Aminoglutethimide Treatment Research Articles

Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals

In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

Hepatic effects of aminoglutethimide: A model aromatic amine

Primary aromatic amine drugs are structural alerts in drug development because of their association with a high incidence of idiosyncratic drug reactions (IDRs). If biomarkers could be found that predict IDR risk, it would have a major impact on drug development. Previous attempts to do this through screening of hepatic gene expression profiles in rodents treated with aromatic amine drugs found limited changes. Of the drugs studied, aminoglutethimide (AMG) induced the most changes, and this led to a more comprehensive study of its effects on the liver. Brown Norway rats treated with AMG for up to 14 days showed only a transient elevation of glutamate dehydrogenase. Pathway-specific PCR arrays found few AMG-induced gene changes associated with an immune response and, of these changes, the majority were involved with innate immunity such as Tlr2, Ticam2, CD14, and C3. AMG treatment also led to significant changes in the apoptosis and mitochondrial panel of genes. It was recently found that AMG does induce significant changes in the bone marrow of rats, and agranulocytosis is a common IDR caused by AMG. In contrast, liver injury is not a common IDR associated with AMG. Therefore, the liver may be able to effectively deal with AMG reactive metabolites, and changes observed in this study may be involved in adaptation. Myeloperoxidase is also known to be able to oxidize aromatic amines to reactive metabolites, and these observations suggest that metabolism outside of the liver may be important for the mechanism of aromatic amine-induced IDRs.

Effect of Aminoglutethimide on Neutrophils in Rats: Implications for Idiosyncratic Drug-Induced Blood Dyscrasias

Aminoglutethimide (AMG) is an aromatic amine aromatase inhibitor associated with a high incidence of idiosyncratic blood dyscrasias, especially agranulocytosis. Animal models of idiosyncratic drug reactions (IDRs) represent essential tools to study these reactions; however, there is currently no valid model of idiosyncratic drug-induced agranulocytosis. Although AMG does not cause agranulocytosis in most animals or humans, drugs associated with serious IDRs generally cause a higher incidence of mild reactions that resolve despite continued treatment. Therefore, the effects of AMG on neutrophils and bone marrow in rats were studied to understand the mechanisms of more serious IDRs. An increase in peripheral blood neutrophils occurred as early as 24 h after AMG treatment with minimal changes to the total leukocyte count. Further investigation using 5-bromo-2'-deoxyuridine (BrdU) found an increased release of neutrophils from the bone marrow. Histologically, this corresponded to an increase in myeloid cells in the bone marrow, which was confirmed by differential staining with CD45 and CD71. AMG treatment stimulated an increase in colony forming unit granulocyte-macrophage and colony forming unit granulocyte ex vivo. There was also a marked increase in the number of activated neutrophils in the circulation expressing the extravasation marker CD62L. These findings indicate that AMG affects neutrophil production, release, and function. Similar effects on neutrophil kinetics in clozapine-treated rats have previously been found, and transient neutrophilia has been observed in patients taking other drugs associated with idiosyncratic agranulocytosis; therefore, the changes observed with AMG may be biomarkers to predict the risk that a drug will cause agranulocytosis.

The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis

Cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone. Cyp11a1 expression is increased in activated T cells. We sought to determine the role of Cyp11a1 activation in the development of peanut allergy and TH cell functional differentiation. A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on T(H)1, T(H)2, and T(H)17 differentiation were determined. Cyp11a1 gene silencing was performed with Cyp11a1-targeted short hairpin RNA. Peanut sensitization and challenge resulted in diarrhea, inflammation, and increased levels of Cyp11a1, IL13, and IL17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG treatment decreased IL13 and IL17A mRNA expression in the small intestine without affecting Cyp11a1 mRNA or protein levels. In vitro the inhibitor decreased IL13 and IL17A mRNA and protein levels in differentiated T(H)2 and T(H)17 CD4 T cells, respectively, without affecting GATA3, retinoic acid-related orphan receptor γt (RORγt), or T(H)1 cells and IFNG and T-bet expression. Short hairpin RNA-mediated silencing of Cyp11a1 in polarized T(H)2 CD4 T cells significantly decreased pregnenolone and IL13 mRNA and protein levels. Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T(H)2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.

