Ethoxyresorufin Deethylation Research Articles

Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants

The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide.

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.

Induction of hepatic drug-metabolizing enzymes by cyclic fatty acid monomers in the rat

The effects of cyclic monomers on the activities of several drug-metabolizing enzymes were evaluated. Female Wistar rats were fed, for 4 wk, a semi-synthetic diet containing different quantities of cyclic monomers isolated from linseed oil heated at 275°C for 12 hr under nitrogen. Microsomal proteins and cytochrome c were significantly increased in rats fed a diet containing 0.1 or 1% cyclic monomers. Aminopyrine demethylation, a model reaction preferentially induced by phenobarbital, was increased by this treatment. NADPH-cytochrome P-450 reductase was also stimulated. Moreover, ethoxyresorufin deethylation, known to be greatly increased by methylcholanthrene-type inducer was only increased threefold by this treatment. The activity of p-nitrophenol UDP-glucuronosyl transferase decrease while the conjugation of bilirubin was stimulated. These results suggest that cyclic monomers isolated from heated linseed oil show some characteristics of phenobarbital-type inducers.

Differential induction of mixed-function oxidase (MFO) activity in rat liver and intestine by diets containing processed cabbage: Correlation with cabbage levels of glucosinolates and glucosinolate hydrolysis products

Both white and Savoy-type cabbage added to a semi-purified diet at 25% dry weight and fed to rats ad lib. for 5 days significantly induced ethoxyresorufin (ERR) deethylation in the small and large intestine. Savoy cabbage also induced hepatic activity and, in general, exhibited a greater inducing effect than white cabbage. These enzyme-inducing effects were altered when the cabbage had been processed. The content of intact glucosinolate was greater in Savoy than in white cabbage. The indole glucosinolate (glucobrassicin) content of both types of cabbage was approximately halved by cooking but was unaffected by fermentation, whilst homogenization of Savoy cabbage led to the total disappearance of intact glucosinolates. Levels of the indole glucosinolate breakdown products ascorbigen and indole-3-carbinol were highest in homogenized cabbage, and ascorbigen levels were also higher in cooked than in fresh cabbage of either type. When added to the semi-purified diet and fed ad lib. to rats for 5 days, indole-3-carbinol was a potent inducer of hepatic ERR deethylation and cytochrome P-450 activity, but had much less effect in the intestine. Other glucobrassicin metabolites, diindolylmethane and indole-3-acetonitrile, also had some inducing effect in the liver but no effect in the intestine, while ascorbigen significantly induced ERR deethylation in the small and large intestine but had no effect on hepatic MFO activity.

Inhibition of cytochrome P-450 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin

The kinetic characteristics and mechanism of flavonoid inhibition of cytochrome P-450-mediated reactions were examined in rat liver microsomes, using the naturally occurring flavonoid, quercetin (3,3′,4′,5,7-pentahydroxyflavone). Quercetin inhibited the O-deethylation of ethoxyresorufin in β-naphthoflavone-induced microsomes by 15–80% at concentrations of 10–250 n m. The pattern of inhibition was dependent on quercetin concentration. Quercetin also inhibited p-nitroanisole demethylation and benzo( a)pyrene hydroxylation, but did not change the proportions of the individual benzo( a)pyrene metabolites in comparison to controls. Specific steps in the P-450 reaction pathway were tested for sensitivity to quercetin inhibition. The K m values of the P-450 substrates tested were increased in the presence of quercetin; competition for and/or alteration of the substrate binding site contributes to the mechanism of inhibition. In experiments under anaerobic, carbon monoxide-saturated conditions, quercetin did not inhibit cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase. The cumene hydroperoxide-supported O-deethylation of ethoxyresorufin was inhibited by quercetin (15–60% inhibition at concentrations of 50–300 n m), suggesting that quercetin may interfere with the formation or breakdown of the oxygenated heme complex. Stoichiometry experiments established that quercetin is a potent uncoupler of P-450 reactions, elevating the rates of H 2O 2 formation almost twofold. Structure/activity studies indicated that certain other naturally occurring flavonoids were at least as potent inhibitors of ethoxyresorufin deethylation as quercetin. These findings are of interest in light of the significant dietary exposure of the human population to the flavonoids.

