Cis-stilbene Oxide Research Articles

Olefins epoxidation by Ph 4PHSO 5 catalyzed by manganese porphyrins: A novel mechanistic proposal

The epoxidation of three olefins, cyclooctene, styrene and cis-stilbene by Ph 4PHSO 5, catalyzed by Mn(TMP)Cl, has been studied in dichloroethane at 0°C. In the absence of imidazole the reaction does not proceed. When the axial ligand is present the oxidation provides the corresponding epoxides. Cis-stilbene stereospecifically yields cis-stilbene oxide. The epoxidation rates linearly depend on Ph 4PHSO 5 and catalyst concentrations. By increasing the axial ligand concentration the rates increase up to a plateau. This occurs when all the manganese-porphyrin has been complexed by imidazole to form the bis-coordinated adduct. Also by increasing the olefin initial concentration the rates reach a plateau. This behavior, which might be taken as evidence of the formation of a Michaelis-Menten type intermediate, is alternatively rationalized here on the basis of a balance of the rates of the various steps of the overall process. In particular it is envisaged that at high olefin concentration the oxygen transfer to the double bond from the oxo-manganese porphyrin becomes at least as fast as the oxygen transfer from Ph 4PHSO 5 to the manganese porphyrin.

Kinetic parameters of lymphocyte microsomal epoxide hydrolase in carbamazepine hypersensitive patients: Assessment by radiometric HPLC

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 K m , V max, and intrinsic clearance ranged from 6.1–89.9 μM, 3.0–23.2 pmoles diol formed/min/mg protein, and 0.147–0.493 μl/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.

Induction of metallothionein synthesis by glutathione depletion after trans- and cis-stilbene oxide administration in rats

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, injection 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.

Carbon-Carbon Bond Formation by Reductive Coupling with Titanium(II) Chloride Bis(tetrahydrofuran)

Titanium(II) bis(tetrahydrofuran) 1, generated by the treatm ent of TiCl4 in THF with two equivalents of n-butyllithium at -78 °C, has been found to form carbon-carbon bonds with a variety of organic substrates by reductive coupling. Diphenylacetylene is dimerized to exclusively (E,E)-1,2,3,4-tetraphenyl-1,3-butadiene; benzyl bromide and 9-bromofluorene give their coupled products, bibenzyl and 9,9'-bifluorenyl, as do benzal chloride and benzotrichloride yield the 1,2-dichloro-1,2-diphenylethanes and 1,1,2,2-tetrachloro-1,2-diphenylethane, respectively. Styrene oxide and and cis-stilbene oxide undergo deoxygenation to styrene and fra/«-stilbene, while benzyl alcohol and benzopinacol are coupled to bibenzyl and to a mixture of tetraphenylethylene and 1,1,2,2-tetraphenylethane. Both aliphatic and aromatic ketones are smoothly reductively coupled to a mixture of pinacols and/or olefins in varying proportions. By a choice of experimental conditions either the pinacol or the olefin could be made the predom inant product in certain cases. The reaction has been carried out with heptanal, cyclohexanone, benzonitrile, benzaldehyde, furfural, acetophenone, benzophenone and 9-fluorenone. In a remarkable, multiple reductive coupling, benzoyl chloride is converted into 2,3,4,5-tetraphenylfuran in almost 50% yield. The stereochemical course of two such couplings, that of diphenylacetylene to yield exclusively (E,E)-1,2,3,4-tetraphenyl-l,3-butadiene and that of acetophenone to produce only racemic-2,3-diphenyl-2,3-butanediol, is interpreted to conclude that the couplings proceed via two electron transfer pathways (TET) involving titanium (IV) cyclic intermediates of the titanirene and the oxatitanacyclopropane type, respectively.

