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The effect of Amphenone B, an inhibitor of corticoid synthesis, on thyroxine (T4)-induced metamorphosis was studied in toad tadpoles kept in thiourea. Amphenone injections retarded T4-induced tail resorption markedly. The effect of Amphenone was nullified by aldosterone and corticosterone added to the water in which tadpoles were kept. Steroidogenic cells of adrenals in Amphenone-injected animals were enlarged markedly as compared with those in the saline-injected tadpoles or the Amphenone-injected tadpoles which were supplemented with corticoids. The results strongly suggest that endogenous corticoids act together with thyroid hormone to accelerate metamorphosis.

In short termexperimentsinthedog2-methyl–l,2-bis(3-pyridyl)-l-propanonc (SU 4885) in high dosage totally suppressed the secretion of steroids by the adrenal gland. In low dosage there was a shift from cortisol and corticosterone to 11-desoxycortisol (Reichstein's Compound S) and desoxycorticosterone suggesting a selective inhibition of hydroxylation at C11. In contrast amphenone suppressed total steroid secretion (i.e., of cortisol and corticosterone) with no evidence for a selective action on 11-hydroxylation.

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The effect of Amphenone B on development of renal hypertension was studied in rats whose kidneys were encapsulated with latex envelopes. The compound was administered in the diet for 13 weeks at dose levels of 0.1 and 0.3% by weight. Both dose levels of Amphenone B were equally effective in preventing rise of systolic blood pressure to the level of untreated, renal-encapsulated controls although both doses of the drug allowed modest rises of pressure to occur. Growth was inhibited by the drug. Amphenone B depressed thyroid activity as assessed by increased release rate of radioactive iodide from the thyroid gland; decreased resting metabolic rate; decreased growth; decreased food intake; and increased thyroid size. The protection against development of hypertension afforded by this compound may be related, at least in part, to its antithyroid activity.

SummaryWhen compared with appropriate controls, it appears as if rats treated with Amphenone “B” show a decrease in function of the hypertrophied thyroid gland as measured by oxygen consumption but a probable increase in cortisone-like function of the hypertrophied adrenal gland as measured by electroshock seizure threshold, thymus involution, reaction to 24-hour fast? and histological study of the adrenal.

Amphenone B was added in concentrations of 0.1 to 0.5% to a number of synthetic diets and to a fox chow diet. These were then fed to young male rats to determine the effects on adrenal weight and on the cholesterol content of the adrenals, liver, and plasma. The rats fed amphenone mixed with synthetic diets showed a greater increase in adrenal size and adrenal cholesterol than those fed amphenone mixed with the chow diet. The liver and plasma cholesterol values of rats fed amphenone on synthetic diets were also increased appreciably while those of rats fed amphenone in the chow diet were near normal. Synthetic diets containing erucic acid and amphenone appeared to have the greatest effect on adrenal size and cholesterol content but other synthetic diets in some cases caused a greater increase in liver and plasma cholesterol concentrations. An attempt to examine the effect of injected amphenone on cholesterol levels in rats fed different diets was limited by the fact that high doses of amphenone cause prolonged anesthesia.

SummarySuppression of corticosteroid production by inhibitors effected a tenfold increase in susceptibility of mice to death from endotoxin shock. Amphenone B and metyrapone caused some increase in susceptibility, but if given more than 1 hr before the endotoxin afforded protection to the test animals. Aminoglutethimide exerted a deleterious effect for at least 24 hr. The effect of the aminoglutethimide was reversed by pregnenolone, progesterone and deoxycorticosterone, although the LD50 remained lower than in the control animals. Corticosterone, cortisone and hydrocortisone afforded marked protection in both the endotoxin treated mice as well as the endotoxin-inhibitor treated mice. Andros-tenedione caused a decrease in the LD50 value in both the control and aminoglutethimidetreated mice. By contrast 17-a-hydroxyprogesterone afforded no protection against endotoxin in the control mice and a significant, but miniscule, degree of protection in the inhibitor-treated mice.This investigation was supported by ...

Die Hoden geschlechtsreifer Rattenbocke wurden ohne Vorbehandlung nach Hypophysektomie, nach Verabfolgung von Choriongonadotropin (Primogonyl) und von Amphenon B elektronenmikroskopisch untersucht. Das Cytoplasma normaler Zwischenzellen besitzt ein blasiges, glattes ER, zahlreiche Mitochondrien vom gemischten Typ mit elektronendichten Einschlussen, freie Ribosomen, einzelne Fetttropfen und eine typische, gut ausgebildete Golgizone. Das blaschenformige ER wird offenbar von den Golgihohlraumen abgeschnurt. Nach Hypophysektomie sind kaum Veranderungen wahrzunehmen. Nach Choriongonadotropin nimmt die Zahl und Grose der Blaschen zu, die Mitochondrien einzelner Zellen schwellen und die Mitochondrieneinschlusse verschwinden. Nach Amphenon B-Gabe geht das vesikulare ER in ein tubulares uber. Die Golgihohlraume zeigen keine Erweiterungen. Diese Veranderungen werden mit den biochemisch fasbaren Hemmungen oder Stimulierung einzelner Syntheseschritte des Testosterons in Zusammenhang gebracht.

