transferase (GST) M1 is a member of the GST mu family of cytosolic enzymes that have been hypothesized to catalyze the conjugation of glutathione to a large number of hydrophobic substances, including carcinogens such as polynuclear aromatic hydrocarbons present in tobacco smoke, leading to their excretion. Epidemiologic and experimental evidence suggests that the risk of cervical cancer is related to both human papillomavirus (HPV) infection and cigarette smoking. We compared the enzymatic activities and mRNA levels of GSTs in GSTM1-positive human cervical keratinocytes (HCKs) that had been transfected with HPV16 with those in the parental cells. The GSTM1 activity toward the substrate trans-stilbene oxide was 5- to 7-fold lower than in the parental cells. The relative mRNA level in HCK transfected with HPV16 E6/E7, as quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) with normalization against endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, was 6% that of the parental cells. It was 16 and 82%, respectively, in cells that were transfected with HPV16 E6 alone or HPV16 E7 alone. When quantified by competitive RT-PCR using an exogenous nuclease-resistant synthetic cyclophilin RNA transcript as control, the mRNA level in HCK transfected with HPV16 E6 was approximately 10-fold lower that that in the parental cells. It was approximately 5- to 7-fold lower in the HPV16 E7 or HPV16 E6/E7 cells. Our results suggest that viral infections, through the modulation of cellular xenobiotic-metabolizing enzymes, may play a role in the ability of cells to handle environmental carcinogens.

Topics: Carcinogens, Environmental; Cells, Cultured; Cervix Uteri; Disease Susceptibility; Enzyme Induction; Female; Gene Expression Regulation; Gene Expression Regulation, Viral; Glutathione Transferase; Humans; Isoenzymes; Keratinocytes; Male; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Penis; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stilbenes; Transfection

The natural polyphenolic compound resveratrol (trans-3,4', 5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 +/- 0.65 to 2.92 +/- 0.30 pmol.mg protein-1) and prostaglandin E2 (from 11.46 +/- 1.15 to 8.02 +/- 0.80 nmol.mg protein-1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 +/- 0.5 microM (5-lipoxygenase), 40 +/- 5.0 microM (15-lipoxygenase), 35 +/- 4.0 microM (cyclooxygenase activity of prostaglandin H synthase) and 30 +/- 3.0 microM (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.

Topics: Apoptosis; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Cyclooxygenase Inhibitors; Humans; K562 Cells; Lipoxygenase; Lipoxygenase Inhibitors; Oxidative Stress; Prostaglandin-Endoperoxide Synthases; Resveratrol; Stilbenes

The effect of smoking on DNA adduct formation in lymphocytes was analysed in individuals with low (deficient) and high (non-deficient) glutathione S-transferase (class Mu) activity. DNA adduct levels in lymphocytes were determined by the highly sensitive nuclease P1-enhanced 32P-postlabeling assay. The lymphocyte DNA adducts/10(8) nucleotides of smokers deficient in glutathione S-transferase Mu activity (n = 12) were significantly higher than those of smokers non-deficient (n = 9) in glutathione S-transferase Mu activity. The DNA adduct levels of the lymphocytes inversely correlated with glutathione S-transferase Mu activity. A correlation was found between DNA adduct levels and daily cigarette consumption. Results of the present study suggest that individuals deficient in glutathione S-transferase Mu activity may be at greater risk of DNA damage.

Topics: Adult; Autoradiography; DNA Adducts; DNA Damage; Glutathione Transferase; Humans; Lymphocytes; Male; Middle Aged; Phosphorus Radioisotopes; Smoking; Stilbenes; Tobacco Smoke Pollution; Toxicity Tests

Both trans- and cis-stilbene oxide (TSO and CSO) markedly induced heme oxygenase-1 (HO-1) at the transcriptional level in rat liver. HO-1 induction by TSO and CSO was preceded by glutathione (GSH) depletion in the liver. Pretreatment of rats with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, enhanced GSH depletion evoked by either TSO or CSO and augmented the increase in HO-1 mRNA. In contrast, pretreatment with perfluorodecanoic acid (PFDA), which reduced hepatic GSH S-transferase activity, prevented TSO- and CSO-mediated GSH depletion and abolished HO-1 induction. In addition, TSO and CSO enhanced c-jun but not c-fos mRNA, which is in parallel with the HO-1 mRNA change. These findings indicate that the oxidative stress evoked by GSH depletion after the treatment of rats with stilbene oxides could stimulate both HO-1 and c-jun gene expression. Pretreatment with either BSO or PFDA also affected the induction of CYP2B1/2 mRNA and apoprotein by TSO or CSO, suggesting that not only the change of heme pool size but also some other unknown factor or factors may be involved in the regulation of the CYP2B1/2 and HO-1 gene expression. cis-Stilbene (CS), a parent compound of CSO, also induced HO-1 mRNA, together with hepatic GSH depletion, but trans-stilbene (TS) failed to elevate HO-1 mRNA under the experimental conditions. In addition, CS increased CYP2B1/2 mRNA, whereas TS did not. These results suggest that CS could be rapidly oxidized by cytochrome P-450 (P-450) to CSO, leading to GSH depletion in the liver. Such differences in the hepatic metabolic pathways of CS and TS are attributable to the differential effects on HO and P-450 induction by these compounds. Like other phenobarbital-type P-450 inducers, TSO and CSO also induced CYP2C6 and 3A2 apoproteins in rat liver. Stilbene oxide reduced CYP2E1 mRNA and apoproteins for CYP2E1 and 2C11. All of these findings indicate that stilbene compounds have unique effects on hepatic HO-1 and P-450 regulation in rats.

Topics: Animals; Blotting, Northern; Cytochrome P-450 Enzyme System; Enzyme Induction; Glutathione; Heme Oxygenase (Decyclizing); Liver; Male; Oxidative Stress; Rats; Rats, Wistar; RNA, Messenger; Stilbenes

7,12-dimethylbenz(a)anthracene (DMBA) causes necrosis in endocrine organs. DMBA metabolism in follicles and corpora lutea from porcine ovaries was demonstrated not only in the microsomal but also in the mitochondrial fraction, in contrast to what has been found in the rat ovary. Maximal activities were present in these fractions of the corpus luteum, with specific activities of 5.9 and 2.2 pmol/min x mg protein, respectively. DMBA metabolism in mitoplasts, i.e. mitochondrial inner membranes, proved to be more than 10-fold higher than the corresponding activity in the mitochondrial fraction. The purities of the subcellular fractions were assessed by measurements of marker enzymes. 17-42% of the mitochondrial DMBA metabolism was concluded to be due to microsomal contamination. In the mitoplast fraction such contamination was only 0.18-2.8%. Ellipticine and alpha-naphthoflavone reduced the metabolism of DMBA in the luteal microsomal fraction by 95 and 77%, respectively. In mitochondria the inhibition by these agents was 63 and 30%, respectively. Indomethacine and estradiol decreased microsomal DMBA metabolism by 53 and 52%, respectively. In mitochondria the inhibition was 52 and 23%, respectively. None of these inhibitors affected the DMBA metabolism by the mitoplast fraction.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Biotransformation; Corpus Luteum; Cytochrome P-450 Enzyme System; Female; Microsomes; Mitochondria; Monoamine Oxidase; NADH, NADPH Oxidoreductases; Ovarian Follicle; Ovary; Stilbenes; Swine

The efficacy of two free radical scavengers, selenium and zinc, and a microsomal epoxide hydrolase-inducing agent, cis-stilbene oxide on the acute toxicity of T-2 toxin, a potent cytotoxic trichothecene, was investigated. Mice were pretreated daily for 3 consecutive days with either zinc sulfate (4.4 mg/kg, intraperitoneally [i.p.]), sodium selenite (1, 2, and 3 mg/kg i.p.) or cis-stilbene oxide (50 mg/kg i.p.). A full 24-hr after the final dosing with these agents, mice were given T-2 toxin (2, 2.5, or 3 mg/kg i.p.). The acute lethal toxicity of T-2 toxin (2.5 mg/kg) was reduced by administration of only sodium selenite (3 mg/kg) and cis-stilbene oxide (50 mg/kg). No significant effect on weight gain was observed.

Topics: Animals; Dose-Response Relationship, Drug; Epoxide Hydrolases; Free Radical Scavengers; Male; Mice; Mice, Inbred Strains; Microsomes; Poisoning; Selenium; Stilbenes; T-2 Toxin; Zinc

The differential induction of hepatic cytochrome P-450 (P450) was studied in Eisai-hyperbilirubinuria rats (EHBR/Eis). This rat is a mutant that has as high a concentration of bilirubin in the urine as in the plasma. A single administration of trans-stilbene oxide (TSO, 2 mmol/kg), a phenobarbital (PB)-type P450 inducer, did not increase total P450, the CYP2B1/2 or the CYP2C6 in EHBR/Eis liver. TSO was able to induce delta-aminolevulinic acid synthetase and heme oxygenase, rate-limiting enzymes in heme biosynthesis and degradation, respectively, in both EHBR/Eis and Sprague-Dawley rat (SDR), the strain from which EHBR/Eis is derived. TSO also produced similar effects on glutathione depletion and on the activities of other drug-metabolizing enzymes in both strains. A 23-fold increase in CYP2B1/2 mRNA in the SDR liver was observed 24 hr after TSO treatment. In the EHBR/Eis strain, however, TSO increased CYP2B1/2 mRNA only 2-fold. In addition, repeated injection of TSO failed to induce P450 isozymes, CYP2B1/2, CYP2C6 or CYP3A2 in EHBR/Eis. On the other hand, there was essentially no difference in the induced levels of CYP1A1/2 apoprotein and mRNA between twins of SDR and EHBR/Eis livers treated with 3-methylcholanthrene or 1-benzylimidazole. The increased levels of both CYP2B1/2 apoprotein and mRNA from EHBR/Eis liver treated with TSO and 1-benzylimidazole were much smaller (2.5- and 5-fold increases, respectively) than from the SDR liver (17.5- and 15-fold increases, respectively). Although PB expressed CYP2B1/2 apoprotein and mRNA to a similar extent in both homozygous and heterozygous EHBR/Eis livers, CYP3A2 and CYP2C6 were less responsive to PB in homozygous EHBR/Eis. Repeated treatment with TSO induced these isozymes in heterozygote but not in homozygote. These findings suggest that the suppressed expression of PB-inducible P450 isozyme genes in the EHBR/Eis liver may be a general phenomenon associated with PB-type inducers. Therefore, EHBR/Eis may be experimentally useful for studying the mechanism of P450 induction by PB and PB-type inducers.

Topics: Animals; Cytochrome P-450 Enzyme System; Hyperbilirubinemia; Immunoblotting; Liver; Male; Microsomes; Phenobarbital; Rats; Stilbenes; Time Factors

Microsomal epoxide hydrolase (HYL1) is a single-gene enzyme responsible for the hydrolysis of epoxides derived from the oxidative metabolism of xenobiotics. Variation in HYL1, therefore, may be an important determinant of drug toxicity. We have investigated HYL1 enzyme kinetics in six different species including man, for which a liver bank genotyped for polymorphisms in exons 3 and 4 of the HYL1 gene was used. Activity was measured by radiochromatography with high specific activity radiolabeled substrates, cis-stilbene oxide (CSO) and carbamazepine 10,11-epoxide (CBZ-E). In addition, naphthalene was used to investigate the hydrolysis of an epoxide (naphthalene 1,2-epoxide [N-E] generated in situ. There was marked species variation in enzyme activity that was substrate dependent. CSO was rapidly hydrolyzed by microsomes from all species, the rank order of specific activity being human > rabbit > dog > rat > hamster > mouse. In contrast, hydrolysis of CBZ-E was only observed with human liver microsomes. CBZ-E was only a weak (IC50 = 1 mM) inhibitor of CSO hydrolysis. The hydrolysis of N-E, determined as the diol-to-total metabolite ratio, was human > rabbit > dog > hamster > mouse > rat. Intraspecies variation in man was 4-fold, 7-fold and 2-fold for CSO, CBZ-E and N-E, respectively: none of this variation could be directly accounted for by the HYL1 polymorphisms in exons 3 and 4. These data emphasize the need for careful toxicokinetic evaluation of species used in the safety evaluation of compounds likely to form epoxide intermediates in vivo.

Topics: Adult; Animals; Carbamazepine; Cricetinae; Cytochrome P-450 Enzyme System; Dogs; Epoxide Hydrolases; Exons; Female; Humans; Kinetics; Male; Mice; Microsomes, Liver; Middle Aged; Naphthalenes; Polymorphism, Genetic; Rabbits; Rats; Species Specificity; Stilbenes

Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one(t-PBO), delta 5-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Brain; Butanones; Coturnix; Cytosol; Dinitrochlorobenzene; Electrophoresis, Polyacrylamide Gel; Epoxy Compounds; Ethacrynic Acid; Etiocholanolone; Female; Glutathione Transferase; Isoelectric Focusing; Kidney; Liver; Lung; Male; Molecular Sequence Data; Nitrobenzenes; Nitrophenols; Protease Inhibitors; Sequence Alignment; Sequence Homology, Amino Acid; Stilbenes; Tissue Distribution

In order to improve the resistance of gamma-radiation sterilization of polypropylene (PP), the formulations of the additives such as antioxidants, crosslinking agent, and trans-stilbene oxide (StO) have been carried out. The irradiated PP and ethylene-propylene copolymer samples with control and additives were characterized by mechanical tester, colorimetry, and Fourier transform infrared spectroscopy with total attenuated reflectance mode (FTIR-ATR). Crosslinking agent and StO formulated PP showed remarkable radiation resistance and minimum discoloration. Also, radiation resistance of ethylene-propylene copolymers with 3% of ethylene contents was better than that of PP homopolymers in the case of no additives. The proposed mechanisms of radiation stabilization with additives are also discussed.

Topics: Antioxidants; Cross-Linking Reagents; Gamma Rays; Materials Testing; Polypropylenes; Radiation Dosage; Spectroscopy, Fourier Transform Infrared; Sterilization; Stilbenes

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.

Topics: Adolescent; Adult; Aged; Anticonvulsants; Carbamazepine; Chromatography, High Pressure Liquid; Drug Hypersensitivity; Enzyme Inhibitors; Epoxide Hydrolases; Female; Glutathione Transferase; Humans; Hydrolysis; Isoenzymes; Kinetics; Lymphocytes; Male; Microsomes; Middle Aged; Radiometry; Stilbenes; Trichloroepoxypropane; Tritium

To investigate the relationship between glutathione (GSH) depletion and metallothionein (MT) synthesis, the effects of substrates and an inhibitor of GSH S-transferases on concentrations of hepatic GSH, zinc (Zn) and MT were studied in rats. Trans-stilbene oxide (TSO) is an inducer of drug metabolizing enzymes and also a substrate of GSH S-transferase, whereby it covalently reacts with and depletes GSH. The hepatic GSH level was decreased to 25% of the control 2 h after injection of TSO, and returned to the control level by 24 h. TSO significantly increased hepatic concentrations of Zn and MT in a dose-dependent manner. Two isoforms of MT (MT-I and MT-II) were increased by TSO; MT-II was the dominant form. Pretreatment with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced MT synthesis itself as well as that induced by TSO and cis-stilbene oxide (CSO). On the contrary, infection into rats of perfluorodecanoic acid (PFDA), an inhibitor of GSH S-transferase, resulted in a decrease in basal levels of Zn, and prevented the increase in MT synthesis by TSO and CSO. These results suggest that the decrease of GSH concentration in the liver which causes oxidative stress conditions may be related to MT induction.