Acute Stress-Induced Facilitation of the Hypothalamic-Pituitary-Adrenal Axis: Evidence for the Roles of Stressor Duration and Serotonin

Stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis stimulates the release of both facilitatory and inhibitory components. We proposed that the transient removal of the inhibitory component, corticosterone, during a stressor would leave the HPA axis in a state of hyper-responsiveness (facilitated state). Consistent with this expectation, we have previously observed that aminoglutethimide (AG)-induced removal of corticosterone during an immobilization stressor resulted in the hypersecretion of both ACTH and corticosterone to a subsequent stressor. In the present study we determined the effect of stressor duration on the magnitude of facilitation. AG plus a 10-min immobilization (IMM₁₀) stress on day 1 resulted in facilitation of the HPA axis. This was reflected in higher ACTH and corticosterone responses to an injection stress on day 2 as compared to appropriate control rats. AG plus a 60-min immobilization (IMM₆₀) stress on day 1 resulted in significantly greater facilitation as compared to the AG+IMM₁₀ pretreatment. It is apparent that facilitation of the HPA axis is dependent on the duration of stress. Stress can alter plasma corticosterone-binding globulin levels and AG administration can cause accumulation of the corticosterone biosynthetic precursor, adrenal cholesterol. In order to rule out these peripheral reasons for the hypersecretion of ACTH and corticosterone in our paradigm, we measured the plasma free fraction of corticosterone and adrenal mitochondrial cholesterol levels on day 2 after different pretreatments on day 1. AG+IMM₆₀ pretreatment caused a significant increase in the plasma free fraction of corticosterone. Hypersecretion of ACTH and corticosterone in this group, despite an enhanced feedback signal, suggests central loci for the origin of facilitation. Also, AG treatment on day 1 did not result in accumulation of free or esterified adrenal cholesterol levels on day 2, and therefore cannot account for the hypersecretion of corticosterone. In our final study we attempted to determine if serotonin released during the first stressor is partially responsible for stress-induced facilitation of the HPA axis. We administered 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a 5HT_1A agonist, either alone or in conjunction with stress and examined the effects of these pretreatments on the magnitude of facilitation. Interestingly, DPAT administered in lieu of stress produced facilitation similar in magnitude to that produced by IMM₁₀. DPAT adminsitered in conjunction with IMM₁₀ augmented stress-induced facilitation. Our results suggest that stress-induced facilitation of the HPA axis is associated with the release of serotonin during stress.

Aminoglutethimide, a Steroidogenesis Inhibitor, Abolishes Hormonal Induction of Ornithine Decarboxylase in Steroidogenic Tissues: Evidence for Its Role as cAMP-Dependent Protein Kinase Inhibitor

Aminoglutethimide (AMG), a potent inhibitor of steroidogenesis used in the treatment of breast cancer and some adrenal pathologies, abolished the induction of ornithine decarboxylase (ODC) elicited by peptide hormones and by dibutyryl-cAMP in steroidogenic tissues. This effect seems to be related to an inhibition of cAMP-dependent protein kinase (IC50 = 287 μM) rather than blockade of the steroidogenic pathway. This inhibition may explain some of the effects observed in AMG treatment which cannot be ascribed to its direct effect on the cytochrome P450scc complex or aromatase. Taking into account that ODC, the rate-limiting enzyme in polyamine synthesis, is elevated in many types of cancer and that overexpression of this enzyme is associated with cell transformation, one may speculate that the inhibitory action of AMG on protein kinase A represents a positive colateral effect of this drug in cancer therapy.

Hormones increase mRNA of cyclic-nucleotide-gated cation channels in airway epithelia

