Characterization of sheep liver and lung microsomal ethylmorphine N-demethylase

Ethylmorphine N-demethylase activity of the sheep liver and lung microsomes was reconstituted in the presence of solubilized microsomal cytochrome P-450, NADPH-cytochrome c reductase and synthetic lipid, phosphatidylcholine dilauroyl. The Km of the lung microsomal ethylmorphine N-demethylase was calculated to be 4.84 mM ethylmorphine from its Lineweaver-Burk graph and lung enzyme was inhibited by its substrate, ethylmorphine, when its concn was 25 mM and above, reaching to 67% inhibition at 50 mM concn. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear. From these graphs, two different Km values were calculated for the liver enzyme as 4.17 mM and 0.40 mM ethylmorphine. Ethylmorphine N-demethylase activities of both liver and lung microsomes were inhibited by NiCl2, CdCl2 and ZnSO4. Ethylalcohol inhibited N-demethylation of ethylmorphine in lung and liver microsomes. Acetone (5%) slightly enhanced the N-demethylase activity of the liver enzyme, whereas 5% acetone completely inhibited the lung enzyme. Phenylmethylsulfonyl fluoride at 0.10 mM and 0.25 mM concn had no effect on liver enzyme activity, while at these concns, it inhibited the activity of the lung enzyme by about 35%.     

The effects of peripubertal testosterone on ethylmorphine demethylase activity in adult rats testectomized neonatally

The physiologic activity of ethylmorphine demethylase (EMDM) is 2.5 times greater in adult male rats than in females. Defeminization, effected neonatally by testicular androgens, causes 65% of the increase while masculinization causes the rest. Castration of the intact adult does not alter the defeminized activity of the enzyme but abolishes its masculinized activity. Testosterone restores the latter. Adults that are not defeminized, because of neonatal castration, have enzymes unresponsive to testosterone. Thus, defeminization seems necessary for the expression of masculine activity. This was studied by castrating males at birth (day 1) and implanting capsules of testosterone propionate on day 35. On day 71, the activity had increased 4.4-fold to physiologic levels, compared with rats receiving no steroid (1.9 nmol.min-1.mg-1). Removal of the implant on day 71 decreased the activity on day 84 to that of the no-steroid rats. Thus, defeminization is neither caused by androgen in peripubertal rats nor needed to effect full physiologic activity of the enzyme. The activation of EMDM by peripubertal testosterone may reflect its regulation of growth hormone; first, testosterone (days 35-71) failed to increase enzyme activity in males hypophysectomized on day 35; second, growth hormone infused (5 micrograms.h-1) over days 71-76 blocked the activation, actually decreasing activity 3.7-fold; third, the activity induced by testosterone was further increased (26%) by growth hormone release-inhibiting hormone (5 micrograms.h-1, days 71-76).     

Age variations in ethylmorphine demethylase and benzopyrene hydroxylase activities in rat liver microsomes

The ethylmorphine demethylase activity of the rat liver microsomes was higher in three-month animals (as compared to one-month), remained at a high level in the 12-month and decreased in old rats. The benzopyrene hydrolase activity increased in three-month animals, decreased in 12-month rats and remained the same with subsequent ageing of the organism. Asynchronicity of variations in the oxidation rate of different xenobiotics with ageing is considered as a result of changes in substrate specificity of the monooxygenase system due to metabolic peculiarities which reflect the age development of the organism and extinction of the reproduction function.     

The additive effects of progestins on testosterone-stimulated hepatic ethylmorphine metabolism and cytochrome P-450 content

The actions of several progestins on mouse liver were studied in terms of their inherent potency and for their ability to modify the biologic activity of testosterone. When hepatic ethylmorphine demethylase activity and cytochrome P-450 content were used as end points, the biological potency of progestins was ranked as follows: cyproterone acetate>progesterone>medroxyprogesterone acetate>hydroxyprogesterone caproate controls. The induced alterations of these parameters were, therefore, unrelated to reported progestational (cyproterone acetate medroxyprogesterone acetate>>hydroxyprogesterone caproate>progesterone) or androgenic (medroxyprogesterone acetate>cyproterone acetate = hydroxyprogesterone caproate = progesterone) actions of these steroids. A similar conclusion was reached when hepatic weight and microsomal protein content were used as end points.

When progestins (0.1–10 mg/day) were administered with testosterone (0.1 mg/day), the effect of both steroids were additive. This is in contrast to their actions on other tissues such as kidney and sub-maxillary gland where progestins potentiate and inhibit androgen action. We conclude from these studies that the mechanism of action of progestins on the liver differs from that on other tissues.     

Xenobiotic-metabolizing enzyme systems in test fish--II. The ethylmorphine N-demethylase activity of guppy (Poecilia reticulata) liver

The oxidative demethylation of the model substrate ethylmorphine has been characterized for the first time in the liver of a fish (Poecilia reticulata). The enzyme showed maximal activity at 35 degrees C and pH values higher than 8. The values of Km and Vmax for the reaction were 0.83 +/- 0.11 mM and 4.64 +/- 0.81 nmol HCHO/(mg microsomal protein) per min. The activity is attributed to the cytochrome P-450-dependent monoxygenase system, since it is inhibited by CO and requires NADPH; moreover it is inhibited competitively by alpha-naphthoflavone and non-competitively by metyrapone. The enzyme activity is induced by a two-week treatment of fish with phenobarbital and may be associated with a protein band of Mr 54,000.     