Topics: Animals; Buthionine Sulfoximine; Decanoic Acids; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorocarbons; Glutathione; Glutathione Transferase; Kinetics; Liver; Male; Metallothionein; Methionine Sulfoximine; Rats; Rats, Wistar; RNA, Messenger; Stereoisomerism; Stilbenes; Zinc

1. The ability of model stable epoxides and metabolites generated by human liver microsomes from benzo[a]pyrene, aflatoxin B1, naphthalene and tamoxifen to produce cytotoxicity and genotoxicity in human peripheral lymphocytes has been investigated. 2. The stable epoxides 1,1,1 trichloropropene-2,3-oxide (100 microM) and trans stilbene oxide (100 microM) as well as metabolites generated from aflatoxin B1 (30 microM) and naphthalene (100 microM) by an extracellular metabolising system were toxic to isolated resting mononuclear leucocytes (MNLs), whereas glycidol (100 microM), benzo[a]pyrene (100 microM) and tamoxifen (50 microM) were not. 3. The stable epoxides 1,1,1 trichloropropene-2,3-oxide (100 microM) and trans stilbene oxide (100 microM) but not glycidol (100 microM) were toxic to dividing lymphocytes only after a 72-h exposure. Tamoxifen (30 microM), aflatoxin B1 (30 microM) and their metabolites were also toxic to dividing lymphocytes. Benzo[a]pyrene (100 microM) and naphthalene (100 microM) were not toxic either in the absence or presence of the extracellular metabolising system. 4. Benzo[a]pyrene (100 microM) and aflatoxin B1 (30 microM) were directly genotoxic to lymphocytes, this genotoxicity was significantly enhanced by the presence of the extracellular metabolising system. This indicates that both intracellular and extracellular bioactivation of these two compounds can produce genotoxicity. In contrast, naphthalene and tamoxifen were non-genotoxic.

Topics: 1-Propanol; Adult; Aflatoxin B1; Benzo(a)pyrene; Carcinogens; Cell Division; Environmental Pollutants; Epoxy Compounds; Estrogen Antagonists; Humans; Male; Microsomes, Liver; Mutagens; NADP; Naphthalenes; Propanols; Sister Chromatid Exchange; Spermatogenesis; Stilbenes; T-Lymphocytes; Tamoxifen; Trichloroepoxypropane

Exposure to more than one toxic compound is common in real life. The resulting toxic effects are often more than the simple sum of the effects of the individual compounds. It is unlikely that it will ever be possible to test all combinations. It is therefore highly desirable to improve or develop means for reasonably approximating predictions of interactions. In order to be valid and extrapolatable, these predictions are most promising if they are mechanism-based. Examples will be given for possibilities of mechanism-based predictions of interactions which exceed trivialities of simple increases by enzyme induction of enzymatic rates of a given biotransformation pathway leading to a toxic metabolite. Instead, examples will be provided where competition between various enzymes for shunting the same substrate into divergent pathways can lead to predictable dramatic changes in toxicity by shifting the metabolic routes under conditions of no significant changes of overall metabolism. Further examples are given on predictable interactions between chemicals which need bioactivation for exerting their toxicity and chemicals which effect hormonal status and other endogenous factors which in turn modify enzymes involved in the control of toxic metabolites.

Topics: Animals; Benzo(a)pyrene; Drug Interactions; Enzyme Induction; Epoxide Hydrolases; Male; Microsomes, Liver; Mutagenicity Tests; Phosphorylation; Rats; Rats, Sprague-Dawley; Salmonella typhimurium; Stilbenes; Xenobiotics

The relationship between the activity of glutathione S-transferases (GSTs), especially the mu isozyme, and the production of responses to nitroglycerin (GTN) was investigated in rabbit aorta. GST mu isozyme activity was measured using trans-stilbene oxide (TSO) as a substrate. Each aorta was divided into four parts, two of which were frozen for enzymatic analyses while the remaining two were used to measure the effects of GTN (0.5 microM), i.e. the increase in cGMP levels and the corresponding relaxation. Thus, all three measures were obtained in each individual rabbit aorta. Eight different rabbits were studied. An excellent correlation was obtained between the rise in cGMP and the mu isozyme activity (r2 = 0.948). A good correlation was also obtained between TSO activity and the relaxation response to GTN. Total GST activity did not correlate well with either cGMP increases or percent relaxation. These observations indicate that the activity of the mu isozyme measured using TSO and not the total GST correlates with the responses to GTN in the in vitro rabbit aorta model.

Topics: Animals; Aorta; Cyclic GMP; Glutathione Transferase; Isoenzymes; Models, Biological; Nitroglycerin; Rabbits; Stilbenes; Vasodilation

The microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of cis-4,4'-dimethylstilbene oxide (1a), cis-4,4'-diethylstilbene oxide (1b), cis-4,4'-diisopropylstilbene oxide (1c), and cis-4,4'-dichlorostilbene oxide (1d) have been investigated using rabbit liver microsomal preparations. The kinetic parameters, Km and Vmax, and the absolute stereochemistry of the reactions have been determined and compared with those of cis-stilbene oxide (1e). All epoxides 1a-d are hydrolyzed by mEH with high product enantioselectivity to give (R,R)-(+)-diols with ee > or = 90%. The presence of the substituents on the phenyl rings markedly reduces the rates of mEH catalyzed hydrolysis with respect to cis-stilbene oxide, by increasing Km and reducing Vmax in the cases of 1a, 1b, and 1d, or reducing only the Vmax in the case of 1c. The very low Vmax, together with a persistent ability to fit into the mEH active site, make all these epoxides, and particularly 1c, inhibitors of cis-stilbene oxide hydrolysis. The kinetic and stereochemical results are interpreted on the basis of the proposed topology of the mEH active site.

Topics: Animals; Binding Sites; Circular Dichroism; Epoxide Hydrolases; Epoxy Compounds; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Male; Microsomes, Liver; Rabbits; Stereoisomerism; Stilbenes; Structure-Activity Relationship

Trans-stilbene oxide, trans-beta-methylstyrene 7,8-oxide, trans-beta-ethylstyrene 7,8-oxide, trans-beta-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his- strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10(6) of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-beta-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.

Topics: Animals; Cell Division; Chalcone; Chalcones; Cricetinae; Cricetulus; Epoxide Hydrolases; Epoxy Compounds; Humans; Lymphocytes; Mutagenicity Tests; Mutagens; Salmonella typhimurium; Sister Chromatid Exchange; Stilbenes; Styrenes

Known inducers of the hepatic glutathione (GSH) S-transferases were tested at the limits of their solubility as inducers of the enzyme in cultured human keratinocytes. Neither phenobarbital, trans-stilbene oxide, propylthiouracil, nor butylated hydroxyanisole increased GSH S-transferase activity or led to the appearance of alpha- or mu-forms of the enzyme, as judged by Western blotting. Only the pi-form of the enzyme was found before and after all treatments. Thus, the enzyme is not inducible in keratinocytes. However, 4 mM propylthiouracil did lead to a 50% increase in GSH reductase activity, and phenobarbital at 4 mM completely abolished GSH peroxidase and GSH reductase activity and led to a significant loss of viability.

Topics: Butylated Hydroxyanisole; Cells, Cultured; Enzyme Induction; Glucosephosphate Dehydrogenase; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Humans; Keratinocytes; Phenobarbital; Propylthiouracil; Stilbenes

Glutathione transferases play an important role in the detoxification of many different endogeneous and exogenous compounds such as metabolites of polycyclic aromatic hydrocarbons (PAH) of cigarette tar. There is evidence that PAH may be atherogenic. The glutathione transferase activity towards trans-stilbene oxide (GST-tSBO) can be separated in blood in GST-positive and GST-negative phenotypes. We have previously suggested that the GST-negative phenotype may be associated with a higher morbidity in intermittent claudication among middle aged smokers. In the present study, GST-tSBO could easily be measured in human, rabbit and bovine arterial smooth muscle cells (SMC) in culture. The level of GST-tSBO was higher in rabbit than in bovine SMC. It was stable in bovine SMC during 5 cell passages and it could be induced twofold by long-time incubation with dimethylsulfoxide-soluble particulate matter from cigarette smoke or 3,4-benzo(a)pyrene. There was a positive correlation between the level of GST-tSBO in blood and in "healthy" arterial and venous tissue from individuals operated with coronary bypass. The enzyme levels in arterial tissue were lower than in venous tissue. GST-tSBO in atherosclerotic segments of human arteries was lower than in "healthy" segments from the same artery. These findings suggest that the arterial wall may have a low defense against toxic compounds that may decrease further as atherosclerosis proceeds. It is concluded that SMC are suitable for the study of the effects of PAH in relation to GST-tSBO and that the enzyme activity in blood will reflect the individual GST-tSBO phenotype also in vascular tissues.

Topics: Adult; Animals; Arteriosclerosis; Cattle; Cells, Cultured; Female; Glutathione Transferase; Humans; In Vitro Techniques; Male; Middle Aged; Muscle, Smooth, Vascular; Phenotype; Polycyclic Compounds; Rabbits; Smoking; Stilbenes

Class mu glutathione S-transferases (GSTs) are important in the detoxication of epoxides generated by oxidative metabolism. Phenobarbital, 3-methylcholanthrene, and pyridine have failed to enhance the expression of class mu GST isozymes in rabbit hepatic tissue (T. Primiano, S. G. Kim, and R. F. Novak, Toxicol. Appl. Pharmacol., 113, 64-73, 1992). Two class mu GST isozymes have been isolated from rabbit hepatic cytosol and purified to homogeneity using S-hexylglutathione-agarose, CM-Sepharose, and PBE94 chromatofocusing chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses showed that both isozymes possessed M(r) values of approximately 25,500 and cross-reacted with class mu-specific GST IgG. Gel filtration analysis revealed that these isozymes were dimers with molecular weights of approximately 45 kDa. The class mu GST isozymes had pIs of 7.8 and 7.2 as determined by nonequilibrium pH gel electrophoresis. The class mu GST 7.8 and 7.2 isozymes exhibited different metabolic activities toward the substrates 1-chloro-2,4-dinitrobenzene, bromosulfophthalein, 1,2-epoxy-3-(p-nitrophenoxy)propane, trans-4-phenyl-3-buten-2-one, p-nitrobenzyl chloride, and 3,4-dichloronitrobenzene. Metabolic activity of the two GSTs toward the substrate 1-chloro-2,4-dinitrobenzene was inhibited by Cibacron blue, triethyltin bromide, S-hexylglutathione, bromosulfophthalein, and indomethacin. The amino acid composition of GST mu 7.8 and 7.2 was determined and found to be very similar to those of purified rat class mu GST isozymes. N-terminal analysis of the first 21 residues of the pI 7.8 class mu GST isozyme revealed that it had 71 and 81% sequence identity with the Yb1 and Yb2 subunits, respectively. Similarly, N-terminal analysis of the first 21 residues of the pI 7.2 class mu GST isozyme revealed a 75% sequence identity with either the rat Yb1 or Yb2 subunit. Examination of class mu GST expression in rabbit hepatic cytosol following treatment with a series of known inducers including phenobarbital, 3-methylcholanthrene, isosafrole, pyrazine, trans-stilbene oxide, butylated hydroxyanisole, and tert-butylhydroquinone was accomplished. The data show that these agents not only failed to enhance class mu GST expression, but that 3-methylcholanthrene and isosafrole caused suppression of class mu GSTs. These results provide evidence for the existence of two closely related class mu GST isozymes in rabbit hepatic tissue and suggest that the mo

Topics: Amino Acid Sequence; Animals; Antioxidants; Cytosol; Enzyme Induction; Glutathione Transferase; Hydroquinones; Isoenzymes; Liver; Male; Molecular Sequence Data; Rabbits; Stilbenes; Substrate Specificity

Glutathione S-transferase (GST) enzymes detoxify carcinogens in tobacco smoke. Interindividual variation in GST function may be related to differences in risk for smoking-related cancer. Leukocytes from 50% of Caucasians lack GST activity toward trans-stilbene oxide (TSO), due to a deletion of the gene for the GST-mu enzyme. Presence of GST-TSO activity in leukocytes has been associated with low risk for lung cancer among cigarette smokers. We sought to determine whether GST activity in lung tissue is determined by the same gene polymorphism and whether it is associated with risk for lung cancer. Subjects were cigarette smokers, identified at the time of lung resection or autopsy in Seattle hospitals. Uninvolved lung tissue was obtained from 35 patients with lung carcinoma and 43 control patients and assayed for GST-mu activity with TSO, for the presence of the GST-mu gene product with an immunological assay, and for the GST-mu gene with Southern blotting. Mailed questionnaires were used to collect information on subjects' smoking histories and exposures which might alter enzyme activity. Interindividual results from the three assays correlated well. Smokers with high GST-TSO enzyme activity present in their lung tissue had a lower risk for lung carcinoma than did smokers with no or low activity (relative risk = 0.30; 95% confidence interval, 0.11-0.79), as did smokers with GST-mu antigen identified in lung tissue versus those with no antigen (relative risk = 0.30; 95% confidence interval, 0.11-0.79). Smokers with both maternal and paternal copies of GST-mu DNA (n = 7) had a lower cancer risk than smokers lacking GST-mu DNA (n = 30; relative risk = 0.35; 95% confidence interval, 0.06-2.10). High GST-mu activity appeared to be associated with a greater decrease in lung cancer risk among 38 heavy cigarette smokers (relative risk = 0.15; 95% confidence interval, 0.03-0.64) than among 38 light smokers (relative risk = 0.61; 95% confidence interval, 0.14-2.60). Presence or absence and number of copies of the GST-mu gene appear to determine activity of the GST-mu enzyme in lung. Smokers with the GST-mu enzyme have approximately one-third of the risk for lung carcinoma of smokers without the enzyme.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Blotting, Southern; Female; Genetic Predisposition to Disease; Glutathione Transferase; Humans; Individuality; Isoenzymes; Leukocytes; Lung; Lung Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Predictive Value of Tests; Reproducibility of Results; Risk Factors; Smoking; Stilbenes

A cDNA clone (PLGSTA) of 896 bp, containing an open reading frame encoding a 225-amino-acid polypeptide of M(r) 25,723, was isolated from a cDNA library constructed in lambda gt11 from the liver of a marine teleost flatfish, the plaice (Pleuronectes platessa). The identification of this cDNA as that coding for the subunit of the major cytosolic glutathione S-transferase of plaice liver, GST-A, was supported by its heterologous expression in and purification of its protein product from Escherichia coli. The recombinant-derived protein exhibited identical M(r) and immunoreactivity and a similar substrate specificity to GST-A previously isolated from plaice liver. Comparison of the deduced amino acid sequence of the plaice GST-A polypeptide with the primary structures of GSTs from other Phyla revealed that it showed the greatest similarity to plant, insect and mammalian Theta class GSTs. Southern blot analysis of plaice DNA hybridized to the PLGSTA cDNA showed a banding pattern indicative of the presence of a single gene. Northern blot analysis of a variety of plaice tissues showed hybridizing bands of approx. 1100 nucleotides in all tissues tested, with the highest relative amounts in liver and intestinal mucosa. A marked increase in hybridization intensity was observed in hepatic RNA samples from plaice treated with trans-stilbene oxide, suggesting that GST-A is induced by epoxides in this species.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA; Fishes; Glutathione Transferase; Insecta; Isoenzymes; Liver; Mammals; Molecular Sequence Data; Plants; RNA, Messenger; Sequence Alignment; Species Specificity; Stilbenes

Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione transferase subunits 1, 2, 3, 4, 6, 7 and 8 in the liver and adrenal of intact and hypophysectomized male and female Sprague-Dawley rats. A sexual dimorphism in the levels of several of these isoenzymes and in their responses to hypophysectomy was demonstrated. In the liver of sham-operated females and males there are differences in glutathione transferase activities and isoenzyme pattern. H.p.l.c. analysis showed higher levels of subunits 1, 3 and 4 in male rats compared with females. In contrast with the pronounced sex differences in sham-operated rats, the isoenzyme patterns of hypophysectomized males and females were very similar. In the adrenal glands, however, a sexual dimorphism became apparent only after hypophysectomy, when the level of subunit 4 was increased 14-fold in the female, whereas the corresponding increase in the male rat was only 2.7-fold. The hepatic pattern of glutathione transferase subunits could be altered by continuous infusion of growth hormone to both sham-operated and hypophysectomized rats of both sexes. This treatment feminized the isoenzyme pattern in sham-operated males and a similar effect was obtained upon treating hypophysectomized rats with thyroxine, cortisone acetate and a continuous infusion of growth hormone.

