Influence of Metyrapone on Development of Heterodera glycines

Metyrapone, an inhibitor of steroid synthesis, affected the survival and rate of development of Heterodera glycines. Metyrapone in aqueous tartaric acid solvent influenced sex ratios. The effect on sex ratios was mediated through the host, whereas the effect on survival was apparently effected directly. Tartaric acid increased larval penetration of soybean roots.     

Glucuronate, the precursor of vitamin C, is directly formed from UDP-glucuronate in liver

The conversion of UDP-glucuronate to glucuronate, usually thought to proceed by way of glucuronate 1-phosphate, is a site for short-term regulation of vitamin C synthesis by metyrapone and other xenobiotics in isolated rat hepatocytes. Our purpose was to explore the mechanism of this effect in cell-free systems. Metyrapone and other xenobiotics stimulated, by approximately threefold, the formation of glucuronate from UDP-glucuronate in liver extracts enriched with ATP-Mg, but did not affect the formation of glucuronate 1-phosphate from UDP-glucuronate or the conversion of glucuronate 1-phosphate to glucuronate. This and other data indicated that glucuronate 1-phosphate is not an intermediate in glucuronate formation from UDP-glucuronate, suggesting that this reaction is catalysed by a 'UDP-glucuronidase'. UDP-glucuronidase was present mainly in the microsomal fraction, where its activity was stimulated by UDP-N-acetylglucosamine, known to stimulate UDP-glucuronosyltransferases by enhancing the transport of UDP-glucuronate across the endoplasmic reticulum membrane. UDP-glucuronidase and UDP-glucuronosyltransferases displayed similar sensitivities to various detergents, which stimulated at low concentrations and generally inhibited at higher concentrations. Substrates of glucuronidation inhibited UDP-glucuronidase activity, suggesting that the latter is contributed by UDP-glucuronosyltransferase(s). Inhibitors of beta-glucuronidase and esterases did not affect the formation of glucuronate, arguing against the involvement of a glucuronidation-deglucuronidation cycle. The sensitivity of UDP-glucuronidase to metyrapone and other stimulatory xenobiotics was lost in washed microsomes, even in the presence of ATP-Mg, but it could be restored by adding a heated liver high-speed supernatant or CoASH. In conclusion, glucuronate formation in liver is catalysed by a UDP-glucuronidase which is closely related to UDP-glucuronosyltransferases. Metyrapone and other xenobiotics stimulate UDP-glucuronidase by antagonizing the inhibition exerted, presumably indirectly, by a combination of ATP-Mg and CoASH.     

Developmental changes caused by metyrapone and azadirachtin in Spodoptera littoralis (Boisd.) (Lep., Noctuidae) and Galleria mellonella (L.) (Lep., Pyralidae)

Larvae of Spodoptera littoralis and Galleria mellonella reared on standard laboratory food were used in experiments with metyrapone and azadirachtin. Metyrapone in S. littoralis caused high elevation of metabolic rate which resulted in a significant increase of body-mass. This phenomenon was associated with precocious transformation to pupal stage. The same results were obtained after application of azadirachtin or neem oil. Restriction of reproduction was also observed in S. littoralis . Both these compounds influenced body pigmentation: the treated larvae was light green or light gray. Synthesis of melanin was inhibited. In Galleria mellonella , the effect was not as prominent.     

Metyrapone modulation of tyrosine aminotransferase induction by dexamethasone in cultured hepatocytes

Metyrapone, an inhibitor of cytochrome P-450-dependent monooxygenases, enhanced the induction of tyrosine aminotransferase by dexamethasone in primary cultures of hepatocytes, while it had no effect on the basal level of the enzyme activity in the absence of the hormone. The amplification of the hormonal induction of tyrosine aminotransferase activity was strictly correlated with the concentration and with the inhibitory action of the compound on cytochrome P-450. The phenomenon occurred even at the maximally effective concentrations of dexamethasone, thus showing that metyrapone is a 'Glucocorticoid Potency Amplifier'. The dexamethasone activity amplification by metyrapone could be the consequence of a modulation of the glucocorticoid biotransformations due to the cytochrome P-450 inhibitor.     

Tetrahydrofurane - an inhibitor for ethanol-induced liver microsomal cytochrome P450.