AMPHENONE “B”3 (Allen and Corwin, 1951), a substituted desoxy- benzoin, given orally or subcutaneously, produces marked enlargement of rat thyroid and adrenal glands (Hertz, Allen and Tullner, 1950; Hertz, Tullner and Allen, 1951; Hogness, Williams and Lance, 1952). The thyroid enlargement is associated with reduction in I131 concentration (Hogness, Williams and Lance, 1952) and a lowered metabolic rate (Holtkamp, Heming and Sax, 1952). The thyromegalic effects are not found in hypophysectomized rats (Hertz, Allen and Tullner, 1950) or in those given supplementary thyroid extract (Hertz, Tullner and Allen, 1951). The adrenomegaly produced by Amphenone “B”is associated with increase in cholesterol concentration (Hogness et al., 1952) without evidence of decreased adrenal function (Hertz et al., 1951; Hogness et al., 1952; Heming et al., 1952). As in the case of the thyroid, adrenal enlargement is not found in hypophysectomized animals (Hertz et al., 1950) or in those given cortisone (Hertz et al., 1951).

We have investigated the effect of paraquat (methyl viologen) on lipid peroxidation in bovine adrenal cortex mitochondria. Incubation of a buffered aerobic mixture of mitochondria in the presence of Fe2+ or NADPH resulted in the formation of lipid peroxides whose accumulation could be followed at 532 nm as malondialdehyde. Fe2+ stimulates lipid peroxidation in normal mitochondria and those in which enzymes have been inactivated with heat. In contrast, NADPH has a stimulatory effect only in normal mitochondria, but not in heat-treated mitochondria. These results indicate that NADPH-dependent lipid peroxidation is an enzymatic process. Paraquat strongly inhibits this enzymatic lipid peroxidation, but has no effect on the non-enzymatic Fe2+-dependent process. The chemiluminescence that accompanies the NADPH-dependent lipid peroxidation is also markedly decreased in the presence of paraquat. Superoxide dismutase, which removes Superoxide anion efficiently, does not inhibit malondialdehyde production. The mechanism of the inhibition of the lipid peroxidation by paraquat has been examined. Paraquat has no effect on NADPH-2,6-dichlorophenolindophenol reductase and on NADPH-cytochrome c reductase activities in bovine adrenal cortex mitochondria. However, paraquat strongly inhibits the NADPH-dependent reduction of cytochrome P-450. These results suggest that the inhibitory effect of paraquat on NADPH-dependent lipid peroxidation in adrenal cortex mitochondria is due to a decrease in the level of reduced cytochrome P-450 probably by diverting electrons from cytochrome P-450. Cytochrome c, which can compete with P-450 for available electrons from adrenodoxin, like paraquat had an inhibitory effect on NADPH-dependent lipid peroxidation. Lipid peroxidation was also strongly inhibited by steroid hydroxylase inhibitors e.g., amphenone B, aminoglutethimide and metyrapone.

Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

In concert with studies of the effects of various pharmacologic inhibitors of corticosteroidogenesis on adrenocortical morphology, U-8113, an analog of amphenone B, was administered daily to Sprague-Dawley rats for 7, 14, 21 or 30 day. The primary morphological responses involved increases in adrenal weight, width of zona fasciculata, width of zona reticularis, intracellular lipids, mitochondrial size, mitochondrial vacuolation and crystalline-like inclusions, small coated vesicles, lysosomes, autophagic vacuoles and cholesterol ester clefts. In particular, the increases in lysosomes, coated vesicles and autophagic vacuoles containing morphologically altered mitochondria were considered reflective of mechanisms designed to maintain cellular integrity amidst functional impairment. Lipid analysis revealed marked increases in cholesterol esters and phospholipids, supportive of morphological observations. When permitted a 14 day recovery period following either 14 or 30 days of inhibitor therapy, most fine structural alterations and lipid derangements were diminished, and the cells approximated normal parameters.

PREVIOUS publications have described the chemical synthesis (Allen and Corwin, 1950) and some of the biological properties of Amphenone-B.* This substance has progestational activity in the rabbit (Allen, Hertz and Tullner, 1950) and has an anesthetic effect in the rat, rabbit and dog (Hertz, Allen, Tullner and Westfall, 1952). Amphenone-B also has folliculoid activity (Hertz, Allen and Tullner, 1950) in the castrated female rat. It has been reported, in addition, that the rat responds to the compound with a marked hyperplasia of the thyroid, a goitrogenic action which is probably related to its aromatic amino groups (Hertz et al., 1951). But of all the effects in its bizarre spectrum of endocrine action, the most interesting and unexpected is the profound hypertrophy of the adrenal glands. Just as the goitrogenic activity can be suppressed by the simultaneous administration of thyroxine, the hypertrophy of the adrenals can be suppressed by the administration of cortisone (Hertz, Tullner, and Allen, 1951).