Topics: Adrenal Glands; Animals; Cortisone; Cytosol; Female; Glutathione Transferase; Growth Hormone; Hypophysectomy; Isoenzymes; Liver; Macromolecular Substances; Male; Rats; Rats, Inbred Strains; Reference Values; Sex Characteristics; Stilbenes; Thyroxine; Time Factors

Glutathione S-transferase (GST) class Mu activity was determined in 145 unrelated hospital patients in Berlin by measuring their conjugation activity towards the specific substrate trans-stilbene oxide (TSO) with two substrate concentrations (50 and 250 microM) in homogenates prepared from lymphocytes. Eighty individuals (55.2%) had an activity lower than 10 pmol/min/10(6) lymphocytes and were classified as GST class Mu deficient. In 142 of 145 cases, phenotype was confirmed by the results of a genotyping procedure using the polymerase chain reaction technique. Two fragments of 273 and about 650 bp including one and two introns, respectively, could always be amplified from genomic DNA in individuals with high GST class Mu activity and could not be amplified in persons with impaired glutathione-TSO conjugation activity. This indicates that persons with low activity carry a large deletion mutation within the GST class Mu gene. The enzymatically determined antimode between low and high activity determined as 10 pmol/min/1 million lymphocytes in the assay with 50 microM TSO could be clearly confirmed by genotyping.

Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Berlin; Chromosome Deletion; Female; Germany; Glutathione; Glutathione Transferase; Humans; Isoenzymes; Lung Neoplasms; Lymphocytes; Male; Middle Aged; Molecular Sequence Data; Mutation; Phenotype; Polymerase Chain Reaction; Stilbenes

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

Topics: Epoxide Hydrolases; Epoxy Compounds; Fatty Acids; Glycine max; Kinetics; Membranes; Seeds; Stilbenes; Subcellular Fractions; Substrate Specificity

Glutathione transferases (GST) are detoxifying enzymes who act with many endogenous and exogenous substances such as polycyclic aromatic hydrocarbons (PAH). The GST activity towards trans-stilbene oxide (GST-tSBO) is inherited in an autosomal dominant fashion and can be separated in high (GST-positive) and low (GST-negative) phenotypes when measured in blood. Human fibroblast cultures were established from males matched for age, smoking habits and clinical manifestations of atherosclerosis. Matched pairs of GST-negative and GST-positive fibroblasts were studied. There was a very strong correlation between the levels of GST-tSBO in peripheral blood and in cultured fibroblasts within the same individual. When fibroblasts were exposed to benzo(a)pyrene (BP) or dimethylbenzanthracene (DMBA) GST-negative cells produced relatively more collagen than GST-positive cells. GST-negative fibroblasts showed a greater cell death than GST-positive fibroblasts as well among controls as after exposure to PAH. It is concluded that lack of GST-tSBO is easily discriminated in cultured skin fibroblasts. GST-negative and GST-positive fibroblasts showed different susceptibility towards some toxic stimuli that might be of importance in atherogenesis.

Topics: Aged; Cell Death; Cells, Cultured; Collagen; DNA; Fibroblasts; Glutathione Transferase; Humans; Male; Middle Aged; Polycyclic Compounds; Skin; Stilbenes

Glutathione transferase (GT) activity towards trans-stilbene oxide (tSBO), benzo[a]pyrene-4,5-oxide (B[a]PO) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in human liver and lymphocytes. GT-tSBO activity is catalyzed by GT mu which has polymorphic expression in human lymphocytes. Our results show that activity of GT-tSBO in lymphocytes correlates with its activity in liver (r = 0.7, P less than 0.001). GT activity towards BPO (GT-BPO) also correlated with GT-tSBO in lymphocytes and liver. However, interindividual variation of GT-BPO is less than that of GT-tSBO, suggesting that BPO may not be as specific a substrate for GT mu and therefore other GT isozymes may contribute to BPO conjugation. Conjugation of CDNB by GT was not different using cytosols from either high or low GT mu individuals. The functional significance of the GT-mu polymorphism was evaluated by measuring its effect on benzo[a]pyrene (B[a]P)- and aflatoxin B1 (AFB1)-DNA adduct formation in vitro. Human liver cytosols prepared from persons having low or high GT-tSBO activity were incubated with human liver microsomes, calf thymus DNA and B[a]P or AFB1. HPLC analysis revealed that the major B[a]P adduct was dG(N2)-7 beta, 8 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). BPDE-dG adducts were decreased equally by cytosols from either low or high conjugators. In contrast, AFB1-DNA binding was inhibited to a greater extent in high conjugators than low conjugators. HPLC analysis demonstrates that adducts formed were AFB1-FAPyr and AFB1-N7-Gua. The correlation between AFB1-DNA adduct concentrations and GT mu activity was highly significant with a correlation coefficient of r = 0.88 at P less than 0.001. These results suggest that GT mu plays an important role in detoxifying DNA reactive metabolites of AFB1 and this enzyme may be a susceptibility marker for AFB1 related liver cancer. Moreover, our data demonstrate that lymphocytes are a reliable surrogate tissue for detecting liver GT mu polymorphisms.

Topics: Adolescent; Adult; Aflatoxin B1; Aged; Aged, 80 and over; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Chromatography, High Pressure Liquid; Cytosol; Dinitrochlorobenzene; DNA; Female; Glutathione Transferase; Humans; Liver; Liver Neoplasms; Lymphocytes; Male; Middle Aged; Polymorphism, Genetic; Pyrimidines; Stilbenes

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.

Topics: Amino Acid Sequence; Antibodies; Bile Acids and Salts; Chromatography; Glutathione Transferase; Hemin; Humans; Hydrogen Peroxide; Isoelectric Focusing; Isoenzymes; Kinetics; Leukotriene A4; Leukotrienes; Molecular Sequence Data; Muscles; Stilbenes; Substrate Specificity

Kochia foliage that had tested positive to Dragendorff's reagent (presumptive alkaloids) and had elicited chronic toxicosis when fed to rats was fed to sheep to characterize early stages of kochia toxicosis and evaluate treatments that might improve tolerance. Twelve fine-wool lambs (46 +/- 9 kg BW) were fed chopped kochia hay (35%) mixed with chopped alfalfa hay (65%) for 4 wk. The kochia diet had 14.3% CP and 39.9% ADF. Dry matter intake averaged 3.4% of BW/d. Body weight did not change during 4 wk and blood serum components were not changed from values at the onset. Thereafter, kochia was increased to 50% of diet for five more weeks, during which four treatments were imposed randomly (three lambs/treatment): 1) none; 2) N-acetyl-L-cysteine plus trans-stilbene oxide, 21 and 52 mg/kg of BW, respectively, given i.p. twice weekly; 3) retinyl palmitate, 275 mg, plus alpha-tocopherol, 300 mg/lamb dosed i.m. twice weekly; and 4) zinc sulfate mixed in the feed to provide 500 mg daily. Kochia contained 4.8% oxalate. The diet with 50% kochia had 16% CP and 36% ADF, and digestibility coefficients were 59% for DM, 72% for CP, and 59% for ADF. After 5 wk, blood glucose was elevated slightly, total bilirubin was increased about 1.5-fold (P less than .05), alanine aminotransferase was elevated slightly (P less than .05), and inorganic phosphorus and urea (blood urea N) were diminished (P less than .05); other serum components, including calcium, were unchanged from initial levels (P greater than .10). Treatments had negligible effects for modifying serum signs of mild chronic toxicosis associated with kochia hay fed as 50% of diet.

Topics: Acetylcysteine; Animal Feed; Animals; Blood Chemical Analysis; Female; Male; Nutritive Value; Plant Poisoning; Plants, Toxic; Sheep; Sheep Diseases; Stilbenes; Vitamin A; Vitamin E; Zinc

Livestock grazing lush Kochia scoparia (L.) Schrad, sometimes experience BW loss, hyperbilirubinemia, photosensitization, and polyuria. Animals fed kochia hay may exhibit milder or negligible signs of toxicosis but fail to utilize nutrients efficiently. To characterize early aspects of kochia toxicosis and to evaluate prospective treatments, 12 wether lambs (34 +/- 3 kg) were fed prebloom kochia hay (83% OM, 15% CP, and 6.3% total oxalate) and treated as follows: 1) no treatment; 2) drenched daily with aqueous ZnSO4 to provide 30 mg of Zn/kg of BW); 3) injected i.p. twice weekly with N-acetyl-L-cysteine (CYS) in saline (21 mg/kg of BW) plus trans-stilbene oxide (TSO) in corn oil (27 mg/kg of BW); and 4) treated as 2) plus 3). Treatments were imposed factorially (2 x 2) with three lambs per treatment. Kochia intake (ad libitum) averaged .57 kg/d (1.7% of BW) for 80 d, and digestibility of DM and CP were 44 and 59%, respectively, at wk 4, but BW loss was severe (6 to 11 kg/lamb). After 14 d, serum insulin and prolactin were decreased (P less than .05) below initial values (.48 to .11 and 102 to 28 ng/ml, respectively). Serum somatotropin increased (P less than .05) from 4.5 to 6.8 ng/ml at 4 wk. Serum total bilirubin increased threefold at 3 wk (P less than .05) and declined slightly thereafter through 10 wk. Early changes in serum enzymes reflected mild hepatotoxicosis without cholestasis, whereas histopathology (at 80 d) showed diffuse hepatocyte swelling and nephrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcysteine; Analysis of Variance; Animal Feed; Animals; Blood Chemical Analysis; Growth Hormone; Hormones; Insulin; Liver; Male; Nitrogen; Nutritive Value; Plant Poisoning; Plants, Toxic; Prolactin; Sheep; Sheep Diseases; Stilbenes; Sulfates; Zinc; Zinc Sulfate

Chick liver glutathione peroxidase activity was separated into selenium-dependent and selenium-independent enzyme fractions and the effects of chemicals on the activities of each fraction were examined. Clofibrate induced the selenium-dependent enzyme, while phenobarbital, butylhydroxyanisole and trans-stilbene oxide elevated selenium-independent peroxidase activity. A third enzyme, which showed glutathione peroxidase activity toward both hydrogen peroxide and cumene hydroperoxide, was found.

Topics: Animals; Benzene Derivatives; Butylated Hydroxyanisole; Chickens; Chromatography, Gel; Clofibrate; Glutathione Peroxidase; Hydrogen Peroxide; Liver; Male; Methylcholanthrene; Phenobarbital; Stilbenes

The effect of hypophysectomy and subsequent treatment with adrenocorticotropic hormone (adrenocorticotropin, ACTH) on the isoenzymes of glutathione transferase in the rat adrenal gland was investigated. A large increase (approx. 11-fold) in the level of transferase subunit 4 was observed in hypophysectomized animals by immunoblotting. When the activity of glutathione transferase 4-4 was measured in adrenal cytosol using trans-stilbene oxide as a selective substrate, a 15-fold increase was noted. Lack of the pituitary hormone ACTH is apparently related to this increase, since treatment of hypophysectomized animals with ACTH for 2 weeks partially down-regulated subunit 4. Glutathione transferase subunits 3 and 8 in the adrenal were also increased in amount by hypophysectomy, but not at all to the same extent. The activity of glutathione transferase 4-4 was elevated also in the liver and ovary (5 and 1.5 times respectively) after hypophysectomy. These elevated enzyme levels were, however, not affected by ACTH treatment. This down-regulation of glutathione transferases in the rat adrenal by ACTH may be related to the fact that, under normal conditions, this organ is highly susceptible to the toxic effects of various polycyclic hydrocarbons, whereas under circumstances where there is no ACTH production, as in hypophysectomized rats, the adrenal is resistant to these same hydrocarbons.

Topics: Adrenal Glands; Adrenocorticotropic Hormone; Animals; Blotting, Western; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Female; Glutathione Transferase; Hypophysectomy; Isoenzymes; Liver; Ovary; Rats; Rats, Inbred Strains; Stilbenes

The identification of genetic traits that predispose individuals to environmentally induced cancers is one of the most important problems in cancer risk assessment. Genetic deficiency in the mu-isozyme of the glutathione (GSH) S-transferases (EC 2.5.1.18) has recently been associated with increased lung cancer risk. To test whether this association could arise from a metabolically mediated sensitivity to mutagenic substrates, cytogenetic damage in lymphocytes from 21 isozyme-deficient and 24 nondeficient individuals was induced. Cells were treated with trans-stilbene oxide, an excellent substrate for GSH S-transferase mu, or cis-stilbene oxide, a poor substrate for the isozyme. Sister chromatid exchange induction was measured as an indicator of cytogenetic damage. A trimodal distribution of trans-stilbene oxide-induced sister chromatid exchanges was observed in the population, including resistant, moderate, and highly sensitive groups. Glutathione S-transferase mu deficiency was associated with both moderate and high sensitivity to trans-stilbene oxide-induced damage but had no effect on cis-stilbene oxide-induced sister chromatid exchange. The results indicate that GSH S-transferase mu, a proposed marker of cancer susceptibility, is also a marker of susceptibility to the induction of cytogenetic damage by a certain class of mutagens. The differential effects of the cis- and trans-isomers of stilbene oxide illustrate that the stereoselectivity of GSH S-transferase mu toward various alkene epoxide substrates can be an important factor affecting individual sensitivity to DNA-damaging epoxides.