Synthesis of chiral diferrocenyl dichalcogenides and their application to asymmetric nucleophilic ring opening of meso-epoxides

Four novel optically active bis[(R,S)- and (S,R)-2-(1-dimethylaminoethyl)ferrocenyl] dichalcogenides [abbreviated as (R,S)- and (S,R)-(Fc*E)2(E = S and Te)], each of which possesses two axial and two central elements of chirality, have been prepared by lithiation of commercially available chiral (1-dimethylaminoethyl)ferrocenes, followed by treatment with elemental sulfur or tellurium and air oxidation, in 42–65% isolated yields. The structure of the di-(S,R)-ferrocenyl disulfide has been fully characterized by X-ray crystallography. Ferrocenyl chalcogenide anions (Fc*E–) produced by reduction of the above (Fc*E)2 and the known (Fc*Se)2 with LiAlH4 in tetrahydrofuran are found to act as useful stereoselective nucleophiles for ring opening of meso-epoxides such as cyclohexene oxide, cyclopentene oxide, cyclooctene oxide and cis-stilbene oxide to give the corresponding ferrocenyl β-hydroxyalkyl chalcogenides in high yields with up to 71% diastereoisomeric excess (de). Although the diastereoselectivity depends on the combination of chalcogen atom and meso-epoxide, it is generally higher with E = S and Se than with E = Te. Selenoxide elimination of (S,R)-2-(1-dimethylaminoethyl)ferrocenyl 2-hydroxycyclohexyl selenide (66% de), obtained by the ring opening of cyclohexene oxide with (S,R)-(Fc*Se)2, gives (S)-cyclohex-2-enol with 60% ee. The S-configuration suggests that the ring opening, by the nucleophilic attack of the chiral ferrocenyl chalcogenide anion on the epoxide carbon, mainly occurs via the least hindered transition state considering the interactions between the epoxide, the 1-dimethylaminoethyl moiety and the ferrocene.

Kinetics and stereochemistry of the microsomal epoxide hydrolase‐catalyzed hydrolysis of cis‐stilbene oxides

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.

Reconsidered mechanism in Ru III(hedta)-catalyzed epoxidation of stilbenes

The cis-stilbene oxide/ trans-stilbene oxide isomer assignment given formerly in Inorg. Chim. Acta, 193 (1992) 217 should be reversed. This assignment was based on the 1H NMR spectra of the stilbene oxides given in The Varian High Resolution NMR Spectral Catalog, Vol. 2, which is in error. The large amount of trans-oxide versus cis-oxide (5.5:1) from the cis-stilbene epoxidation by [Ru III(hedta)]/t-BuOOH, and 100% trans-oxide from trans- stilbene, is reinterpreted as indicative of a radicaloid intermediate in ⩾85% of the reaction channels. Comparisons are made with 15 ruthenium oxo catalysts which epoxidize stilbenes. Two categories are observed which are (A) stereoretentive Ru IVO catalysts or sterically hindered Ru VIO porphyrin oxidants, and (B) those which give isomer mixtures for Z-olefins and which are less hindered Ru VO and Ru IVO catalysts. The mechanistics aspects of these epoxidations are discussed.

Epoxide hydrolase in the bulb mite Rhizoglyphus robini: properties and induction

Using styrene oxide as substrate, most of the epoxide hydrolase (EH) activity monitored in the bulb mite Rhizoglyphus robini was associated with the microsomal compartment. The microsomal and cytosolic EHs did not display any significant preference in hydrating trans stilbene oxide (TSO) and cis stilbene oxide (CSO). The microsomal EH, which has a Km value of 5×10-5M and pH optimum of 7.8, was sensitive to ethanol and its activity was inhibited to a moderate extent by 4-fluorochalcone oxide, TSO, CSO and trans-chalcone oxide at a level of 10-4M. Microsomal EH was considerably induced (4–5-fold) in mites feeding garlic and onion, or ingesting TSO-impregnated filter papers. Other epoxides like CSO, 2,4-dichlorostilbene oxide, methyl chalcone oxide and heptachlor epoxide displayed moderate induction levels (1.4–2.6-fold). Of the toxicants assayed only sodium phenobarbital was a potent inducer. Lindane, malathion and DDT did not stimulate EH activity and 3-methyl-cholanthrene was even inhibitory. A decrease in EH activity was observed with a number of phytochemicals tested such as sinigrin, flavone, menthol, trans-β-carotene, chalcone, allyl sulphide and trans-cinnamic acid.

Selective induction of rat liver phase II enzymes by N-heterocycle analogues of phenanthrene: a response exhibiting high correlation between UDP-glucuronosyltransferase and microsomal epoxide hydrolase activities.