Correlation of hepatic cytosolic androgen binding proteins with androgen induction of hepatic microsomal ethylmorphine N-demethylase in the rat

Previous reports have demonstrated the presence of moderate to high affinity binding for androgens in the cytosol of livers from male rats. This binding was significantly lower in female rats or in immature rats of either sex. The hepatic androgen binding protein, which sedimented at approx. 4 S on sucrose density gradients, has been called a receptor which mediates the actions of androgens in the liver. The experiments in the present study were designed to evaluate the hepatic androgen binding protein for characteristics which have been attributed to receptors in other tissues and to correlate the presence of androgen binding with androgen induction of hepatic drug metabolism. In the current studies, we have shown that cytosol from the livers of male rats bound [3H]dihydrotestosterone [( 3H]DHT) and translocated this steroid ligand to the nucleus in a time and temperature dependent manner. Cytosol prelabeled with [3H]DHT, when passed over a column of denatured DNA cellulose, eluted in three radioactive peaks. Two of these peaks were absent when cytosol from livers of female or hypophysectomized males was used. In addition, the presence of high concentrations of hepatic androgen binding correlated well with the ability of androgen to induce ethylmorphine N-demethylase, a marker of microsomal cytochrome P-450-dependent drug metabolism. Values for both parameters were higher in males than in either females or hypophysectomized males. Testosterone treatment induced both parameters in ovariectomized females and 17 beta-estradiol repressed both in males. However, testosterone treatment failed to induce hepatic androgen binding in hypophysectomized males and immature males, both of which are also unresponsive to androgen induction of drug metabolism. The results suggest that one or more hepatic cytosolic androgen binding proteins possess several characteristics associated with steroid receptors in reproductive tract tissue. Furthermore, this binding may be implicated as a mediator for the androgen induction of at least one component of hepatic drug metabolism.     

Comparison of human fetal hepatic and adrenal cytochrome P450 activities with some major gestational steroids and ethylmorphine as substrates.

The immunoidentified human fetal liver and adrenal microsomal contents of cytochromes P450IIIA and P450XVIIA1 were compared to the metabolism of steroids and ethylmorphine. In fetal liver microsomes, 16 alpha-hydroxylation of dehydroepiandrosterone (DHA) was catalyzed at a high rate in almost all investigated specimens and accompanied by a high ethylmorphine N-demethylase activity. Progesterone 16 alpha- and 17 alpha-hydroxylation was found only in the livers with the highest DHA 16 alpha-hydroxylation activities, while 21-hydroxylation of progesterone was catalyzed only occasionally in these samples. In fetal adrenal microsomes, 21-hydroxylation of progesterone to 11-desoxycorticosterone (DOC) and 11-desoxycortisol (DOCOL) was catalyzed. In contrast to fetal liver, the adrenals also catalyzed the 17 alpha-hydroxylation of pregnenolone and the formation of DHA from 17 alpha-OH-pregnenolone. 16 alpha-hydroxylation of DHA and ethylmorphine N-demethylation were modest in the adrenals. P450IIIA/HLp was immunoidentified in all investigated liver specimens except two (18/20) in which no ethylmorphine N-demethylation or 16 alpha-hydroxylation of DHA was found. P450XVIIA1 bands were observed in 8/20 blots of liver specimens, but there was no correlation between the density of these bands and the 17 alpha-hydroxylation of progesterone. All 11 fetal adrenal samples catalyzed DHA 16 alpha-hydroxylation, although only 8 were positive for P450IIIA/HLp. All investigated adrenals were positive in regard of the P450XVIIA1 band, except one (8/9) with a low 17 alpha-hydroxylation of progesterone. All adrenal specimens catalyzed 21-hydroxylation of progesterone and contained P450C21 bands in immunoblots and all samples catalyzed the formation of DOC and DOCOL from progesterone. Our findings in the fetal livers show a correlation between the DHA 16 alpha-hydroxylation and immunoidentified P450IIIA/HLp bands. In adrenals, there was a correlation between the immunoidentified P450XVIIA1 bands and the 17 alpha-hydroxylation of progesterone.     

Growth hormone depresses ethylmorphine demethylase activity: correlation with decreased levels of fast-turnover cytochrome P-450 in hypophysectomized female rats

The hepatic monooxygenase system was studied in hypophysectomized female rats infused for 5 days with ovine growth hormone (GH). At 7.5 micrograms.h-1 GH decreased the total cytochrome P-450 by 16%; at 10 micrograms.h-1 it reduced both cytochrome P-450 (31%) and the activity of ethylmorphine demethylase (31%). GH did not alter the activities of NADPH cytochrome c reductase or aniline hydroxylase. The lower GH dose decreased the amount of fast- and slow-turnover P-450 by 11 and 38%, respectively, while the higher dose decreased both by 49%. The loss of demethylase activity therefore correlates with the loss of fast-turnover P-450. This component is relatively more abundant in the female (fast: slow turnover of 4.3) than the male (fast:slow turnover of 2.5). GH did not affect the half-lives of the P-450 components, suggesting that it decreases their synthesis. The P-450 concentration in microsomes from GH-treated animals did not increase after incubation with hemin, suggesting that in vivo the hormone does not lower P-450 synthesis via depression of heme. Puromycin mimicked the effect of GH and when given with the hormone their effects on the P-450 levels were multiplicative (p less than 0.05), suggesting different modes of action and that GH does not decrease P-450 by acting at translation.     