Topics: Adult; Aged; Chemical Phenomena; Chemistry; Dose-Response Relationship, Drug; Female; Glutathione Transferase; Humans; Isoenzymes; Lymphocytes; Male; Sister Chromatid Exchange; Stilbenes

Topics: Adult; Carcinogens; Cells, Cultured; Female; Glutathione Transferase; Humans; Inactivation, Metabolic; Isoenzymes; Lymphocytes; Male; Mutagens; Polymorphism, Genetic; Sister Chromatid Exchange; Smoking; Stilbenes

The cytosolic glutathione transferases (GSTs) with basic pI values have been studied in mouse liver after treatment with 2,3-t-butylhydroxyanisole (BHA), cafestol palmitate (CAF), phenobarbital (PB), 3-methylcholanthrene (3-MC) and trans-stilbene oxide (t-SBO). The cytosolic GST activity was induced by all compounds except for 3-MC. Three forms of GST were isolated by means of affinity chromatography and f.p.l.c. The examination of protein profiles and enzymic activities with specific substrates showed that the three GSTs correspond to those found in control animals, i.e. GSTs MI, MII and MIII. The class Mu GST MIII accounted for the major effect of induction, whereas the class Alpha GST MI and the class Pi GST MII were unchanged or somewhat down-regulated. The greatest induction was obtained with BHA, PB and CAF. The activities of other glutathione-dependent enzymes were also studied. An increase in glutathione reductase and thioltransferase activities was observed after BHA, PB or CAF treatment; glyoxalase I and Se-dependent glutathione peroxidase were depressed in comparison with the control group in all cases studied.

Topics: Animals; Butylated Hydroxyanisole; Cytosol; Diterpenes; Enzyme Induction; Glutaredoxins; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Isoenzymes; Lactoylglutathione Lyase; Liver; Methylcholanthrene; Mice; Organ Size; Oxidoreductases; Phenobarbital; Protein Disulfide Reductase (Glutathione); Stilbenes

A glutathione transferase from human mononuclear leukocytes with a high activity toward trans-stilbene oxide (GT-tSBO) has been studied in liver and blood from fetus and adults and in blood from neonates. Using starch gel electrophoresis, different phenotypes of GST1 have been determined, GST1 0, GST1 1, and GST1 2. As judged from activity measurements and the fact that only those individuals who express the null allele of GST1, the GST1 0, which has a low activity toward trans-stilbene oxide, it is concluded that the hepatic transferase GST1 is identical to GT-tSBO, as well as to hepatic transferase mu. In addition, it has been shown that the different genotypes of GST1 1 (GST1 1-1, GST1 1-0) and GST1 2 (GST1 2-2, GST1 2-0) can be separated by measuring the GT-tSBO activity in whole blood from the same individual. It is also demonstrated that GT-tSBO activity is much lower in fetal liver, approximately 10 times, compared with adult liver, while this activity seems to be unchanged in the blood from fetus and adults, as well as in neonates.

Topics: Adult; Dinitrochlorobenzene; Electrophoresis, Starch Gel; Fetus; Glutathione Transferase; Humans; Infant, Newborn; Leukocytes, Mononuclear; Liver; Phenotype; Stilbenes

Rat hepatoma cells grown intraperitoneally as an ascites tumour were analysed with respect to their contents of cytosolic glutathione transferases. In contrast with normal liver tissue, the hepatoma cells were dominated by the class Pi glutathione transferase 7-7. All the major hepatic enzyme forms were down-regulated to almost undetectable concentrations. Livers of rats bearing ascites-hepatoma cells expressed low, but significant, amounts of protein which, by electrophoretic and immunochemical properties, appeared identical with transferase 7-7. This enzyme is not detectable in normal hepatocytes. Treatment of rats with trans-stilbene oxide induced the expression of transferase 7-7 in the livers of normal rats as well as in hepatoma-cell-bearing animals. In addition, a 2-fold induction of transferase 7-7 was measured in the hepatoma ascites cells. No significant elevation of any other enzyme forms in the hepatoma cells was noted.

Topics: Animals; Cell Line; Cytosol; Glutathione Transferase; Isoenzymes; Liver Neoplasms, Experimental; Rats; Stilbenes; Tumor Cells, Cultured

The effects of 3-methylcholanthrene (3-MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenobarbital, trans-stilbene oxide (TSO), pregnenolone-16 alpha-carbonitrile (PCN), dexamethasone, ethanol, isoniazid and butylated hydroxyanisole (BHA) on hepatic glutathione S-transferase (GST) activities toward six substrates were determined in hamsters. TCDD and 3-MC, which are comparatively poor inducers of GSTs in rats, were most effective in enhancing GST activities in hamster liver. In contrast, TSO, BHA and phenobarbital, which are very effective inducers of hepatic GSTs in rats and mice, were ineffective or poor inducers of GSTs in hamster liver. While dexamethasone increased some GST activities, treatments with PCN, ethanol and isoniazid were without effect. The findings indicate that not only the control activity but also the inducibility of hepatic GSTs are different in hamsters from those in other species.

Topics: Animals; Butylated Hydroxyanisole; Cats; Cricetinae; Dexamethasone; Dogs; Enzyme Induction; Ethanol; Glutathione Transferase; Guinea Pigs; Isoniazid; Liver; Male; Mesocricetus; Methylcholanthrene; Mice; Phenobarbital; Polychlorinated Dibenzodioxins; Pregnenolone Carbonitrile; Quail; Rabbits; Rats; Species Specificity; Stilbenes; Trout

Recently we reported elimination of radioactivity from [14C]heptachlor from body stores of lactating ovines, mainly into excreta rather than milk, contrasting sharply with bovines. To further assess heptachlor metabolism and clearance by ovines, 12 fine-wool wether lambs (41 +/- 3 kg) housed in metabolism stalls were fed pelleted alfalfa hay (96%) plus molasses (3%) ad libitum and were dosed i.p. once with [14C]heptachlor (1.643 mg/kg body wt; sp. act. = .89 microCi/mg). Feces and urine were collected separately and quantitatively. Light mineral oil was mixed with feed (5 g/100 g) of six lambs and trans-stilbene oxide, an inducer of biotransformational enzymes, was administered i.p. (4 g/hd initially; 2 g/hd daily thereafter) through 20 d to three lambs given each mineral oil treatment, in 2 x 2 factorial arrangement. Feces, urine, blood, bile and body tissues were assayed for total 14C activity. Radioactivity (heptachlor and [or] metabolites) eliminated into excreta during 21 d amounted to 34 to 36% of dose administered, of which 67% appeared in urine and 33% in feces. Biological half-time for elimination into excreta was 11.3 d [Kel = -.061/d], similar to 11.7 d we reported for lactating ewes. Clearance from blood had T1/2 = 14 d. Neither mineral oil nor trans-stilbene oxide altered rate or route of 14C activity excreted or concentrations of 14C activity in blood. Results confirmed that ovines eliminate heptachlor much more rapidly than bovines.

Topics: Animals; Animals, Newborn; Heptachlor; Male; Metabolic Clearance Rate; Mineral Oil; Sheep; Stilbenes

Microsomal and cytosolic epoxide hydrolase (mEH and cEH respectively) and glutathione S-transferase (GST) activities were measured in the liver, kidney, and gills of rainbow trout. Assays were optimized for time, pH, and temperature, using trans-stilbene oxide (TSO) and cis-stilbene oxide (CSO) as substrates for cEH and mEH, respectively. Optimal pH values for mEH, cEH, and GST were similar to mammalian values (i.e. 8.5, 7.5, and 9). Temperature optima differed between tissues and cell fractions. Specific activity of cEH-TSO was 3-14 times greater than mEH-CSO for all three tissues, and 8-60 times greater on a tissue weight basis. Liver and, to a lesser extent, kidney mEH were active against benzo[a]pyrene 4,5-oxide, whereas gill mEH was not active against this substrate. Liver cytosolic GST was active against CSO and 1-chloro-2,4-dinitrobenzene (CDNB) but not TSO, whereas gill and kidney cytosolic GST were active only against CDNB. Liver and kidney microsomal GST were active against CDNB, but no activity was found in gill microsomes. The results are discussed in relation to possible endogenous substrates and uninduced xenobiotic metabolizing capacities of different trout tissues.

Topics: Animals; Cytosol; Epoxide Hydrolases; Gills; Glutathione Transferase; Hydrogen-Ion Concentration; Kidney; Microsomes, Liver; Salmonidae; Stilbenes; Temperature; Trout

Glutathione transferase (GT; EC 2.5.1.18) mRNA levels were measured in human liver samples by using mouse and human cDNA clones that encode class-mu and class-alpha GT. Although all the RNA samples examined contained class-alpha GT mRNA, class-mu GT mRNA was found only in individuals whose peripheral leukocytes expressed GT activity on the substrate trans-stilbene oxide. The mouse class-mu cDNA clone was used to identify a human class-mu GT cDNA clone, lambda GTH411. The amino acid sequence of the GT encoded by lambda GTH411 is identical with the 23 residues determined for the human liver GT-mu isoenzyme and shares 76-81% identity with mouse and rat class-mu GT isoenzymes. The mouse and human class-mu GT cDNA inserts hybridize with multiple BamHI and EcoRI restriction fragments in the human genome. One of these hybridizing fragments is missing in the DNA of individuals who lack GT activity on trans-stilbene oxide. Hybridizations with nonoverlapping subfragments of lambda GTH411 suggest that there are at least three class-mu genes in the human genome. One of these genes appears to be deleted in individuals lacking GT activity on trans-stilbene oxide.

Topics: Amino Acid Sequence; Animals; Base Sequence; Chromosome Deletion; Glutathione Transferase; Humans; Isoenzymes; Liver; Molecular Sequence Data; Rats; RNA, Messenger; Stilbenes

A glutathione transferase from human leukocytes (GT-tSBO) with high activity towards trans-stilbene oxide has now been found to correspond to the hepatic class Mu glutathione transferase. This class, Mu, of transferase(s) has been found in high concentration in liver, kidney, and adrenal gland, judged by he activity of GT-tSBO. This activity is only found in some individuals, approximately 46%. By using a cDNA clone derived from class Mu transferase(s) from mouse, it is suggested that this enzyme is not transcribed, depending on a deletion (of a part) of a gene in those individuals that are lacking the activity towards tSBO.

Topics: Chromosome Mapping; Genetic Variation; Glutathione Transferase; Humans; Isoenzymes; Liver; Stilbenes

Topics: Animals; Aroclors; Carboxylic Ester Hydrolases; Chlorodiphenyl (54% Chlorine); Clofibrate; Enzyme Induction; Isoenzymes; Male; Microsomes, Liver; Rats; Stilbenes

Optically pure (+)- and (-)-trans-stilbene oxide (TSO) enantiomers were administered to immature male Sprague-Dawley rats. (+)-TSO was the more potent inducer of liver microsomal cytochrome P-450-dependent monooxygenases. The greater potency of (+)-TSO may be explained on the basis of stereoselective metabolism since a far greater concentration of TSO was found in liver microsomes of (+)-TSO-treated rats. Furthermore, of the enzymes known to metabolize TSO, cytosolic epoxide hydrolase turned over the (-)-TSO enantiomer at a faster rate, consistent with the greater persistence of the (+)-enantiomer. Although this report is of chiral effects in potency of enzyme induction, stereoselective metabolism (i.e. disposition) rather than inherent structural characteristics (recognition) may be responsible for these effects.

Topics: Animals; Cytochrome P-450 Enzyme System; Cytosol; Liver; Male; Microsomes, Liver; Rats; Receptors, Aryl Hydrocarbon; Receptors, Drug; Stereoisomerism; Stilbenes

Solubilized rhesus monkey liver microsomes were used as the starting material for the purification of epoxide (cis-stilbene oxide) hydrolase. Successive chromatography over DEAE-Sephacel followed by CM-cellulose resulted in two peaks of activity, CM A and CM B. Passage of these two eluates over separate hydroxyapatite columns resulted in two peaks of activity from CM A, HA A1, and HA A2, and one peak from CM B and HA B, with respective recoveries of 1, 7, and 0.2% of cis-stilbene oxide hydrolase activities. A similar recovery was found for benzo[a]pyrene-4,5-oxide hydrolase, while trans-stilbene oxide hydrolase activity coeluted only in HA A2. Fraction HA A1 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblots of the three eluates and solubilized microsomes incubated with anti-HA A1 demonstrated a single band at 49 kDa in each fraction. The three eluates were differentially affected by the inhibitors of epoxide hydrolase, trichloropropene oxide and 4-phenylchalcone oxide, and addition of Lubrol PX and phospholipid. Immunoprecipitation of HA A2 resulted in coprecipitation of cis- and trans-stilbene oxide hydrolase activity. Upon immunoprecipitation of solubilized microsomes, all the cis-stilbene oxide and benzo[a]pyrene-4,5-oxide, but only 50-60% of trans-stilbene oxide hydrolase activity was precipitated. These studies support findings with other species that (i) an immunochemically distinct cytosolic-like epoxide hydrolase exists in microsomes, and (ii) microsomal epoxide hydrolase activity can be separated during ion-exchange chromatography giving proteins with similar molecular weights and immunochemical cross-reactivity. The precipitation of cis- and trans-stilbene oxide hydrolase activity in eluate HA A2 provides convincing evidence that these isozymes are not structurally identical.

Topics: Animals; Benzopyrenes; Chromatography; Electrophoresis, Polyacrylamide Gel; Epoxide Hydrolases; Immunoassay; Immunosorbent Techniques; Macaca mulatta; Male; Microsomes, Liver; Molecular Weight; Phospholipids; Polyethylene Glycols; Solubility; Stilbenes

Isozyme 4-4 of rat liver glutathione S-transferase catalyzes the stereoselective addition of glutathione to the oxirane carbon of R-absolute configuration of cis-stilbene oxide, 2, to give 98 +/- 2% of the (1S,2S)-1,2-diphenyl-1-(S-glutathionyl)-2-hydroxyethane product with a turnover number (kc) of 0.22 s-1. The two enantiomers of trans-stilbene oxide, 3, are somewhat poorer substrates for the enzyme. Enantioselective addition of glutathione to 3 proceeds with turnover numbers of 0.12 s-1 and 0.023 s-1 for the (R,R,)- and (S,S)-antipodes, respectively.

Topics: Animals; Glutathione Transferase; Isoenzymes; Liver; Rats; Stereoisomerism; Stilbenes; Substrate Specificity

A glutathione transferase from human mononuclear leucocytes with high activity towards trans-stilbene oxide (GT-tSBO) was purified. GT-tSBO is expressed in only about 50% of the individuals studied. As judged from activity measurements, immunological studies and the fact that only those individuals who express glutathione transferase mu have high activity towards trans-stilbene oxide, it is concluded that the hepatic transferase mu is identical with the glutathione transferase (GT-tSBO) in mononuclear leucocytes.

Topics: Dinitrochlorobenzene; Glutathione Transferase; Humans; Isoenzymes; Liver; Neutrophils; Phenotype; Stilbenes

The structures of many dibenz[a,j]acridine (DBAJAC) metabolites formed in vitro in incubations with liver microsomes prepared from 3-methylcholanthrene-pretreated male Wistar rats have previously been determined; they were trans-DBAJAC-3,4-dihydrodiol, trans-DBAJAC-5,6-dihydrodiol, DBAJAC-5,6-oxide, 3-hydroxy-DBAJAC, 4-hydroxy-DBAJAC and several multiply oxidized secondary metabolites. Herein are reported [14-3H]DBAJAC metabolite distributions obtained by h.p.l.c. separation of products produced in incubations with liver and lung microsomes prepared from untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated male Wistar rats. Liver microsomal metabolites were also quantitated in preparations from trans-stilbene oxide-pretreated rats. For all preparations trans-DBAJAC-3,4-dihydrodiol, the candidate proximate carcinogen according to the bay-region theory of carcinogenesis, was the major metabolite (30-40%) while DBAJAC-5,6-oxide and phenols were also quantitatively important. In incubations conducted in the presence of 3,3,3-trichloropropene-1,2-oxide (1.5 mM) formation of dihydrodiol was inhibited by about 85%. DBAJAC-N-oxide was also identified as a minor metabolite (approximately 1%) formed in incubations with phenobarbital-induced and control liver microsomes.