1. Among nitrogen heterocycles based on the planar phenanthrene structure are three (1,7- and 4,7-phenanthroline and phenanthridine) which selectively increase rat hepatic phase II drug metabolizing enzyme activities without increasing cytochrome P450 concentration. Of six monooxygenase activities investigated, only ethoxyresorufin dealkylase was raised but this was only minor. 2. The detergent-activated UDP-glucuronosyltransferase activities towards morphine, 4-nitrophenol, and 1-naphthol were increased up to five-, three- and two-fold of control respectively. Microsomal epoxide hydrolase activity towards cis-stilbene oxide was increased up to three-fold and cytosolic glutathione S-transferase activity towards 1-chloro-2, 4-dinitrobenzene reached twice the control value. 3. Cytosolic 4-nitrophenol sulphotransferase activity was not increased by any compound and like some monooxygenase reactions, was decreased by 4,7- and 1,7-phenanthrolines. 4. 1,10-Phenanthroline and two compounds which lack a heterocyclic nitrogen atom, (phenanthrene and 9-phenanthrol), failed to elicit any induction of enzyme activities. 5. Changes in microsomal epoxide hydrolase activity showed high correlation (r = 0.97) with changes in UDP-glucuronosyltransferase (4-nitrophenol) activity.

RuIII(hedta) as an oxygen atom transfer catalyst in the epoxidation of stilbenes

RuIII(hedta) and RuIII((CH3)2edda)+ (hedta3−=N-hydroxyethylethylenediaminetriacetate; (CH3)2edda2−=N, N− dimethethylethylenediamine-N, N−diacetate) catalyze the epoxidation of cis-stilbene and trans-stilbene using tert- butylhydroperoxide (t-BuOOH) as the oxygen source. Prior spin-trapping studies have documented the existence of LRuIIIO ↔ LRuIVO·− ↔ LRuVO2− character in the species obtained from RuIIIL and to-BuOOH (L=polyam- inopolycarboxylate ligands related to edta4−). The O-atom complex, LRuIIIO, appears responsible for the epoxidation of stilbenes. Yields as high as 63.5% cis-stilbene oxide plus 11.0% trans-stilbene oxide from cis-stilbene and 65.1% cis-stilbene oxide from trans-stilbene (with no trans-stilbene oxide) are formed in the epoxidation reactions. Secondary oxidations of the epoxide products produce between 4 to 8% benzaldehyde depending on conditions. The product distribution using the RuIIIL/t-BuOOH catalyst requires at least three epoxidation pathways: (i) concerted transfer of the oxenoid oxygen to the stilbene nucleophile; this process is favored for cis-stilbene; (ii) an outer-sphere electron transfer from the stilbene to LRuIIIO forming a carbon-centered cation radical adjacent to LRuIIIO·−; this radical pair may couple directly for cis-stilbene or after a rapid isomerization of the trans- stilbene radical; (iii) an acyclic pathway which has both free radical and carbocation resonant character; this allows for isomerism of cis-stilbene to trans-stilbene oxide products. RuIIIO(hedta) is also observed to cleanly oxidize benzaldehyde to benzoic acid, sec-phenetyl alcohol to acetophenone, and benzyl alcohol to benzaldehyde and benzoic acid. Cyclohexene is hydroxylated and further oxidized to 2-cyclohexene-1-one.

Effects of environmentally encountered epoxides on mouse liver epoxide-metabolizing enzymes