Liver microsomes from ethanol-pretreated rats have been compared with microsomes from male and female controls and phenobarbital- and benzpyrene-pretreated rats. The 0-dealkylation activity for 7-ethoxycoumarin was enhanced after all treatments. Metyrapone selectively inhibited the activity after pretreatment with phenobarbital and naphthoflavone blocked the activity after benzpyrene treatment. Ethanol and even more so tetrahydrofurane inhibited specifically the 0-dealkylation in microsomes from ethanol-pretreated rats. Only in these microsomes tetrahydrofurance produced a pronounced ligand-type optical difference spectrum and concomitantly a new low-spin cytochrome P450 species in the EPR-spectrum. According to inhibition experiments, liver microsomes from male and female rats have a different pattern of cytochrome P450 species.     

Influence of glucocorticoid administration and inhibition on fetal baboon pulmonary maturity and the amniotic fluid L/S ratio.

The pregnant baboon feto-placental unit responded to intra-amniotic dexamethasone by a rise in the lecithin-sphingomyelin ratio (L/S ratio) and accelerated pulmonary maturity with evidence of increased amounts of pulmonary surfactant. Metyrapone 11 beta-hydroxylase inhibition of corticoid synthesis prevented the expected rise in the L/S ratio without influencing the normal development of pulmonary maturity. The L/S ratio did not accurately reflect pulmonary maturity when metyrapone was administered to the pregnant baboon. Prudently, model systems such as the baboon should be used for more detailed studies of endocrine effects on pulmonary development because of the reported untoward effects of exogenous steroid administration on the human neonatal nervous system.     

Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450

9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.     

A simple radioimmunoassay for plasma cortisol and 11-deoxycortisol (17, 21-dihydroxy-4-pregnene-3, 20-dione).

A simple procedure for the measurement of cortisol and 11-deoxycortisol in plasma or serum is described. One-half ml. of plasma is extracted with methylene chloride. Separation of cortisol and deoxycortisol is achieved by a Sephadex LH-20 mini-column. The assay itself is achieved by the use of a commercially available cortisol kit employing rabbit anti-cortisol coated tubes. This antibody exhibits a 20% crossreactivity with 11-deoxycortisol. This procedure is extremely useful in the assessment of a pituitary-adrenal reserve in the Metyrapone test.     

Inhibition of rat testicular microsomal steroidogenesis by oxytocin and metyrapone

Capillary gas chromatographic 'steroid profiling' has been utilised to separate and quantify the metabolites (derivatized as methyloximes and/or trimethylsilyl ethers) formed from pregnenolone after incubation with rat testicular microsomes. A wide range of steroid metabolites was found, indicating that both the 5-ene and 4-ene pathways of testosterone biosynthesis were operating, as well as 16 alpha-hydroxylation, 20 beta-reduction and the formation of several C19 steroids (the 16-androstenes). At the concentration used, Metyrapone markedly inhibited 16 alpha- and 17-hydroxylation and side-chain cleavage of 17-hydroxylated C21 steroids. 16-Androstene production was also markedly inhibited and the formation of other metabolites was affected to lesser extents. Oxytocin abolished the formation of all C21 and C19 metabolites of pregnenolone.     

An incidentally discovered case of Cushing's syndrome without clinical signs

This paper describes a middle-aged man in whom an adrenal mass was incidentally discovered by upper abdominal echogram. Physical examination revealed no signs of Cushing's syndrome. The plasma cortisol level at 0800 h was within the normal range, but the diurnal rhythm had disappeared. Plasma ACTH was undetectable throughout the whole day. Urinary 17-OHCS was slightly increased and was not suppressed by 2 mg or 8 mg dexamethasone. Metyrapone test and CRF test revealed no response. A left adrenalectomy was performed and histological diagnosis of the removed tumor was an adrenal adenoma. After operation, oral steroid supplementation was necessary. These data suggest that the autonomous cortisol secretion by the tumor accounted for all his daily cortisol secretion, but it was too small to be clinically functional. We propose that every patient with an incidentally discovered adrenal mass should be subjected to endocrinological evaluations.     

Transport of cortisol,progesterone and cholesterol across isolated mesentery

Summary Isolated rat mesentery, mounted in a diffusion cell, is used as a model for the study of vascular endothelium permeability characteristics. The passage of tracer molecules is measured in the absence of osmotic or hydrostatic pressure gradients across the mesentery. The permeability coefficient of the membrane for cortisol and progesterone is similar. When bound to transcortin, cortisol crosses mesentery at a significantly slower rate. Metyrapone diatartrate increases by 30% the passage of free and of transcortinbound cortisol, but is without effect on the passage of progesterone or glucose in the same conditions. When the transfer of cholesterol across mesentery is studied, a high percentage of the tracer is trapped by the membrane.     