Topics: Acridines; Animals; Biotransformation; Lung; Male; Methylcholanthrene; Microsomes; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains; Stilbenes

The hydrolysis of trans- and cis-stilbene oxide and benzo[a]pyrene-4,5-oxide was measured in cytosol and microsomes of liver, kidney, and testis of control and clofibrate-fed rats. Significant levels of nonprotein sulfhydryls were detected in cytosol from liver (4.6 mM) and testis (1.5 mM). Glutathione was moderately stable in these fractions and interfered with the partition assays as conjugates were retained in the aqueous phase along with diols. When the products were separated by thin-layer chromatography, significant amounts of glutathione-conjugates were found to have been formed in the cytosol of liver and testis. Overnight dialysis or preincubation of cytosol with 0.5 mM diethylmaleate eliminated conjugate formation without affecting diol production. In dialyzed cytosol from clofibrate-fed rats (0.5%, 14 days), the rates of hydrolysis of trans-stilbene oxide were 506, 171, and 96% of controls for liver, kidney, and testis, respectively, and 126% of controls in liver microsomes. Rates of hydrolysis of cis-stilbene oxide were 149, 172, and 96% of controls in microsomes and 154, 124, and 91% of controls in cytosols from livers, kidneys, and testis of clofibrate-fed rats respectively. Hydrolysis of benzo[a]pyrene-4,5-oxide was similar to that of cis-stilbene oxide. Conjugation of the cis-stilbene oxide with glutathione was detected in cytosols from all three tissues with lesser amounts in the microsomes from liver and kidneys. After clofibrate treatment, the rates of this activity were 200, 173, and 95% of controls in cytosol from liver, kidneys and testis, and 203 and 202% of controls in microsomes from liver and kidneys respectively. These results indicate that epoxide hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other evidence which suggests that hydrolysis of cis- and trans-stilbene oxides in cytosol is catalyzed, in part, by distinct enzymes.

Topics: Animals; Benzopyrenes; Clofibrate; Cytosol; Dietary Fiber; Epoxy Compounds; Ethers, Cyclic; Glutathione; Hot Temperature; Isomerism; Kidney; Liver; Male; Microsomes; Microsomes, Liver; Rats; Rats, Inbred Strains; Stilbenes; Testis

Mouse liver light and heavy mitochondrial fractions contain significant epoxide hydrolase activity in addition to that present in the cytosol and microsomes. As the mitochondrial fraction itself contains a number of subfractions, experiments were designed to determine the localization of the epoxide hydrolase activity in these subfractions. Subcellular fractions were prepared using livers from 6- to 8-week-old Swiss-Webster male mice. Using trans-stilbene oxide (TSO) as substrate, the highest activity was localized in the cytosolic fraction, followed by the light mitochondrial fraction. Subfractionation of the light mitochondrial fraction by isopycnic sucrose density gradient resulted in the separation of mitochondria from peroxisomes as monitored by marker enzymes. The separation of these two subcellular organelles was also confirmed by the electron microscopic studies. Distribution of TSO-hydrolase activity in the sucrose density gradient fractions closely resembled the activity distribution of the peroxisomal markers catalase and urate oxidase, but significant activity was also found in mitochondria. Treatment of mice with clofibrate selectively induced TSO-hydrolase in the cytosol without affecting this enzyme activity in the peroxisomal fraction. There was no difference in the distribution pattern of TSO-hydrolase and marker enzymes in sucrose density gradients of mitochondrial fractions from clofibrate-treated and control mice. The epoxide hydrolase activity in the peroxisomes is immunologically similar to, and also has the same molecular weight as, the cytosolic epoxide hydrolase.

Topics: Animals; Centrifugation, Density Gradient; Chemical Precipitation; Clofibrate; Cytosol; Epoxide Hydrolases; Epoxy Compounds; Immunodiffusion; Liver; Male; Mice; Microbodies; Mitochondria, Liver; Monoamine Oxidase; Proteins; Rabbits; Stilbenes; Succinate Dehydrogenase

Male New Zealand white rabbits were treated with various inducers of hepatic metabolism enzymes to characterize the induction of UDP-glucuronyltransferase (UDP-GT) enzymes. Rabbits were pretreated with phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254, ethanol, trans-stilbene oxide, pregnenolone-16 alpha-carbonitrile, or clofibric acid. Hepatic microsomes from treated and control animals were incubated with the GT1-type substrates, p-nitrophenol and 1-naphthol; the GT2-type substrate, morphine; and the steroid substrate, estrone. Compared to the rat, the rabbit was particularly resistant to UDP-GT induction. Ethanol was the most potent inducer for both GT1 and GT2 activities, but it failed to induce steroid (estrone, estradiol, and testosterone) UDP-GT activities. Ethanol pretreatment increased oxazepam-GT but it decreased bilirubin-GT activity. 3-Methylcholanthrene (3MC) and beta-naphthoflavone (BNF) are the prototypic GT1 inducers in the rat, but 3MC caused no induction of GT1 activity and BNF caused induction of both GT1 and GT2 activities in the rabbit. None of the xenobiotic pretreatments increased the hepatic microsomal glucuronidation of estrone. These results demonstrate that the induction of UDP-GT activities, and the use of this phenomenon to classify UDP-GT forms, is somewhat species-specific and cannot necessarily be extrapolated from rats to other species. In addition, the substrate selectivity of ethanol-induced microsomal UDP-GT was established.

Topics: Animals; Energy Intake; Enzyme Induction; Estrone; Ethanol; Glucuronosyltransferase; Male; Microsomes, Liver; Phenobarbital; Rabbits; Stilbenes; Substrate Specificity

Leukocyte isozyme(s) of glutathione transferase (GT-tSBO) has been shown to be dominantly inherited. The frequency of the phenotypes of this isozyme in bronchial carcinoma and control patients matched for age and smoking history is reported here. Control smokers showed an increased likelihood of having GT-tSBO (59%) compared with lung cancer patients (35%). The lack of GT-tSBO was related to the extent of smoking by the lung cancer patients but not to the pathology of the lung tumor. It was concluded that the gene expressing this isozyme(s) may be a host genetic determinant of susceptibility to lung cancer in smokers.

Topics: Aged; Female; Glutathione Transferase; Humans; Isoenzymes; Leukocytes; Lung Neoplasms; Male; Middle Aged; Smoking; Stilbenes

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.

Topics: Animals; Cell Fractionation; Endoplasmic Reticulum; Epoxide Hydrolases; Female; Liver; Male; Mice; Mice, Inbred C57BL; Rats; Rats, Inbred Strains; Sex Factors; Species Specificity; Stilbenes; Subcellular Fractions; Tissue Distribution

1-Nitropyrene is an environmental mutagen and carcinogen which undergoes both oxidative and reductive metabolism. We have previously shown that nitroreduction to N-hydroxy-1-aminopyrene leads to the formation of arylamine--DNA adducts. In the present study, we have investigated the oxidative metabolism of 1-nitropyrene and the subsequent binding of ring-oxidized metabolites to DNA. In vitro incubations were conducted using hepatic microsomes from uninduced rats or from rats pretreated with phenobarbital, Aroclor 1254, 3-methylcholanthrene, or 3-methylcholanthrene and trans-stilbene oxide. H.p.l.c. analysis of the incubation mixtures indicated the presence of the previously reported metabolites, 1-aminopyrene, 3-, 6-, and 8-hydroxy-1-nitropyrene, and 1-nitropyrene trans-4,5-dihydrodiol. In addition, 1-nitropyrene 4,5-oxide, 1-nitropyrene 9,10-oxide, 1-nitropyrene trans-9,10-dihydrodiol and 1-pyrenol were identified. The formation of both K-region dihydrodiols could be increased by trans-stilbene oxide induction of microsomal epoxide hydrase. Formation of the K-region epoxides was greatest using phenobarbital- and Aroclor-induced microsomes and increased with increasing oxygen tension, while 1-pyrenol formation was highest in 3-methylcholanthrene-induced microsomal incubations and was not affected by the oxygen tension. When calf thymus DNA was added to the microsomal incubations, similar levels of DNA-binding occurred in incubations conducted under oxygen, air, argon or anaerobic conditions. H.p.l.c. analysis of the enzymatically hydrolyzed DNA indicated the presence of multiple DNA adducts with the major product coeluting with N-(deoxyguanosin-8-yl)-1-aminopyrene. The K-region oxides bound directly to DNA to give adducts similar to the minor products detected in the microsomal incubations. Incubation of the K-region oxides with the nitroreductase, xanthine oxidase, increased the DNA-binding and resulted in an additional adduct which coeluted with N-(deoxyguanosin-8-yl)-1-amino pyrene. 3-Hydroxy-1-nitropyrene bound extensively to DNA upon nitroreduction by rat liver cytosol or xanthine oxidase, while 6- and 8-hydroxy-1-nitropyrene bound only slightly. None of these oxidized metabolites was activated to DNA-binding species by cytosolic nitroreduction followed by AcCoA-dependent acetylation. The fact that oxidized metabolites of 1-nitropyrene are reduced to DNA-binding derivatives more easily than 1-nitropyrene itself may be important in vivo where 1-nitropy

Topics: Acetylation; Animals; Aroclors; Chlorodiphenyl (54% Chlorine); Chromatography, High Pressure Liquid; DNA; Hydroxylation; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Pyrenes; Rats; Rats, Inbred Strains; Stilbenes; Xanthine Oxidase

A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.

Topics: Animals; Benzopyrenes; Cell Nucleus; Chalcone; Chalcones; Epoxide Hydrolases; Hydrogen-Ion Concentration; Kinetics; Liver; Male; Mice; Mice, Inbred C57BL; Stilbenes; Trichloroepoxypropane

The subcellular distribution of epoxide hydrolase activity towards TSO and HEOM in mouse liver and kidney was investigated using zonal rotor centrifugation. Epoxide hydrolase activity towards TSO was found predominantly in the soluble fraction with peroxisomes accounting for activity in the particulate fractions. Renal particulate activity towards HEOM was found predominantly in the microsomes.

Topics: Animals; Cytosol; Dieldrin; Epoxide Hydrolases; Kidney; Liver; Mice; Microbodies; Microsomes; Stilbenes; Subcellular Fractions; Substrate Specificity

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.

Topics: Animals; Chromatography, Affinity; Clofibrate; Cytosol; Epoxide Hydrolases; Hydrolysis; Isoelectric Focusing; Liver; Male; Mice; Stilbenes; Substrate Specificity

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Chromatography; Cytosol; Electrophoresis, Polyacrylamide Gel; Epoxide Hydrolases; Erythrocytes; Humans; Isoelectric Point; Leukotriene A4; Leukotriene B4; Molecular Weight; Stilbenes; Substrate Specificity

The biliary secretion of methylmercury was investigated in male rats which were given i.p. 400 mumoles/kg azathioprine or 96 mumoles/kg benziodarone 2 hr after the i.v. injection of 5 mumoles/kg MeHgCl. A group of rats were given 400 mg/kg trans-stilbene oxide (TSO) for 4 days before treatment with 10 mumoles/kg MeHgCl. A common link between these three compounds is their interference with ligandin. Azathioprine is a competitive inhibitor of glutathione S-transferase, benziodarone is covalently bound to ligandin and TSO is an inducer of liver ligandin. Although only azathioprine depletes liver GSH stores, both azathioprine and benziodarone inhibited the biliary secretion of methylmercury. As there is published proof that the reaction of MeHg+ with GSH does not require enzymatic help, the inhibitory effect of azathioprine and benziodarone confirms the role of ligandin in the transport of methylmercury or its GSH complex. However, the biliary secretion of methylmercury was increased only slightly by TSO pretreatment, but when 2 hr after the injection of MeHgCl animals received 2 mmoles/kg GSH, secretion increased twice as much in TWO pretreated than in control rats. This indicates the dual dependance of biliary methylmercury secretion on liver GSH and ligandin.

Topics: Animals; Azathioprine; Benzofurans; Bile; Biliary Tract; Glutathione; Glutathione Transferase; Liver; Male; Methylmercury Compounds; Rats; Rats, Inbred Strains; Stilbenes; Time Factors

High glutathione transferase activity towards trans-stilbene oxide has been observed in resting mononuclear leukocytes only in a portion of the individuals examined. Approximately 46% of a population of 248 individuals demonstrated this high activity. In addition, eight families have been investigated in order to elucidate the hereditary transmission of this activity. The results are consistent with a dominant expression of a single gene located on an autosomal chromosome for this high glutathione transferase activity.

Topics: Adolescent; Adult; Aged; Child; Female; Genes, Dominant; Glutathione Transferase; Humans; Male; Middle Aged; Monocytes; Pedigree; Phenotype; Stilbenes

The expression of epoxide hydrolases was studied in cultured rat hepatocytes and hepatoma cell lines. Styrene 7,8-oxide and benzo[a]pyrene 4,5-oxide were used as substrates for microsomal epoxide hydrolase and trans-stilbene oxide for the cytosolic form of this enzyme. In freshly isolated hepatocytes from control rats, microsomal epoxide hydrolase activity was 7.7 and 10.8 nmoles/mg cellular protein/min with benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide as substrates respectively. This enzyme activity increased by more than 2-fold in hepatocytes after 24 hr in culture and remained elevated throughout 96 hr using both substrates. In cultured hepatocytes from rats pretreated in vivo with phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene, both benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide hydrolase activities were increased greater than 1.8 relative to controls. Hepatocytes from 2-acetylaminofluorene-pretreated animals at 24 hr in culture had approximately 9-fold higher activities than control hepatocytes. In marked contrast to microsomal epoxide hydrolase activity, the cytosolic enzyme showed an initial activity of 191 pmoles/mg cellular protein/min in freshly isolated hepatocytes, decreased by 75% after 24 hr in culture, and was barely detectable at 96 hr. A similar trend was apparent in hepatocytes from the pretreated animals. In vitro treatment of hepatocytes with trans-stilbene oxide and phenobarbital increased microsomal epoxide hydrolase, while this activity was refractory to 2-acetylaminofluorene treatment. Styrene 7,8-oxide hydrolase activity was increased in the McA-RH-7777 rat hepatoma cell line by phenobarbital, trans-stilbene oxide and 2-acetylaminofluorene treatment. Similarly, benzo[a]pyrene 4,5-oxide hydrolase activity was also increased in this cell line by treatment with phenobarbital and trans-stilbene oxide but not by 2-acetylaminofluorene. Microsomal epoxide hydrolase activity in rat H4-II-E hepatoma cells was refractory to induction, except by trans-stilbene oxide treatment, which caused a 70% increase in benzo[a]pyrene 4,5-oxide hydrolase activity.