Male mice were treated (i.p.) for 3 days with 15 different environmentally encountered epoxides, and the effects of these compounds on liver microsomal and cytosolic epoxide hydrolase (mEH and cEH), glutathione S-transferase (mGST and cGST) and carboxylesterase (mCE) activities were determined. The epoxides included the pesticides: heptachlor epoxide, dieldrin, tridiphane, and juvenoid R-20458; the natural products: disparlure, limonin, nomilin, and epoxymethyloleate; the endogenous steroids: lanosterol epoxide, cholesterol-α-epoxide, and progesterone epoxide; and the industrial or synthetic epoxides: epichlorohydrin, araldite, trans-stilbene oxide, and 4'-phenylchalcone oxide. The pesticide epoxides were the most effective inducers of liver weight, microsomal protein, and the enzyme activities measured, with mEH and cEH activities towards cis-stilbene oxide (mEHcso and cEHcso), cGST activities towards four of five substrates, and mCE towards clofibrate (mCEclof) and p-nitrophenylacetate (mCEpna) increased following treatment with most of the pesticides. The synthetic epoxides increased some of the same activities, while the natural products, except for increases in cGST activities, and endogenous steroid epoxides were generally not inductive. cEH activity towards trans-stilbene oxide (cEHtso) was increased only following treatment with the peroxisome proliferator, tridiphane, but decreased following treatment with several of the epoxides, while microsomal cholesterol epoxide hydrolase (mEHchol) was increased only moderately by disparlure. Microsomes could effectively conjugate glutathione to chlorodinitrobenzene (mGSTcdnb) and cis-stilbene oxide (mGSTcso). These two activities were differentially induced by a few of the epoxides, suggesting that they may be selective substrates for different isozymes of mGST. Correlation coefficients were determined for the relative response of liver weight, subfraction protein, and enzyme activities. A relatively high correlation was found between the response of liver weight and cytosolic hydrolysis of trans-stilbene oxide ( r = 0.73) and cis-stilbene oxide ( r = 0.62), and cytosolic glutathione conjugation of dichloronitrobenzene ( r = 0.66) and trans-stilbene oxide ( r = 0.75). In addition, relatively high correlations were found between the different cGST activities, in particular for dichloronitrobenzene with trans-stilbene oxide ( r = 0.89). These studies show that there exists a wide variation in the response of xenobiotic-metabolizing enzymes to environmentally encountered epoxides and that a fairly strong correlation exists between the increases in liver size and increases in certain cytosolic enzyme activities; they also suggest further studies concerning the possibility of an additional isozyme of mGST.

Comparative studies of the effects of stilbene compounds on hepatic ornithine decarboxylase and S-adenosylmethionine decarboxylase induction in rats

trans-Stilbene oxide (TSO, 2 mmol/kg, ip.) induced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) to 60-fold and 5-fold of the controls, respectively, in the liver of rats. Parallel to ODC induction, there was a marked increase in putrescine content to 50-fold of the control levels. cis-Stilbene oxide (CSO), a stereoisomer of TSO, also produced the induction of ODC and SAMDC and the increase in putrescine content. There was no difference in the ability to induce ODC and SAMDC between TSO and CSO with respect to the extents of induction and the time needed to reach maximal levels. trans-Stilbene (TS), a mother compound of TSO, did not show such an effect on ODC, while cis-stilbene (CS) induced both ODC and SAMDC. Treatment with glutathione inhibited TSO- and CSO-mediated induction of ODC and SAMDC. These findings add new information concerning the abilities of TSO, CSO and CS on hepatic polyamine metabolism.

Epoxide hydrolase activities in Drosophila melanogaster

Epoxide hydrolase (EH) activity in Drosophila melanogaster cell fractions was characterized using juvenile hormone (JH III) and cis- stilbene oxide (CSO) as substrates. A comparison of detergents indicated that 0.3% Lubrol PX was relatively effective for solubilizing EH activity from the 20,000 and 100,000 g pellets. The effect of inhibitors, pH, temperature, salt and organic solvents on EH activity depended on the substrate and cell fraction tested, which suggested there were multiple activities present. For initial purification, polyethylene glycol was useful for precipitating EH activity from the 100,000 g supernatant.

Expression of arylhydrocarbon hydroxylase, epoxide hydrolases, glutathione S-transferase and UDP-glucuronosyltransferases in H5-6 hepatoma cells

1. 1. The presence of arylhydrocarbon hydroxylase (cytochrome P-450 IA1 dependent), glutathione S-transferase, two distinct forms of epoxide hydrolases and UDP-glucuronosyltransferases was detected in H5-6 hepatoma cell homogenates using model substrates, selective inhibitors and specific antibodies. 2. 2. The activity of arylhydrocarbon hydroxylase decreased strongly at the first days after plating and remained at a minimal value (1.5 pmol/min per mg) after 5 days of culture. 3. 3. The hydratation of trans-stilbene oxide catalyzed by the soluble form of epoxide hydrolase was very low (11.0 pmol/min per mg), whereas the hepatoma cells contained appreciable amounts of the membrane-bound epoxide hydrolase and glutathione S-transferase measured with cis-stilbene oxide as substrate (maximal specific activity: 1.46 and 2.73 nmol/min per mg, respectively). 4. 4. These cells also glucuronidated 1-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. 5. Addition of fenofibrate (70 μM) into the culture medium for 1–3 days failed to significantly stimulate the activity of cytosolic epoxide hydrolase. Only bilirubin glucuronidation increased 2-fold after 2 days of presence of the drug.