Sex- and strain-dependent hepatic microsomal ethylmorphine N-demethylation in mice: the roles of type I binding and NADPH-cytochrome P-450 reductase

The roles of type I binding and NADPH-cytochrome P-450 reductase in ethylmorphine demethylation were investigated in two strains of mice, using sex differences in these activities as a tool. In the CPB-SE strain, females metabolize ethylmorphine faster than males. Sex differences in cytochrome P-450 content and endogenous NADPH-cytochrome P-450 reductase activity were too small to account for this. On the other hand, the differences in the magnitudes of type I spectra and ethylmorphine-induced enhancement of cytochrome P-450 reduction were considerable larger than those in the rates of demethylation. All parameters, except endogenous cytochrome P-450 reduction, were modified in a similar way by testosterone pretreatment: in females they were depressed to the male level, whereas in males they remained unchanged. Castration had no effect in females and enhanced the activities in males. The CPB-V strain exhibited little or no sex differences in ethylmorphine demethylation, cytochrome P-450 content and endogenous cytochrome P-450 reduction. Testosterone pretreatment had little or no influence on these activities. Type I binding and reductase stimulation, however, showed sex differences, comparable to those observed in the CPB-SE strain, which were also abolished by testosterone. A relationship between reductase stimulation and type I binding was observed, which was, apparently, independent of sex or strain. It is concluded that androgen primarily influences the amount of cytochrome P-450-substrate complex formed, but that the reduction of this complex is not rate-limiting in the demethylation of ethylmorphine.     

Changes in certain kinetic properties of hepatic microsomal aniline hydroxylase and ethylmorphine demethylase associated with postnatal development and maturation in male rats

1. Changes in certain kinetic properties (V(max.) and apparent K(m)) of hepatic microsomal mixed-function oxidases have been studied as a function of postnatal development and maturation in male rats. 2. Microsomal cytochrome P-450 content changed only slightly between 1 and 12 weeks of age. 3. Aniline hydroxylase activity (V(max.)) increased abruptly between 1 and 2 weeks of age to greater than adult activities and then returned to a plateau value between 4(1/2) and 12 weeks of age. Ethylmorphine demethylase activity remained low and relatively constant between 1 and 3 weeks of age and then increased markedly ( approximately 100%) between 3 and 4(1/2) weeks. 4. The apparent Michaelis constant (K(m)) for aniline hydroxylation increased almost linearly with time between 1 and 6 weeks of age and tended to reach a plateau value thereafter. The apparent K(m) for ethylmorphine demethylation increased between 1 and 3 weeks of age and then decreased abruptly to a constant value between 6 and 12 weeks. 5. The data indicate that developmental changes in the activity of these microsomal oxidases do not correlate temporally with each other or with changes in microsomal cytochrome P-450 content. 6. The most dramatic changes in enzyme activity were associated with early development (1-3 weeks) and weaning (3-4 weeks). 7. Changes in weight of seminal vesicle, a criterion of sexual maturation in male rats, were most prominent between 6 and 8 weeks of age and thus appeared to be separated in time from the prominent changes in enzyme activity.     

A kinetic study comparing the light-reversal properties of carbon monoxide inhibition of ethylmorphine, benzphetamine, and 7-ethoxycoumarin dealkylation with those of hydroxylation of 17-hydroxyprogesterone and testosterone

The light-reversal properties of carbon monoxide (CO) inhibition of the dealkylation of benzphetamine, ethylmorphine, and 7-ethoxycoumarin by microsomes from phenobarbital (PB)-induced rat livers were compared with those of the 6 beta-, 7 alpha-, and 16 alpha-hydroxylations of testosterone by the same rat hepatic microsomes and C-21 hydroxylation of 17-OH progesterone by steer adrenal microsomes. CO inhibited all reactions studied to essentially the same degree. The significant finding was that the dealkylations were reversed most effectively by light of wavelengths between 440 and 445 nm, rather than around 450 nm, the optimal wavelength for steroid hydroxylations. Moreover, the dealkylations required several-fold higher light intensities for equivalent light reversal. These studies suggest that the heme protein-CO complex responsible for dealkylations has a spectrum corresponding to the shape of the pass band of the 445-nm filter, whereas that of the steroid hydroxylations has its light-reversal maximum at 450 nm and appears to be broader. The measurable differences in the light-reversal properties between the monooxygenations of two groups of substrates, (i) dealkylations and (ii) hydroxylations of lipid substrates, furnish biophysical properties that allow a better characterization of microsomal monooxygenases which should be of value in forwarding progress in the study of these systems.