Topics: 2-Acetylaminofluorene; Animals; Cells, Cultured; Enzyme Induction; Epoxide Hydrolases; Hydroxyacetylaminofluorene; Liver; Liver Neoplasms, Experimental; Male; Phenobarbital; Rats; Rats, Inbred Strains; Stilbenes

We have shown earlier that there are large, hereditary interindividual differences in the cytosolic glutathione transferase activity towards trans-stilbene oxide in human mononuclear leukocytes. In the present study we ask whether these differences reflect the presence or absence of a particular isozyme(s) of glutathione transferase. First, in order to measure the high glutathione transferase activity optimally it was necessary to modify our previous assay by increasing the concentration of reduced glutathione from 3 to 5 mM and of the substrate from 50 to 250 microM. It was then found that the low activity demonstrates an apparent Km for trans-stilbene oxide of 28.3 microM, whereas the corresponding value for the high activity was 127 microM. Secondly, it was found that while glutathione transferase activity towards trans-stilbene oxide in different individuals segregated into three groups, low, high and very high, glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene in these same mononuclear leukocyte fractions formed only a single group with no tendency towards such segregation. Thirdly, the SDS-poly-acrylamide gel electrophoretic pattern obtained with the supernatant fraction from mononuclear cells demonstrating high glutathione transferase activity towards trans-stilbene oxide contained a band of 25 000 molecular weight which was either absent or present at a much lower level in cells demonstrating low activity. We conclude that high activity towards trans-stilbene oxide in circulating, resting human mononuclear cells is catalyzed by a particular isozyme(s) of glutathione transferase. cis-Stilbene oxide, styrene oxide and, possibly, benzo[a]pyrene 4,5-oxide are also substrates for this isozyme(s).

Topics: Dinitrochlorobenzene; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase; Humans; Individuality; Isoenzymes; Kinetics; Leukocytes; Stilbenes; Substrate Specificity

We have characterized certain catalytic properties of cytosolic epoxide hydrolases purified from untreated and clofibrate-treated mouse liver. The enzyme activity was found to be sensitive to oxygen, but nitrogen-saturated buffers containing dithiothreitol maintained high activity for at least 12 h at 0 degrees C. Linearity of the hydration of trans-stilbene oxide with time and protein was established, the pH optimum was broad (6.5 to 7.4) and the temperature optimum was close to 50 degrees C for both forms. The activity was independent of ionic strength, with the exception of the control form in the absence of dithiothreitol, where a lower activity was observed at low ionic strength. The activity decreased when ethanol was replaced by acetone or acetonitrile as solvent for the substrate. Tetrahydrofuran was found to be highly inhibitory, while dimethylsulfoxide had less pronounced effects. The apparent Km values were 4.9 microM, 73 microM and 1980 microM for the control form with trans-stilbene oxide, cis-stilbene oxide and styrene oxide as substrates, respectively. The Km values for the enzyme from clofibrate-treated mice were in the same range, although the V values were higher for all three substrates with this form. The highest turnover was found for trans-beta-propylstyrene oxide as substrate, followed by trans-beta-ethylstyrene oxide. Little or no activity was observed with benzo[a]pyrene 4,5-oxide or cholesterol 5,6 alpha-oxide. The enzymes were found to be sensitive to 5,5'-dithiobis(2-nitrobenzoic acid) and a phenylmercuric salt. alpha-Naphthoflavone, beta-naphthoflavone and chalcone derivatives also inhibited the activity, while none of the compounds known to activate microsomal epoxide hydrolase activated the cytosolic forms.

Topics: Animals; Catalysis; Clofibrate; Cytosol; Enzyme Activation; Epoxide Hydrolases; Kinetics; Liver; Male; Mice; Mice, Inbred C57BL; Solvents; Stilbenes; Substrate Specificity

The effect of hepatic glutathione (GSH) depletion and enzyme induction on hepatic glutathione S-transferase (GST) activity, biliary excretion of GSH, methyl mercury, cadmium and zinc was studied in rats. The GSH depletors, methyl iodide and diethyl maleate, did not influence hepatic GST activity but, depending on the substrate used, benzo(a)pyrene, phenobarbital, pregnenolone-16 alpha-carbonitrile (PCN) and trans-stilbene oxide (TSO) increased it by 16-33, 44-89, 53-97 and 208-279%, respectively. GSH depletors decreased (-88%), benzo(a)pyrene and TSO did not affect, phenobarbital and PCN increased (+113 and +149%) the transport of GSH into bile. The biliary excretion of methyl mercury, cadmium and zinc was reduced by GSH depletors (-97, -74 and -93%), and enhanced by phenobarbital (+139, +280 and +220%) and PCN (+150, +121 and +160%). Treatment with benzo(a)pyrene and TSO did not affect the excretion of methyl mercury and zinc into bile, but decreased that of cadmium. These results do not provide evidence for the role of hepatic GST but strongly support the importance of biliary GSH excretion in the hepatobiliary transport of methyl mercury, cadmium and zinc. It is assumed that phenobarbital and PCN enhance the biliary excretion of these metals by increasing the transport of GSH, the carrier molecule, from liver to bile.

Topics: Animals; Bile; Cadmium; Enzyme Induction; Glutathione; Glutathione Transferase; Liver; Male; Methylmercury Compounds; Phenobarbital; Pregnenolone Carbonitrile; Rats; Rats, Inbred Strains; Stilbenes; Zinc

The effects of dietary clofibrate on the epoxide-metabolizing enzymes of mouse liver, kidney, lung and testis were evaluated using trans-stilbene oxide as a selective substrate for the cytosolic epoxide hydrolase, cis-stilbene oxide and benzo[a]pyrene 4,5-oxide as substrates for the microsomal form, and cis-stilbene oxide as a substrate for glutathione S-transferase activity. The hydration of trans-stilbene oxide was greatest in liver followed by kidney greater than lung greater than testis. Its hydrolysis was increased significantly in the cytosolic fraction of liver and kidney of clofibrate-treated mice and in the microsomes from the liver. Isoelectric focusing indicates that the same enzyme is responsible for hydrolysis of trans-stilbene oxide in normal and induced liver and kidney. Clofibrate induced glutathione S-transferase activity on cis-stilbene oxide only in the liver. Hydrolysis of both cis-stilbene oxide and benzo[a]pyrene 4,5-oxide was highest in testis followed by liver greater than lung greater than kidney. Hydration of cis-stilbene oxide was induced significantly in both liver and kidney by clofibrate but that of benzo[a]pyrene 4,5-oxide was induced only in the liver. These and other data based on ratios of hydration of benzo[a]pyrene 4,5-oxide to cis-stilbene oxide in tissues of normal and induced animals indicate that there are one or more novel epoxide hydrolase activities which cannot be accounted for by either the classical cytosolic or microsomal hydrolases. These effects are notable in the microsomes of kidney and especially in the cytosol of testis.

Topics: Animals; Benzopyrenes; Clofibrate; Diet; Epoxide Hydrolases; Glutathione Transferase; Hydrogen-Ion Concentration; Kidney; Liver; Lung; Male; Mice; Microbodies; Stilbenes

Chalcone oxides and several isosteric compounds have been prepared to examine the importance of the alpha,beta-epoxyketone moiety in the inhibition of the hydrolysis of [3H]-trans-stilbene oxide to its meso-diol by mouse liver cytosolic epoxide hydrolase (cEH). Inhibition of microsomal EH and glutathione S-transferase were also examined. For cEH, replacement of the carbonyl by methylidene reduces inhibitor potency by a factor of 44, while replacement of the epoxide ring with a cyclopropyl ring reduces inhibition by a factor of 450. A 2'-hydroxyl also reduces cEH inhibition by 100 times. These observations are consistent with a model of the active site in which the carbonyl is hy-hydrogen-bonded to an acidic site presumed to be involved in initiating epoxide hydrolysis. The chalcone oxides thus bind tightly but are not readily turned over as substrates.

Topics: Animals; Binding Sites; Chalcone; Chalcones; Cytosol; Epoxide Hydrolases; Epoxy Compounds; Ethers, Cyclic; Glutathione Transferase; Liver; Mice; Microsomes, Liver; Stilbenes; Structure-Activity Relationship

Epoxide hydrolase in human adrenal gland was characterized with respect to catalytic properties and subcellular distribution. With human adrenal microsomes and the substrates styrene-7,8-oxide, cis-stilbene oxide, estroxide and androstene oxide the specific activities were between 1.9 and 19.0 nmol/min/mg protein. With styrene-7,8-oxide as substrate the apparent Km-value was 0.98 mM and the pH optimum was 9.2. Subcellular fractionation revealed that the bulk of the activity was confined to the endoplasmic reticulum. Different compounds known to influence rodent microsomal epoxide hydrolase activity were also tested on the human adrenal enzyme. 1,1,1-Trichloropropene-2,3-oxide (TCPO) and cyclohexene oxide (CHO) inhibited the activity while benzil and clotrimazole stimulated the activity. Partial purification of human adrenal epoxide hydrolase indicates that its molecular weight is about 51 000 and that its concentration relative total protein in the human adrenal microsomes is about 10%.

Topics: Adrenal Glands; Catalysis; Electrophoresis, Polyacrylamide Gel; Epoxide Hydrolases; Epoxy Compounds; Humans; Hydrogen-Ion Concentration; Microsomes; Stilbenes; Subcellular Fractions; Substrate Specificity

Total liver poly(A+)-RNA isolated from untreated, and 3-methylcholanthrene-, trans-stilbene oxide-, and phenobarbital-treated rats has been translated in the rabbit reticulocyte lysate system in order to determine the effect of these xenobiotics on the level of translationally active DT-diaphorase mRNA. The in vitro translation systems were subjected to immunoprecipitation with rabbit IgG raised against purified DT-diaphorase and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the radiolabeled, immunoprecipitated product as DT-diaphorase was confirmed by limited peptide mapping using Staphylococcus aureus V-8 protease. These quantitation results demonstrate that 3-methylcholanthrene leads to an eight-fold elevation in functional DT-diaphorase mRNA at 8 h after a single administration of 3-methylcholanthrene; whereas, trans-stilbene oxide and phenobarbital produced only a modest elevation, two- to three-fold, in the functional DT-diaphorase mRNA level. These data indicate that the increase in the level of DT-diaphorase after 3-methylcholanthrene administration noted previously [B. Höjeberg, K. Blomberg, S. Stenberg, and C. Lind (1981) Arch. Biochem. Biophys. 207, 205-216] can be totally accounted for by an elevation in the mRNA level specific for this protein.

Topics: Animals; Cell-Free System; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Immunochemistry; Liver; Methylcholanthrene; NAD(P)H Dehydrogenase (Quinone); NADH, NADPH Oxidoreductases; Phenobarbital; Quinone Reductases; Rabbits; Rats; RNA, Messenger; Stilbenes

The major form of microsomal cytochrome P-450 induced by trans-stilbene oxide in the liver of male Sprague-Dawley rats was purified and characterized, and compared with the isolated cytochrome P-450 B2 forms from phenobarbital- and 3-methylcholanthrene-pretreated animals. The apparent subunit molecular weight of the trans-stilbene oxide-induced cytochrome was found to be 53 000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the absorbance maximum of the carbon monoxide complex of the ferrous cytochrome was 450 nm. Reconstitution of the N-demethylase activity towards three different substrates showed high and similar activities with the cytochrome P-450 B2 forms from trans-stilbene oxide or phenobarbital-treated rats, with one exception. Amino-acid analysis also showed a very high degree of similarity between these two forms. Upon proteinase treatment with three different proteinases the trans-stilbene oxide-induced cytochrome demonstrated in each case a peptide pattern identical to that obtained with the phenobarbital-induced B2 form. Furthermore, both forms are completely immunologically cross-reactive. We therefore conclude from these experiments that the liver microsomal P-450 B2 from trans-stilbene oxide and phenobarbital-treated rats are very closely related, if not identical.

Topics: Amino Acids; Animals; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Immunodiffusion; Isoenzymes; Male; Methylcholanthrene; Microsomes, Liver; Molecular Weight; Phenobarbital; Rats; Rats, Inbred Strains; Stilbenes

The effect of the phase II enzyme inducer, trans-stilbeneoxide (TSO; 400 mg/kg i.p. on 4 consecutive days), on the clearance of intravenously given [14C]heptachlor (2 mg/kg) was investigated in male Sprague-Dawley rats. TSO or vehicle treatment was started 4 days before (group I), immediately after (group II), or 4 days following (group III) heptachlor injection. Rats were sacrificed 6 days after the last TSO or vehicle dosage. In all 3 groups TSO increased cumulative excretion of heptachlor derived material in feces significantly (P less than 0.01) but had no effect on total urinary excretion. Pretreatment (group I) of rats with TSO resulted in elevated fecal excretion of 14C already on the first day after dosing with heptachlor, whereas the effect of the treatment was not apparent in group II and III rats until after 2 days and reached its maximum 3 days after the start of TSO administration. At termination, tissue radioactivity was significantly (P less than 0.05) lower in fat, kidney, brain, skin, heart and muscle, but not in serum, erythrocytes (group II and III), liver (group II and III), and spleen of TSO treated rats as compared to controls. The body burden calculated from the sum of 14C remaining in tissues was reduced to about 27% of control values. The toxicity of TSO may preclude it from considerations as a useful therapeutic agent in the treatment of chlorinated cyclodiene insecticide-exposed individuals. However, this experiment further emphasizes the importance of pharmacokinetic considerations when examining interactions in the disposition of xenobiotics.

Topics: Animals; Body Burden; Drug Evaluation, Preclinical; Feces; Heptachlor; Kinetics; Male; Rats; Rats, Inbred Strains; Stilbenes; Time Factors; Tissue Distribution; Urine

The effects of treating male Sprague-Dawley rats with phenobarbital, 3-methylcholanthrene or trans-stilbene oxide on cytosolic glutathione transferase and microsomal epoxide hydrolase activities in the liver, intestine, kidney, lung, testis, adrenal, spleen, heart and brain have been investigated. Studies on the time-courses of induction in liver demonstrate that these are complete after five days' treatment at the doses used. Phenobarbital induces both cytosolic glutathione transferase and microsomal epoxide hydrolase activities significantly only in liver and intestine. 3-Methylcholanthrene induces these activities in liver only. Trans-Stilbene oxide induces both of these activities in liver and kidney, and cytosolic glutathione transferase activity in adrenal as well.

Topics: Animals; Cytosol; Epoxide Hydrolases; Glutathione Transferase; Male; Methylcholanthrene; Microsomes; Phenobarbital; Rats; Rats, Inbred Strains; Stilbenes; Time Factors; Tissue Distribution

Topics: Animals; Enzyme Induction; Female; Glutathione Transferase; Liver; Mice; Mice, Inbred Strains; Placenta; Pregnancy; Pregnancy, Animal; Stilbenes

A highly sensitive assay for the epoxide hydrolase activity associated with the preneoplastic antigen (PNA) has been developed based on the synthesis of cis-stilbene oxide labeled with tritium at approximately 15 Ci/mmol. This assay allows the detection of elevated epoxide hydrolase activity in the serum of humans and rodents as well as in the culture medium bathing isolated hepatocytes. The integrity of the enzymatic assay was confirmed in rodents by precipitating the serum PNA activity using an antibody raised against the rat microsomal epoxide hydrolase. Methodology for the detection of PNA in serum will facilitate evaluation of this antigen as a marker for hepatic neoplasia in man and in experimental animals.