Epoxide hydrolase activity on juvenile hormone in Manduca sexta

As suggested by their mechanism-derived IUPAC nomenclature, epoxide hydrolases (EH) are characterized by the hydrolysis of epoxides. In this study EH activity from M. sexta was monitored in cell fractions from fat body and integument using juvenile hormone III (JH) as a substrate. Experiments were conducted to identify a suitable detergent and detergent concentration for solubilization of microsomal and mitochondrial juvenile hormone epoxide hydrolase (JHEH) activity. Triton X-100 efficiently released total and specific JHEH activity from membranes. A pH optimum range was determined for EH activity in cell fractions from different tissue sources. Potential inhibitors of JHEH activity were tested. One of the better inhibitors was a glycidol analog of JH. EH activity on JH III was compared to activity on JH III bisepoxide, cis-stilbene oxide, and trans-stilbene oxide. JHEH tolerance to detergents, salt, and elevated temperature was investigated. As observed in a similar study on D. melanogaster, the effect of inhibitors, substrates, detergents, salt and temperature suggests the presence of EH isozymes. Polyethylene glycol and ammonium sulfate were used to precipitate JHEH activity from solubilized microsomes and cytosol.

Rat and human liver cytosolic epoxide hydrolases: evidence for multiple forms at level of protein and mRNA.

Two forms of human liver cytosolic epoxide hydrolase (cEH) with diagnostic substrate specificity for trans-stilbene oxide (cEHTSO) and cis-stilbene oxide (cEHCSO) have been identified, and cEHCSO was purified to apparent homogeneity. The enzyme had a monomer molecular weight of 49 kDa and an isoelectric point of 9.2. Pure cEHCSO hydrolyzed CSO at a rate of 145 nmole/min/mg. TSO was not metabolized at a detectable level, and like cEHTSO, the enzyme was about three times more active at pH 7.4 than at pH 9.0. Unlike cEHTSO, cEHCSO was efficiently inhibited by 1 mM 1-trichloropropene oxide (90.5%) and 1 mM STO (92%). Similarly, liver cEH purified 541-fold from fenofibrate induced Fischer 344 rats was shown to be a native 120 kDa dimer of two 61 kDa subunits. The enzyme expressed maximum activity of 205 nmole/min/mg at pH 7.4 toward the diagnostic substrate TSO with an apparent Km of 1.7 microM. In Western blots, polyclonal antibodies against rat liver cEH were shown to recognize a single 61 kDa protein band from liver cytosol of rat, mouse, guinea pig, Syrian hamster, and rabbit. This antibody precipitated neither human liver cEHTSO or cEHCSO. Antibodies against rat liver microsomal epoxide hydrolase reacted with cEHCSO in the Western blot and on immunoprecipitation. Using antibodies against rat liver cEH, 24 positive clones were picked upon colony blot screening of a pEX 1/E. coli POP 2136 expression library.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivity and crystal structure of 10,11 -dihdro-10,11-epoxy-5H-dibenzo[a,d]cycloheptene. A comparison with cis-stilbene oxide

The kinetics and product distributions for the HClO4 catalysed hydrolysis of 10,11-diydro-10,11-epoxy-5H-dibenzo[a,d]cycloheptene1 and of cis-stilbene oxide 6 in tetrahydrofuran–water (8:2), have been investigated by HPLC. The former epoxide gives 9,10-dihydroanthracene-9-carbaldehyde 4 and the trans- and cis-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-10,11-diols, 2 and 3, in he ratio 6:6:1. cis-Stilbene oxide reacts wit a rate constant ca. ten times lower, giving mostly (±)-1,2-diphenylethane-1,2-diol. These differences can be explained by the crystal structure of 1, which shows considerable ring strain due to the enlargement of the bond angles at C(10) and C(11). This structure also suggests an explanation for the much lower rate of the microsomal epoxide hydrolase catalysed hydration of 1 relative to 6.

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxy- resorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo( a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of transstilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation ( r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal expoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal β-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.

Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri: Baseline levels and optimization of assay conditions

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.