Effects of vanadate on intracellular reduction equivalents in mouse liver and the fate of vanadium in plasma, erythrocytes and liver

Intraperitoneal injection of 180 μ mol sodium vanadate/kg body wt in mice inhibits the in vivo metabolism of drugs by the hepatic monooxygenase, as measured by exhalation of 14CO 2 after treatment with appropriately labeled [ 14C]methacetin. Determinations of hepatic GSH, NADPH and NADH after vanadate injection show an initial and transient decrease of GSH (10 min, 20%), followed by a transient decrease of NADPH (30 min, 23%), followed by a decrease of NADH (40 min, 23% and 60 min, 26%). Rat liver organ spectrophotometry in the dual-wavelength mode shows an immediate response of the NAD(P)H level, which decreases transiently after addition of vanadate. Furthermore, EPR and AAS measurements indicate that vanadium occurs in the plasma in the oxidation states +IV and +V, whereas in intracellular compartments in liver and erythrocytes vanadium exists practically only in the +IV form (vanadyl). The duration of the inhibition for about 2h coincides well with the transient concentration of vanadate in plasma, which decreases more rapidly than vanadyl. Maximal drug inhibition is associated with the phase of rapid formation of vanadyl in the liver. The experiments are in accordance with the hypothesis that vanadate inhibits the cytochrome P-450 dependent oxidative drug metabolism by diversion of reducing equivalents away from cytochrome P-450. Further evidence for such a hypothesis is provided by in vitro experiments in microsomes. Vanadate causes a dose dependent decrease of ethoxyresorufin deethylation which is reversible by the addition of NADPH.

Fluorescence-microscopic measurement of intracellular cytochrome P-450 enzyme activity (ethoxyresorufin O-de-ethylation) in unfixed liver section.

A fluorescence-microscopic method has been developed for measurement of the intracellular kinetics of the cytochrome P-450 reaction, ethoxyresorufin O-de-ethylation, in individual hepatocytes in unfixed non-frozen liver sections obtained from control or 3-methylcholanthrene-pretreated mice. De-ethylation was enhanced in the presence of salicylamide, but inhibited in the presence of alpha-naphthoflavone, A total of 21 reaction rate curves (fluorescence of the metabolite, resorufin, versus time) were constructed for different individual hepatocytes and groups of two or three hepatocytes, in a total of 12 sections from four control and eight 3-methylcholanthrene-induced mice. No two reaction curves were identical, but the curves were classified, by similarity of curve shape and fluorescence intensity, into three control categories and four 3-methylcholanthrene-induced categories. It was considered that the data represented a total of seven types of hepatocytes, which differed in their ethoxyresorufin de-ethylase reaction characteristics. Hepatocytes in the sections from 3-methylcholanthrene-pretreated mice showed higher de-ethylase activities than the hepatocytes from control mice, but the increase in activity covered a wide range of values, from 3-fold to 17-fold higher than the mean control value, suggesting that 3-methylcholanthrene did not induce ethoxyresorufin de-ethylation to the same extent in all hepatocytes.

Atypical inductive properties of rifampicin

Hepatic and microsomal parameters, metabolism of cytochrome P-450-dependent substrates and spectral properties of cytochrome P-450 have been used in mice in order to classify rifampicin as an inducer by comparing it to phenobarbitone and 3-methylcholanthrene. Rifampicin significantly enhanced relative liver weight, cytochrome P-450 content, microsomal protein, weight of the 100,000 g pellet and shortened the zoxazolamine paralysis time. Compared on the basis of microsomal protein, of five substrate reactions only the ethylmorphine demethylation was enhanced; ethoxycoumarin deethylation and biphenyl 1–2-hydroxylation were unaltered; ethoxyresorufin de-ethylation and biphenyl-4-hydroxylation were decreased. The CO-cytochrome P-450 absorption maximum showed a blue shift of about 0.5 nm after only 1 day of rifampicin pretreatment, while there was no significant blue shift after 1 day of 3-methylcholanthrene pretreatment. Rifampicin has been shown to be an inducer in NMRI mice. Although resembling phenobarbitone it seems, however, to be a similarly atypical inducer as it is in man.

Oxidative biotransformation of benzo(a)pyrene by human lung microsomal fractions prepared from surgical specimens.

1. Microsomal fractions were prepared from 15--50 g specimens of human lung tissue (mostly alveolar) obtained at surgical resections of 13 middle-aged male patients suffering from different pulmonary tumours. Marker enzyme assays indicated that the frations contained about 25% of the endoplasmic reticulum of the homogenate and about 10% of its mitochondrial membranes. 2. The content of cytochrome b5 corresponded to that of rodent lung microsomes, whereas the apparent content of cytochrom P-450 was much lower. 3. The extent of benzo(a)pyrene metabolism varied 13-fold between individuals in the group and was not detectable in about 40% of the cases. 4. The dihydrodiols as % of total metabolites formed was higher than in laboratory animals, the 7,8-dihydrodiol in most cases amounting to more than 40% of total dihydrodiols. 5. The apparent rate of hydroxylation was stimulated by 1 mM 2-diethylaminothyl 2,2-diphenylvalerate and by 1 mM 1,2-oxy-3,3,3-trichloropropane, but inhibited moderately by 0.1 mM metyrapone and extensively by 0.05 mM 7,8-benzoflavone. 6. Ethoxyresorufin deethylation qualitatively paralleled benzo(a)pyrene hydroxylation among individuals.