Previous studies have shown that the mRNA of cyclic-nucleotide-gated nonselective cation (CNG) channels is expressed in rat airway epithelia and that these channels contribute to sodium-mediated short-circuit currents in cultured rat tracheal epithelia. Patch-clamp studies from human A549 cells indicate that these channels contribute to cGMP-stimulated L-cis-diltiazem- and dichlorobenzamil-inhibited whole-cell sodium currents. This study demonstrates that mRNA for primary and secondary subunits of CNG channels, halphaCNG1 and hbetaCNG1 respectively, are expressed in several human airway cell lines, including normal and cystic fibrosis bronchial airway cells, in normal and cystic fibrosis tracheal airway cell lines and nasal polyp tissue from a cystic fibrosis patient. The mRNA of ralphaCNG1 in rat lung increased in response to increased circulating glucocorticoids and decreased in animals with lowered circulating glucocorticoids after aminoglutethimide treatment. Likewise the mRNA of halphaCNG1 increased in the presence of glucocorticoids in cultured alveolar airway cells. The mRNA of alphaCNG1 in rat lung was also increased in response to a low-salt diet and lowered in animals fed a high-salt diet. Likewise the mRNA of alphaCNG1 was increased in response to increased aldosterone and decreased in animals given spironolactone. These results suggest that mRNA for alphaCNG1 increases in response to elevated glucocorticoids or mineralocorticoids. Because alphaCNG1 is a functional sodium entry channel in both rat and human airway epithelial cells, if channel protein is also elevated this channel could mediate an increase in sodium absorption across lung epithelia in response to circulating hormones.

Estrogen-mediated mitochondrial cholesterol transport and metabolism to pregnenolone in the rabbit luteinized ovary.

We investigated the mechanisms of luteotropic actions of estradiol on steroidogenesis. To this end, we examined, in vitro, the metabolism of cholesterol from endogenous or exogenous sources for pregnenolone production in rabbit luteinized ovarian cell mitochondria isolated from pseudopregnant animals in various states of stimulation by estradiol. We found that estradiol-mediated regulation of mitochondrial cholesterol metabolism for pregnenolone production differs from the mechanisms of regulation reported for steroidogenic protein/polypeptide hormones in the following respects: 1) in the estradiol-sensitive, luteinized-ovary, rabbit model, temporary blockage of cytochrome P-450 cholesterol side-chain cleavage enzyme by aminoglutethimide treatment in vivo has no effect on mitochondrial pregnenolone production in vitro after the aminoglutethimide is removed, indicating no additional capacity for upstream cholesterol storage; 2) preincubating mitochondria at 37 degrees C fails to increase subsequent pregnenolone synthesis in response to the addition of isocitrate; and 3) exogenously added cholesterol does not readily enter the steroidogenic pool of cholesterol unless the endogenous cholesterol pool is first depleted. These new observations indicate that estradiol increases the usable steroidogenic cholesterol pool in rabbit ovarian mitochondria. Also, 1) they are consistent with a putative requirement for the participation of one or more estrogen-sensitive protein factors to enhance cholesterol trafficking to the inner mitochondrial membrane, and 2) they complement the observation of estrogen-dependent expression of steroidogenic acute regulatory protein in rabbit luteal tissue.

Viscosity of rat adrenocortical lipids in different functional states: morphological characteristics.

Impaired adrenocortical steroidogenic activity is often concomitant with morphologically and physiologically altered lipids in the cells of the adrenal cortex. The physical state of these lipid droplets and the morphological characteristics of crystal-shaped bodies were studied in different functional states of adrenocortical cells. In the perinatal period when steroidogenesis is suppressed by a negative feedback mechanism, crystal-shaped bodies (i.e. rectangular, electron-lucent formations, either alone or in clusters, surrounded by lysosomal matrix or in close proximity of lysosomes) were frequently observed in the inner zona fasciculata and zona reticularis. In experimentally suppressed adrenocortical activity, following the administration of dexamethasone, aminoglutethimide or cycloheximide, almost identical crystal-shaped bodies were frequently observed in adrenocortical cells. These crystal-shaped bodies appear to be cholesterol, as revealed by the digitonin reaction at the electron microscopic level. Following stimulation of the zona fasciculata by ACTH treatment for 14 days, a marked increase in the fluidity of the lipid droplets was observed in the thermotropic phase transitions with the polarizing microscope. In contrast, following aminoglutethimide treatment, the fluidity of the lipid droplets decreased. The thermotropic phase transitions of normal and neoplastic human adrenal cells, namely adrenocortical tumours causing Conn's or Cushing's syndrome, were also investigated. When hormone biosynthesis was enhanced, the appearance of birefringence and multiple phase transitions of lipid droplets was demonstrable in the low-temperature range.

Serum elimination half-life of tamoxifen and its metabolites in patients with advanced breast cancer.