Western painted turtles ( Chrysemis picta belli ) were adrenalectomized to show if adrenocortical steroids are necessary for electrolyte homeostasis. Two weeks after removal of the adrenal glands plasma Na had decreased by 13.7% whereas plasma K had increased by 22.1%. There were no significant changes in plasma Ca, Mg, or Cl. Plasma osmolarity increased by 4.4% while the blood hematocrit decreased by 31.9%. Muscle Na and Cl decreased by 27.1% and 26.7%, respectively, but there was no change in muscle K or in percentage of muscle water. Electrolyte changes observed 3 weeks after hypophysectomy were similar to those observed 2 weeks after adrenalectomy Treatment with Amphenone B or betamethasone was followed by some electrolyte changes, but injection of Metopirone failed to impair electrolyte balance. The only measurable response with aldactone was a significant increase in plasma calcium. Hepatic glycogen concentrations decreased after hypophysectomy and adrenalectomy and increased after treatment of intact turtles with betamethasone; Amphenone B had no effect.

SummaryAmphenone “B” induces a marked hypertrophy of both the thyroid and adrenal in the rat. Simultaneous administration of thyroxin and Amphenone “B” prevents the thyroid hypertrophy without reducing the adrenal enlargement. Combined cortisone and Amphenone “B” administration prevents the adrenal enlargement without altering the thyroid hypertrophy. Thus, Amphenone “B” exerts its adrenal and thyroid effect by acting through two distinct end—points in the anterior pituitary.

Summary(1) Enlargement of rat adrenal and thyroid glands following the administration of Amphenone “B”, observed by Hertz, Allen, and Tullner, was confirmed. (2) The adrenal enlargement was associated with an increase in adrenal cholesterol concentration. At the same time, the adrenals remained capable of secreting sufficient steroid to produce a marked fall in circulating eosinophiles. Amphenone “B” may exert its adrenomegalic effect by direct stimulation of the adenohypo-physis to produce ACTH rather than indirectly as seen, for example, in thiouracil-induced goiter. (3) In addition to thyroid enlargement, a marked decrease in I131 concentration in the gland was observed. This could be due to a thiouracil-type of action, although the effects noted within the time relationships of our experiments were similar to those produced by TSH and hence possibly the result of increased secretion of radioiodine rather than a decreased uptake. (4) Decrease in size of the testes was observed in animals given Amphenon...

A simple aerobic and anaerobic (N 2 plus TTC) perfusion technique was employed to study the intermediary metabolism of intact calf adrenals and the effect of disparate chemical and physical agents on their metabolic activity and steroidogenic function. The results demonstrated that Meyerhof-Embden and Krebs-Johnson carbohydrate intermediates were rapidly oxidized in contrast to amino and fatty acids. Studies with various selective enzyme inhibitors confirmed the role of the glycolytic and terminal aerobic pathways. The nature and intensity of the effects of these agents on the steroidogenic processes paralleled those on the metabolic activity with the striking exception of Amphenone B, which produced only a minor decrease in the oxidative metabolism and one of 75% in corticoid output. A 24-hr. incubation at 8 °C. and exposure to γ-radiation (1900–2700 r.) revealed a similar dissociation between their effects on the metabolic and steroidogenic activities of the adrenal. Thus, while a pronounced decline in metabolic activity was invariably associated with a diminution in biosynthetic function, the latter can be markedly depressed without a corresponding depression in the former.

The precise pathogenesis of ascites in cirrhosis of the liver remains obscure. Portal hypertension, 1 , 2 decreased plasma osmotic pressure due to hypoalbuminemia, 3 , 4 increased lymphatic exudation from the liver, 5 , 6 increase of antidiuretic hormone, 7 and increase of sodium-retaining adrenocortical hormone 8 are among the factors considered in the pathogenesis of ascites. The marked decrease of sodium excretion and the presence of increased amounts of aldosterone in the urine of patients with cirrhosis and ascites 9 point to this sodiumretaining hormone as the possible culprit in the pathogenesis of ascites. The complexity of the pathogenesis of ascites is demonstrated schematically in Figure 1. Amphenone B (3,3-bis[ p -dimethylaminophenyl]butanone-2-dihydrochloride) (Fig. 2) was synthesized by Allen and Curwin, 11 in 1950, and has been shown to have principally an inhibiting effect on thyroid and adreno-cortical function. 12,13 Among its other effects are a progesterone- or estrogen-like effect on the female genitalia, with uterine enlargement 12 and a depressing effect on the central