Topics: Acetaminophen; Adult; Animals; Antigens, Neoplasm; Epoxide Hydrolases; Female; Humans; Liver Neoplasms; Male; Middle Aged; Precancerous Conditions; Rabbits; Rats; Rats, Inbred Strains; Stilbenes

The effect of dietary trans-stilbene oxide (TSO) on hepatic and renal xenobiotic metabolizing-enzyme activities and bromobenzene-induced toxicity was quantified in adult male Sprague-Dawley rats. Rats were fed a regular diet or the same diet supplemented with 2.5 g TSO/kg diet for 10 days. TSO treatment did not alter hepatic or renal arylhydrocarbon hydroxylase activity, but significantly increased glutathione S-transferase and uridine diphosphoglucuronyl transferase activities in both organs. In addition, TSO increased hepatic, but not renal, epoxide hydrolase activity. The same treatment did not produce adverse effects on renal or hepatic functions, but markedly potentiated bromobenzene hepatotoxicity. A single dose of bromobenzene (0.2 ml/kg) caused a slight increase in serum glutamic pyruvic transaminase (SGPT) activity and minor hepatic necrosis in animals fed the control diet; the same dose of bromobenzene markedly increased SGPT activity and produced severe hepatic necrosis in the TSO-fed animals.

Topics: Animals; Bromobenzenes; Kidney; Liver; Male; Rats; Rats, Inbred Strains; Stilbenes

Topics: 2-Acetylaminofluorene; Animals; Enzyme Induction; Enzymes; Liver; Male; Methylcholanthrene; Pharmaceutical Preparations; Phenobarbital; Rats; Stilbenes

The effects of phenobarbital, trans-stilbene oxide, and 3-methylcholanthrene on epoxide hydrolase (EC 3.3.2.3) within centrilobular, midzonal, and periportal hepatocytes were investigated employing rabbit anti-serum produced against rat hepatic microsomal epoxide hydrolase in unlabeled antibody peroxidase-anti-peroxidase and indirect fluorescent antibody-staining techniques. In livers of control rats, midzonal and periportal hepatocytes bound the anti-epoxide hydrolase to similar extents while centrilobular hepatocytes bound approximately 25% more antibody. 3-Methylcholanthrene did not cause significant alterations in immunohistochemical staining for epoxide hydrolase within any region of the liver lobule, whereas phenobarbital and trans-stilbene oxide produced significant alterations in both the intensity and pattern of intralobular staining for the enzyme. After 4 days of phenobarbital pretreatment, anti-epoxide hydrolase binding to hepatocytes was slightly, but significantly, elevated, especially within midzonal regions. After 7 days of phenobarbital pretreatment, anti-epoxide hydrolase binding was increased by approximately 65% within midzonal regions and by approximately 41 and 24%, respectively, within centrilobular and periportal regions. In livers of trans-stilbene oxide-pretreated rats, anti-epoxide hydrolase binding was increased by approximately 80% within both the midzonal and periportal regions and by approximately 43% within centrilobular regions. These immunohistochemical findings demonstrate that phenobarbital and trans-stilbene oxide both induce epoxide hydrolase nonuniformly within the liver lobule. However, while phenobarbital induces the enzyme to the greatest extent within midzonal hepatocytes and to the least extent within periportal hepatocytes, trans-stilbene oxide induces epoxide hydrolase equally within midzonal and periportal hepatocytes.

Topics: Animals; Epoxide Hydrolases; Fluorescent Antibody Technique; Histocytochemistry; Male; Methylcholanthrene; Microsomes, Liver; Organ Specificity; Phenobarbital; Rats; Stilbenes

Topics: Animals; Cytosol; Enzyme Induction; Glutathione Transferase; Isoelectric Focusing; Isoenzymes; Liver; Macromolecular Substances; Male; Rats; Rats, Inbred Strains; Stilbenes

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.

Topics: Animals; Cytochrome P-450 Enzyme System; Drug Interactions; Enzyme Induction; Female; Methylcholanthrene; Microsomes, Liver; Oxygenases; Phenobarbital; Pregnenolone Carbonitrile; Rats; Rats, Inbred Strains; Spironolactone; Stilbenes

Trans-Stilbene oxide (400 mg/kg) produced a 500% increase in the microsomal epoxide hydratase activity in rat and mouse with little change in the soluble enzyme activity. However, in guinea pig, the soluble epoxide hydratase activity increased by about 33% with only a small increase (47.6%) in the microsomal enzyme activity. The soluble glutathione S-transferase activities were also induced in both rat and mouse, with little change in that of the guinea pig. Increasing dosage of trans-stilbene oxide from 400 mg/kg to 1000 mg/kg had little effect on the above enzyme activities. That the guinea pig was not relatively refractory to all inducing agents was shown by the fact that phenobarbital (100 mg/kg) and 3-methylcholanthrene (25 mg/kg) produced relatively similar increases in the activities of aniline hydroxylase and P-aminopyrine N-demethylase in rat, mouse and guinea pig. However, these inducers produced only a 15-20% stimulation in the soluble glutathione S-transferase and microsomal epoxide hydratase activities in guinea pig, when compared to a 50-80% increase in rat and mouse, suggesting a general resistance to induction by the phase II enzymes in guinea liver. In all three animal models, the inducer markedly increased the microsomal total phospholipid content, although the sphingomyelin content itself was decreased. In both rat and mouse, the microsomal cholesterol content was significantly decreased while that in guinea pig was unaffected. Possible factors responsible for the observed species differences are discussed.

Topics: Animals; Enzyme Induction; Epoxide Hydrolases; Glutathione Transferase; Guinea Pigs; In Vitro Techniques; Male; Mice; Microsomes, Liver; Rats; Rats, Inbred Strains; Solubility; Species Specificity; Stilbenes

The metabolism of testosterone and androstenedione by liver microsomes was investigated after treatment of rats with trans-stilbene oxide, phenobarbital, or 3-methylcholanthrene. Conditions for linearity of the assay with time and amount of cytochrome P-450, as well as saturating substrate concentrations, were established. The metabolites were separated by thin-layer chromatography and quantitated by scintillation counting. The rates of formation of different testosterone and androstenedione metabolites after induction with trans-stilbene oxide or phenobarbital were similar, indicating that these xenobiotics induce the same isozyme of cytochrome P-450. This conclusion was further supported using inhibitors of cytochrome P-450 (SKF-525A, metyrapone and alpha-naphthoflavone) and with immunoinhibition by antibodies directed towards the phenobarbital-inducible form of cytochrome P-450. After treatment with trans-stilbene oxide or phenobarbital, the specific rates of formation of the 6 beta- and/or 2 beta-hydroxy metabolites and of 17 beta-hydroxy-4-androstene-3,16-dione were increased. In contrast, administration of 3-methylcholanthrene led to decreases in the specific rates of formation of almost all testosterone and androstenedione metabolites investigated. However, all three of these inducers cause increases in the total liver metabolism of testosterone and androstenedione. These increases are 2--30-fold in the case of trans-stilbene oxide, 3--46-fold for phenobarbital and 1--4-fold after treatment with 3-methylcholanthrene. The possible physiological significance of these effects is as yet unknown.

Topics: Androstenedione; Animals; Benzoflavones; Cytochrome P-450 Enzyme System; Hydroxylation; Kinetics; Male; Methylcholanthrene; Metyrapone; Microsomes, Liver; Phenobarbital; Proadifen; Rats; Rats, Inbred Strains; Stilbenes; Testosterone

The initial rates of hydration of sixteen epoxides in the presence of cytosolic and microsomal fractions of mouse liver were determined. 1,2-Disubstituted trans-epoxides were found to be excellent, selective substrates for the cytosolic epoxide hydrolase, while 1,2-cis-epoxides were poorly hydrated when one or more substituents was a phenyl moiety. Epoxides of cyclic systems including benzo[alpha]pyrene 4,5-oxide, and two cyclodiene analogs were hydrated almost exclusively by the microsomal epoxide hydrolase while monosubstituted epoxides were hydrated by both systems. Some epoxides which were mediocre substrates proved to be reasonable inhibitors of the cytosolic epoxide hydrolase, indicating that the structural requirements for substrate binding and turnover are different. Some reagents known to interact with sulfhydryl groups, including styrene oxide, proved to be good inhibitors. This work facilitates the design of radiochemical and spectrophotometric assays for both major forms of epoxide hydrolase as well as prediction of potential intrinsic substrates. Also such data may be meaningful in assessing the risk involved in human exposure to epoxidized xenobiotics.

Topics: Animals; Chemical Phenomena; Chemistry; Chromatography, Gas; Cytosol; Epoxide Hydrolases; Epoxy Compounds; Kinetics; Liver; Male; Mice; Microsomes, Liver; Stilbenes; Structure-Activity Relationship; Substrate Specificity

Cytochromes P-450 and epoxide hydrolase in hamsters were studied by using two-dimensional gel electrophoresis of hepatic microsomes from untreated animals and those treated with phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, trans-stilbene oxide, and pregnenolone-16 alpha-carbonitrile. Coelectrophoresis with corresponding microsomes from rats and in situ peptide mapping were used to identify resolved microsomal polypeptides as cytochromes P-450 or epoxide hydrolase. Two forms of hepatic microsomal epoxide hydrolase were shown to exist in hamsters; these evidenced extensive structural homology with the corresponding enzyme in rats and were induced by the same xenobiotics. At least eight inducible polypeptides in microsomes from hamsters were tentatively identified as cytochromes P-450. Two of these were electrophoretically identical and structurally related with previously characterized forms of the enzyme in rats. Homologues of several major cytochromes P-450 induced by pregnenolone-16 alpha-carbonitrile and/or phenobarbital in the rat were apparently not present in the hamster. In most cases, putative forms of inducible cytochrome P-450 in the hamster existed at significant levels in microsomes from untreated animals whereas in rats the levels of most inducible forms of the enzyme were low in control microsomes, being more strictly dependent on xenobiotic pretreatment. In contrast with epoxide hydrolase, the molecular complexity of hepatic cytochrome P-450 seems to be comparable for rats and hamsters, but the structure and control of these hemoproteins appear to have markedly diverged.

Topics: Animals; Benzoflavones; beta-Naphthoflavone; Cricetinae; Cytochrome P-450 Enzyme System; Enzyme Induction; Epoxide Hydrolases; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Pregnenolone Carbonitrile; Rats; Species Specificity; Stilbenes

The cytosolic epoxide hydrolase (EH-LC) was observed in rhesus monkey liver cytosol, and in both normal and neoplastic human liver. Microsomal epoxide hydrolase (EH-LM) was detected not only in the microsomes of normal and neoplastic human liver and normal rhesus monkey liver, but also in the cytosol of these tissues. No apparent differences were observed between the EH-LM in liver cytosol and that in microsomes. No major differences were observed between the levels of EH-LM in the cytosol of normal and that in neoplastic human liver.

Topics: Animals; Cytosol; Epoxide Hydrolases; Female; Humans; Infant; Liver; Liver Neoplasms; Macaca mulatta; Male; Microsomes, Liver; Middle Aged; Species Specificity; Stilbenes; Styrenes; Tissue Distribution

Using trans-stilbene oxide as substrate, the subcellular distribution of epoxide hydrolase was investigated in livers from DBA/2 mice. The highest specific activities were found in cytosolic and light mitochondrial fractions. Isopycnic subfractionation of the light mitochondrial fraction showed that the organelle-bound trans-stilbene oxide hydrolase is localized in peroxisomes.

Topics: Animals; Epoxide Hydrolases; In Vitro Techniques; Liver; Male; Mice; Mice, Inbred DBA; Microbodies; Organoids; Stilbenes

High doses of cephaloridine produce necrosis of renal proximal tubular cells and this nephrotoxicity has been shown to be reduced by piperonyl butoxide (a mixed-function oxidase inhibitor) in rats and rabbits, and potentiated by phenobarbital (a mixed-function oxidase inducer) in rabbits but not rats. Phenobarbital is known to increase rabbit but not rat renal mixed-function oxidase activities; however, several other compounds such as polybrominated biphenyls (PBB), trans-stilbene oxide (TSO) and beta-naphthoflavone (BNF) have been shown to induce renal enzyme activities in rats. Thus, it was of interest to determine the effects of PBB, TSO and BNF on cephaloridine toxicity in Fischer 344 rats. Nephrotoxicity was estimated by measuring alterations in the kidney-to-body weight ratio, blood urea nitrogen and accumulation of p-aminohippurate (PAH) and tetraethylammonium by renal cortical slices. Hepatotoxicity was quantified as changes in serum glutamic pyruvic transaminase (SGPT) activity. Cephaloridine produced only minor changes in SGPT activity. Animals fed diet supplemented with 100 ppm of PBB for 10 days became less susceptible to cephaloridine nephrotoxicity. Similarly, pretreatment of animals with TSO (300 mg/kg) or BNF (100 mg/kg) for 4 days decreased cephaloridine toxicity. Thus, these results suggest that induction of renal drug-metabolizing enzyme activities by these 3 inducers may enhance some detoxification pathway(s) which convert cephaloridine to a non-toxic metabolite(s). Alternatively, treatments with these inducers may alter cephaloridine pharmacokinetics and decrease renal cortical accumulation of cephaloridine.

Topics: Animals; Benzoflavones; beta-Naphthoflavone; Cephaloridine; Drug Interactions; Enzyme Induction; Kidney; Phenobarbital; Piperonyl Butoxide; Polybrominated Biphenyls; Rabbits; Rats; Rats, Inbred F344; Rats, Inbred Strains; Stilbenes

Administration of trans-stilbene oxide, and new type of inducer of drug-metabolizing enzymes, to rats was found to increase hepatic microsomal UDP-glucuronyl transferase activity with both p-nitrophenol and chloramphenicol as substrate. In Triton X-100 activated microsomes the increase with p-nitrophenol as substrate was to approx. 250% of the control value, while the corresponding value for chloramphenicol was about 600%. These observations indicate that trans-stilbene oxide causes a mixed type 'induction' of UDP-glucuronyl transferase(s), i.e., changes in activity which resemble both those seen after induction with phenobarbital and after treatment with 3-methylcholanthrene. We have also shown that the activity of UDP-glucose dehydrogenase, the enzyme which produces UDP-glucuronic acid, is increased to about 300% of the control after administration of trans-stilbene oxide. The time course of this increase and of the return to control activity after cessation of treatment, the dose-response of this increase and the structural features of the trans-stilbene oxide molecule which are essential for the increase have all been examined. The other two enzymes involved in the conversion of glucose 6-phosphate to UDP-glucuronic acid, namely, phosphoglucomutase and UDP-glucose pyrophosphorylase, were found to be only slightly affected (a 30-60% increase) by treatment with trans-stilbene oxide. After induction with trans-stilbene oxide the hepatic level of UDP-glucuronic acid was unchanged.

Topics: Animals; Enzyme Induction; Glucuronates; Glucuronosyltransferase; Male; Mixed Function Oxygenases; Oxidoreductases; Pharmaceutical Preparations; Phosphoglucomutase; Proteins; Rats; Rats, Inbred Strains; Stilbenes; Uridine Diphosphate Glucose Dehydrogenase

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.