Toluene monooxygenase from the fungus Cladosporium sphaerospermum

Assimilation of toluene by Cladosporium sphaerospermum is initially catalyzed by toluene monooxygenase (TOMO). TOMO activity was induced by adding toluene to a glucose-pregrown culture of C. sphaerospermum. The corresponding microsomal enzyme needed NADPH and O(2) to oxidize toluene and glycerol, EDTA, DTT, and PMSF for stabilization. TOMO activity was maximal at 35 degrees C and pH 7.5 and was inhibited by carbon monoxide, Metyrapone, and cytochrome c. TOMO preferred as substrates also other aromatic hydrocarbons with a short aliphatic side chain. Its reduced carbon monoxide difference spectrum showed a maximum at 451 nm. A substrate-induced Type I spectrum was observed on addition of toluene. These results indicated that TOMO is a cytochrome P450. TOMO and its corresponding reductase were eventually purified by a simultaneous purification revealing apparent molecular masses of 58 and 78 kDa, respectively.     

Effects of inhibitors of steroid biosynthesis on prostaglandin formation in rabbit ovarian follicles

Rabbit ovarian follicles were incubated without stimulation, with LH and with LH + an inhibitor of steroid biosynthesis. Formation of prostaglandins PGE and PGF and of progesterone and estradiol was measured in these incubates. It was found that aminoglutethimide phosphate (AGP) inhibited the LH stimulated biosynthesis of both prostaglandins and steroids. However U 30870 and Metyrapone, while completely inhibiting the LH stimulated biosynthesis of progesterone and estradiol respectively, had no effect on the formation of prostaglandins. Further, the inhibition of prostaglandin formation by AGP could not be reversed by exogenous steroids. It, therefore, appears that the effect of AGP on prostaglandin biosynthesis may not be related to its effect on steroid biosynthesis. However, the response of rabbit follicles to AGP is contrary to that reported for rat follicles and indicates different control mechanisms for prostaglandin formation in the follicles of the two species.     

中国希瓦氏菌D14^T的厌氧腐殖质呼吸

实验证明,希瓦氏菌新种(ShewanellacinicaD14T)在厌氧条件下可以利用多种有机酸盐和甲苯等环境有毒污染物作为电子供体,以腐殖质作为唯一末端电子受体进行厌氧呼吸(即醌呼吸)。电子在细胞膜呼吸链的传递过程中,偶联能量的产生来支持菌体的生长,1mmol/LAQDS可支持细胞增殖约60倍。电子供体的氧化和唯一电子受体腐殖质还原之间存在着动态的偶联过程,随着电子供体量的增加腐殖质还原的量也随之增加。典型呼吸链抑制剂诸如:抑制Fe-S中心的Cu2 ,甲基萘醌类似物标桩菌素,抑制甲基萘醌氧化型向还原型转化的双香豆素和细胞色素P450的专一抑制物甲吡酮等对腐殖质的还原有着极为显著的抑制作用,为进一步证明希瓦氏菌(Shewanellacinica)D14T可利用腐殖质进行厌氧呼吸提供了有力的佐证。而D14T在进行腐殖质呼吸的同时,对于甲苯,苯胺等环境有毒物质的有效降解则具有着重要的环境学意义。     

Effects of inhibitors of steroid biosynthesis on prostaglandin formation in rabbit ovarian follicles

Rabbit ovarian follicles were incubated without stimulation, with LH and with LH + an inhibitor or steroid biosynthesis. Formation of prostaglandins PGE and PGF and of progesterone and estradiol was measured in these incubates. It was found that aminoglutethimide phosphate (AGP) inhibited the LH stimulated biosynthesis of both prostaglandins and steroids. However U 30870 and Metyrapone, while completely inhibiting the LH stimulated biosynthesis of progesterone and estradiol respectively, had no effect on the formation of prostaglandins. Further, the inhibition of prostaglandin formation by AGP could not be reversed by exogenou steroids. It, therefore, appears that the effect of AGP on prostaglandin biosynthesis may not be related to its effect on steroid biosynthesis. However, the response of rabbit follicles to AGP is contrary to that reported for rat follicles and indicates different control mechanisms for prostaglandin formation in the follicles of the two species.     

Assay and properties of rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase

An in vitro assay has been developed for the rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase). The subcellular location and some properties of the enzyme are described. 1,25-Dihydroxyvitamin D3 produced from incubations of yolk sac homogenates was extracted, purified by Sephadex LH-20 chromatography and straight- and reverse-phase high-performance liquid chromatography (HPLC), and measured by a competitive binding assay using chick intestinal receptor. The reaction is linear with time for up to 45 min at a substrate concentration of 80 microM and 4-6 mg/mL microsomal protein. The enzyme, located in the microsomes, requires molecular oxygen and NADPH. Metyrapone (1 X 10(-3) M) was found to inhibit 1-hydroxylation, but a 90% carbon monoxide-10% oxygen atmosphere did not, leaving open the question of involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, inhibited 1 alpha-hydroxylation.     

Metyrapone block of thyroid-induced cardiomegaly

Cardiac wet and dry weight, adrenal weights, blood pressure and food consumption were measured in control male rats, rats fed 0.2% dried thyroid powder in the food, rats fed metyrapone (an inhibitor of adrenocortical 11-hydroxylase, Metopirone CIBA, 50 mg/rat per day) and both metyparone + dried thyroid. Metyparone markedly inhibited thyroid-induced cardiomegaly as well as the increase in adrenal weight. Myocardial dry weight and blood pressure differences between the various groups were not significant. The results supply further evidence of the existence of a relationship between the adrenal cortex and thyroid-induced cardiomegaly. Metyrapone also completely inhibited the post-thyroid drop in body weight.     

Metyrapone in long-term management of Cushing's disease.

Metyrapone was used in the long-term management of 13 patients with pituitary-dependent bilateral adrenal hyperplasia (Cushing''s disease). The total length of treatment ranged from two to 66 months, with a mean of 21 months. The clinical features of the disease rapidly improved on metyrapone and this improvement was maintained. Although plasma ACTH concentrations rose in all patients, the increase was insufficient to overcome the adrenal blockade induced by the drug. Eight of the 13 patients had additional external pituitary irradiation as definitive treatment of their disease and one underwent a transfrontal hypophysectomy. Radiotherapy cured one patient, and after three years metyrapone was withdrawn. Slight hirsuties was noted in four of the seven women who received the drug for six months or more. A fifth woman had more severe hirsuties and this led to bilateral adrenalectomy. Other than hirsuties, side effects were few and the routine use of metyrapone is recommended as an adjunct to more definitive treatment in all patients who present with Cushing''s syndrome, irrespective of aetiology.     

The demethylation of guaiacol by a new bacterial cytochrome P-450

Spectroscopic studies were carried with a cytochrome P-450 in Moraxella sp., strain GU2, that could grow on guaiacol or 2-ethoxyphenol as the sole source of carbon and energy. The dissociation constant of the guaiacol-cytochrome complex was estimated to 0.15 microM, as determined in vivo or using the cell soluble extract. Cytochrome P-450 could also bind 2-ethoxyphenol, 2-propoxyphenol, and 2-butoxyphenol, and the dissociation constants have been determined in each case. Metyrapone depressed the degradation of guaiacol by whole bacteria, and was bound competitively to guaiacol with a constant of about 0.8 mM. Some catechol was excreted by the bacteria when growing on either guaiacol or 2-ethoxyphenol. Catechol and the other product of guaiacol demethylation, formaldehyde, were further oxidized by the bacteria. All the data available so far are consistent with cytochrome P-450 in Moraxella GU2 as a hydroxylase for the guaiacol side chain, behaving as a nonspecific O-dealkylase with broad specificity for guaiacol and homologous compounds with a longer carbon part in the side chain.     

Fate of juvenile hormone in mammalian cell culture.

The metabolism of juvenile hormone (JH) I has been examined in fetal mouse liver cells maintained in culture. Diffusion of the hormone into the cells appears to be passive. The hormone is metabolized essentially to organic-soluble metabolites (diol ester, diol acid and acid) by the action of epoxide hydrase and carboxylesterases. Conjugative reactions play a minor role, less than 3% of the hormone being excreted as conjugates (glucuronides, sulfates and mercapturic acid). About 0.8% of the cellular radioactivity is bound to macromolecules, mainly those of nuclear and mitochondrial origin. Metyrapone and SKF 525-A inhibit covalent binding of the hormone to cytoplasmic macromolecules, which suggests participation of the cytochrome P-450 system in covalent binding of the hormone.