Topics: Animals; Base Sequence; Cloning, Molecular; DNA Restriction Enzymes; DNA, Recombinant; Glutathione Transferase; Liver; Male; Molecular Weight; Nucleic Acid Hybridization; Phenobarbital; Plasmids; Rats; Rats, Inbred Strains; RNA, Messenger; Stilbenes; Transcription, Genetic

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methyl-cholanthrene beta-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT2 substrates) and bilirubin, similar to phenobarbital. (3) Colfibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT1 and GT2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT1 and GT2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450MC and GT1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced GT1 reactions without affecting benzo[a]pyrene monooxygenase.

Topics: Animals; Aroclors; Benzoflavones; Benzopyrene Hydroxylase; Benzopyrenes; beta-Naphthoflavone; Chlorodiphenyl (54% Chlorine); Clofibric Acid; DDT; Enzyme Induction; Glucuronosyltransferase; Male; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains; Stilbenes

Topics: Animals; Cytochrome b Group; Cytochrome P-450 Enzyme System; Cytochromes; Enzyme Induction; Epoxide Hydrolases; Glucuronosyltransferase; Immunochemistry; Male; NADPH-Ferrihemoprotein Reductase; Nuclear Envelope; Phenobarbital; Rats; Rats, Inbred Strains; Stilbenes

The effect of the inducing agent trans-stilbene oxide (TSO) on the metabolism and biliary excretion of [14C]morphine was studied in the isolated in situ perfused rat liver. After administration of morphine by intraportal injection or by the segmented retrograde intrabiliary injection technique, the TSO-treated group showed a marked decrease in the biliary recovery of morphine as its glucuronide conjugate [morphine-3-glucuronide (MG)]. However, recovery of MG in the venous outflow of the single pass perfusate was greatly increased. These findings suggested that TSO treatment enhanced the formation of MG from morphine and changed the primary route of hepatic elimination of MG. TSO treatment also decreased the excretion of morphine (as MG) in the bile of anesthetized renal-ligated rats. This decreased biliary function required several days to develop and appeared closely associated with the inductive effect of TSO. After i.v. administration of [14C]MG itself, biliary recovery was also markedly decreased in TSO-treated rats. It is postulated that the effect of the TSO treatment led to either a decrease in canalicular transport of MG into bile or an increase in the efficiency of transfer of MG to the blood at the sinusoidal side of the hepatocyte. Regardless of the mechanism, the results indicate the need to study compartmentalization of drug transport and metabolism functions.

Topics: Animals; Bile; Carbon Radioisotopes; Enzyme Induction; Glucuronosyltransferase; Liver; Male; Morphine; Morphine Derivatives; Perfusion; Rats; Rats, Inbred Strains; Stilbenes

trans-Stilbene oxide differs from the classical inducers of drug-metabolizing enzymes, phenobarbital and 3-methylcholanthrene, in that it induces the so-called phase II activities, epoxide hydrolase and glutathione S-transferase, to a much larger extent than it induces cytochrome P-450. Nonetheless, the level of cytochrome P-450 in liver microsomes from rats treated with trans-stilbene oxide is increased significantly to twice the control value. The existence of a number of different isozymes of cytochrome P-450 has now been clearly demonstrated and in the present study we have posed the question. What form(s) of cytochrome P-450 is induced by trans-stilbene oxide? A number of criteria including substrate specificity, pattern of benzo(a)pyrene metabolism, sensitivity to inhibitors, substrate binding spectra, ethylisocyanide binding spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and crossed immunoelectrophoresis were used to answer this question. It seems clear that trans-stilbene oxide induces the same form(s) of cytochrome P-450 as phenobarbital.

Topics: 7-Alkoxycoumarin O-Dealkylase; Aminopyrine N-Demethylase; Animals; Benzopyrene Hydroxylase; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Ethylmorphine-N-Demethylase; Male; Microsomes, Liver; Mixed Function Oxygenases; Oxidoreductases; Oxidoreductases, N-Demethylating; Oxygenases; Rats; Rats, Inbred Strains; Stilbenes; Substrate Specificity

The effect of butylated hydroxyanisole (BHA; 600 mg/kg i.p. daily, for 10 days) and trans-stilbene oxide (TSO; 400 mg/kg i.p. daily, for 4 days) on the in vitro hepatic activity of glutathione transferases, the hepatic content of organic anion binding proteins and the plasma disappearance and biliary excretion of sulfobromophthalein (BSP), phenol-3,6-dibromsulphthalein disulfonate and [3H]ouabain was investigated in mice (BHA) and rats (TSO). Both BHA and TSO increased glutathione transferase activity toward BSP (360 and 200%), hepatic ligandin content (160 and 120%) and the biliary excretion of BSP (370 and 85%). BSP-glutathione excretion was enhanced, indicating that BSP conjugation was also stimulated in vivo. In contrast to BSP, biliary excretion of phenol-3,6-dibromsulphthalein disulfonate and organic anion which is not biotransformed but binds to ligandin, was unaltered or slightly increased (29%) after BHA or TSO treatment, respectively. TSO administration also did not affect the excretion of ouabain, a compound that neither binds to ligandin nor is biotransformed before excretion. Induction of ligandin failed to influence the initial disappearance of BSP, phenol-3,6-dibromsulphthalein disulfonate or ouabain from plasma, suggesting that induction had no marked effect on the hepatic uptake of these compounds. These studies suggest that ligandin plays a more important role in the biliary excretion of BSP due to its enzymatic rather than its binding properties.

Topics: Animals; Bile; Butylated Hydroxyanisole; Carrier Proteins; Glutathione Transferase; In Vitro Techniques; Male; Mice; Organ Size; Protein Binding; Rats; Rats, Inbred Strains; Stilbenes; Sulfobromophthalein; Time Factors

trans-Stilbene oxide has been found earlier to be a new type of inducer of drug-metabolizing systems. Here we demonstrate that treatment of rats with this xenobiotic results in an increase in the activity of the cytosolic glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, the first and third enzymes in th pentose phosphate pathway, to 350% and 170% of the control values, respectively. At the time microsomal glucose 6-phosphate dehydrogenase activity was unaffected by administration of trans-stilbene oxide or benzil. The time course and dose-response of the increases in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities have been characterized. The activities of ribulose 5-phosphate 3-epimerase and ribose 5-phosphate activities have been characterized. The activities of ribulose 5-phosphate 3-epimerase and ribose 5-phosphate ketol isomerase, enzymes further along in the pentose phosphate pathway, were not significantly affected by trans-stilbene oxide or benzil. An investigation of the effect of treating rats with different metabolites of stilbene and with other structurally related compounds on hepatic cytosolic glucose 6-phosphate dehydrogenase activity revealed the structural features which are important for increasing this activity. Finally, it was found that administration of trans-stilbene oxide did not affect the activities of glucokinase and phosphoglucose isomerase, the two glycolytic enzymes which can produce glucose 6-phosphate, the link between glycolysis and the pentose phosphate shunt.

Topics: Animals; Carbohydrate Metabolism; Enzyme Induction; Glucosephosphate Dehydrogenase; Male; Pentosephosphates; Phosphogluconate Dehydrogenase; Rats; Rats, Inbred Strains; Stilbenes

A continuous spectrophotometric assay based on the differences in the ultraviolet spectra of trans-stilbene oxide (TSO) and its reaction product 1,2-diphenyl-1,2-ethanediol is described for the measurement of mammalian cytosolic epoxide hydrolase activity. Rates of TSO hydration determined by this method were similar to those obtained by independent analytical methods, and the assay is rapid, reproducible and relatively free from interference.

Topics: Animals; Cattle; Cytosol; Epoxide Hydrolases; Kinetics; Male; Mice; Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Stilbenes

Two-dimensional gel electrophoresis was used to analyze hepatic microsomal polypeptides after treatment of immature, male Long-Evans rats with 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, or trans-stilbene oxide. Epoxide hydrolase and cytochromes P-450a, P-450bLE, P-450c, P-450d, and P-450e were all identified as resolved polypeptides in these electrophoretograms. Idiosyncratic polypeptide patterns characterized the microsomal preparations following treatment of rats with each inducing agent. Immunochemically identical cytochromes P-450bLE and P-450e were always present at the same relative levels even though their total amount varied 3-fold after induction by isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, and trans-stilbene oxide. Cytochromes P-450c and P-450d were coinduced by 3-methylcholanthrene, isosafrole, and Aroclor-1254, but their relative amounts varied. Pregnenolone-16 alpha-carbonitrile treatment resulted in an increase of a single major microsomal polypeptide which was also induced by phenobarbital, isosafrole, SKF-525A, Aroclor-1254, and trans-stilbene oxide. Only one polypeptide was identified as epoxide hydrolase in all of the microsomes analyzed. The results suggest that the levels of cytochromes P-450bLE and P-450e may be subject to coordinate control, whereas the other cytochromes P-450 are independently regulated.

Topics: Animals; Aroclors; Chlordan; Chlorodiphenyl (54% Chlorine); Cytochrome P-450 Enzyme System; Epoxide Hydrolases; Isomerism; Male; Methylcholanthrene; Microsomes, Liver; Peptide Biosynthesis; Phenobarbital; Pregnenolone Carbonitrile; Proadifen; Rats; Safrole; Stilbenes

The effect of trans-stilbene oxide (TSO) induction on the microsome-catalyzed binding of polycyclic aromatic hydrocarbon metabolites to DNA was investigated using two rodent species (Sprague-Dawley rat and C57BL/6N or NMRI Swiss mouse) and 2 different binding substrates (benzo[a]anthracene). It was determined that TSO exerts 2 separate effects on polycyclic aromatic hydrocarbons - it increases the rate of oxidation at the K-region of the molecule due to its induction of specific monooxygenases, and it increases the rate of deactivation of epoxide intermediates by induction of microsomal epoxide hydrolase activity. The importance of these individual effects were determined by inducing monooxygenase activity with BP, altering region specificity and inducing epoxide hydrolase (EH) activity with TSO, assessing the combined inductive effects of TSO and BP, inhibiting EH with 1,1,1-trichloropropene oxide, or increasing its activity by the addition of pure enzyme. This study shows that these effects are similar for both substrates examined, and that the effect of TSO on the binding to DNA of highly carcinogenic bay-region diol-epoxides is multi-faceted, due to its multiple inductive effects.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzo(a)pyrene; Benzopyrenes; DNA; Epoxide Hydrolases; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Microsomes, Liver; Polycyclic Compounds; Rats; Rats, Inbred Strains; Stilbenes

The effect of trans-stilbene oxide (TSO) on organ function and morphology and on drug-metabolizing enzymes was determined in male Sprague-Dawley rats. TSO (300 or 600 mg/kg) was administered i.p., once daily for 5 consecutive days. At a dose of 3400 mg/kg, TSO did no alter body weight, but increased liver weight. The higher dose (600 mg/kg) markedly decreased body weight. TSO treatment (300 mg/kg) induced several drug-metabolizing enzymes. Epoxide hydrolase activity was enhanced in the liver, kidney and lung. In contrast, arylhydrocarbon hydroxylase activity was not significantly altered. Glutathione S-transferase activity, with 1-chloro-2,4-dinitrobenzene as substrate, and uridine diphosphoglucuronyl transferase activity, with p-nitrophenol as substrate, were also increased in the liver and kidney after TSO treatment. It appears that TSO induces hepatic and renal enzyme activities in a similar manner. Treatment with the higher dose of TSO depressed accumulation of p-amino-hippurate by renal cortical slices and increased blood urea nitrogen concentration. Histological examination of kidney sections after treatment with TSO revealed no abnormality. The lower dose led to negligible alteration in liver and the higher dose resulted in mild to moderate hepatic cellular.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Enzyme Induction; Epoxide Hydrolases; Glucuronosyltransferase; Glutathione Transferase; Kidney; Liver; Male; Rats; Rats, Inbred Strains; Stilbenes

Topics: Animals; Cholinesterases; Electron Transport; Epoxide Hydrolases; Fishes; Liver; Microsomes, Liver; Stilbenes

Topics: Androstenedione; Animals; Cytochrome P-450 Enzyme System; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Stilbenes; Testosterone

Cytosolic glutathione reductase activity of rat liver was shown to increase to about 250% of control values after treatment of the animals by intraperitoneal injections of trans-stilbene oxide. The time course and dose-response relationship of the induction brought about by trans-stilbene oxide were determined. The increase of activity was accompanied by a similar increase of protein precipitable by antibodies to rat liver glutathione reductase. These results strongly indicate that increase of glutathione reductase activity in response to treatment with this inducer is the result of true induction. Phenobarbital and 3-methylcholanthrene increased the activities per mg protein in the cytosol fraction by 80 and 24%, respectively. Glutathione reductase was purified to homogeneity from rats treated with trans-stilbene oxide. The molecular and kinetic properties investigated were not significantly different from those of the enzyme from control animals. Selenium-dependent glutathione peroxidase was not induced in the hepatic cytosol of animals treated with trans-stilbene oxide.

Topics: Animals; Cytosol; Enzyme Induction; Glutathione Peroxidase; Glutathione Reductase; Liver; Methylcholanthrene; Peroxidases; Phenobarbital; Rats; Stilbenes

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz-[alpha]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC is faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological tau 1/2 of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benz(a)Anthracenes; Cells, Cultured; Cycloheximide; Dactinomycin; DNA; Enzyme Induction; Epoxide Hydrolases; Kinetics; Liver; Nicotiana; Phenobarbital; Plants, Toxic; Polychlorinated Dibenzodioxins; Protein Biosynthesis; Rats; RNA; Smoke; Stilbenes

Topics: Age Factors; Animals; Benzopyrene Hydroxylase; Enzyme Induction; Epoxide Hydrolases; Female; Male; Methadone; Methadyl Acetate; Microsomes, Liver; Phenobarbital; Rats; Sex Factors; Stilbenes

Topics: Animals; Cytosol; Dose-Response Relationship, Drug; Enzyme Induction; Immunoelectrophoresis; Kinetics; Liver; Male; NADH, NADPH Oxidoreductases; Quinone Reductases; Rats; Stilbenes

Three aspects of the induction of drug-metabolizing enzymes brought about by trans-stilbene oxide have been investigated. (1) The liver hypertrophy in rats treated with trans-stilbene oxide was found to result solely from an increase in the number of cells in this organ, without any increase in the size of each individual cell. (2) Administration of trans-stilbene oxide also produces a 27% increase in the phospholipid content of the hepatic endoplasmic reticulum, i.e., a limited proliferation of this organelle occurs. (3) Furthermore, induction causes changes in the lipid composition of the endoplasmic reticulum. The cholesterol content is decreased, the relative content of sphingo-myelin is also lowered, and a number of changes in the fatty-acid composition occur as well. All of these effects would tend to increase the fluidity of the phospholipid bilayer of the endoplasmic-reticulum membrane and may thus affect drug metabolism.

Topics: Animals; Cell Division; Cholesterol; Endoplasmic Reticulum; Enzyme Induction; Fatty Acids, Unsaturated; Liver; Male; Microsomes, Liver; Phospholipids; Rats; Stilbenes

Topics: Animals; Cytochrome P-450 Enzyme System; Enzyme Induction; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Stilbenes; Substrate Specificity

Topics: Animals; Carbohydrate Dehydrogenases; Enzyme Induction; Liver; Rats; Stilbenes; Time Factors; Uridine Diphosphate Glucose Dehydrogenase

Topics: Amino Alcohols; Benzoin; Biphenyl Compounds; Ethanolamine; Stilbenes