In breast cancer patients discontinuing chronic tamoxifen therapy, the serum elimination of metabolites X, Y and E paralleled that of tamoxifen, whereas that of metabolite Z did not. The serum elimination of tamoxifen and metabolites X and B was increased by amino-glutethimide treatment, whereas that of metabolites Z, Y, and E was not.

Relationship between apolipoprotein E mRNA expression and tissue cholesterol content in rat adrenal gland.

Among extrahepatic tissues the adrenal gland has one of the highest concentrations of apoE mRNA and the highest rate of apoE synthesis. In the present investigation several previously described in vivo treatments were used to assess the relationship between apoE expression and cellular cholesterol in the rat adrenal gland. Treatment of rats with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) to lower serum cholesterol concentration and deplete adrenal gland cholesterol content decreased adrenal gland apoE mRNA concentration. These adrenal responses were blocked by dexamethasone (DEX) suggesting that the effect of 4-APP occurred indirectly via stimulation of the adrenal gland by endogenous adrenocorticotrophic (ACTH). Relative to control rats, DEX treatment increased both adrenal gland cholesterol content and apoE mRNA concentration. Concurrent ACTH and DEX administration reduced both adrenal gland cholesterol content and apoE mRNA concentration relative to DEX-treated rats. ACTH administration also rapidly decreased adrenal gland apoE mRNA concentration and cholesterol content in rats pretreated with DEX. In all the above experiments, adrenal gland cholesterol content and apoE mRNA concentration were positively correlated (r = 0.78, P = 0.0001). In contrast, aminoglutethimide treatment, which blocks adrenal gland steroidogenesis and greatly increases adrenal gland cholesterol content, was without effect on apoE mRNA concentration. ACTH administration to rats treated with DEX + aminoglutethimide resulted in decreased adrenal apoE mRNA despite greatly increased adrenal cholesterol content. This uncoupling of adrenal gland cholesterol content and apoE mRNA concentration suggests that apoE mRNA expression and cellular cholesterol are regulated independently by ACTH.

Evidence That Hydrogen Peroxide Blocks Hormone- Sensitive Cholesterol Transport into Mitochondria of Rat Luteal Cells*

In luteal and granulosa cells, hydrogen peroxide abruptly inhibits activation of adenylate cyclase by receptor-bound gonadotropin and blocks steroidogenesis. In the present studies a post-cAMP site of peroxide action on inhibition of steroidogenesis was investigated. Steroidogenesis, stimulated by dibutyryl or 8-bromo-cAMP, was inhibited by hydrogen peroxide. Yet, cAMP-dependent protein kinase activation in cytosol or intact cells was unaffected by peroxide treatment. Hydrogen peroxide also did not inhibit the activity of cholesterol esterase and acyl coenzyme-A:acyltransferase. Progesterone synthesis was maximally increased 5- to 50-fold with 25- and 22-hydroxycholesterol, respectively. Unlike that seen with cAMP analogs and LH, however, progestin synthesis stimulated by these cell- and mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide. Treatment of animals with amino-glutethimide produces a marked accumulation of steroidogenic cholesterol substrate and a large increase in hormone-independent steroidogenesis in subsequently isolated and washed luteal tissue. In this paradigm, hydrogen peroxide did not inhibit elevated basal progesterone synthesis in luteal cells produced by in vivo aminoglutethimide treatment, yet LH-stimulated steroidogenesis was blocked. However, treatment of luteal cells with hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, an effect partially reversed by the addition of luteal cell cytosol. In summary, while peroxide inhibited cAMP-dependent steroidogenesis, it did not appear to inhibit protein kinase activation or mobilization of cholesterol from intracellular esterified stores. Although peroxide inhibited pregnenolone synthesis, it had no effect on steroidogenesis when substrate was made available by either addition of cholesterol analogs or prior treatment with aminoglutethimide in vivo. We conclude, therefore, that hydrogen peroxide inhibits steroidogenesis by blocking intracellular transport of cholesterol to mitochondria or translocation of cholesterol across the outer mitochondrial membrane.

Mechanism for Control of HydroxymethylglutaryL Coenzyme A Reductase and Cytochrome P-450 Side Chain Cleavage Message and Enzyme in the Corpus Luteum*

The present study was undertaken to investigate the mechanism for control of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and cytochrome P-450 side chain cleavage (P-450scc), enzymes involved in the synthesis and processing of cholesterol in the corpus luteum of pregnant rats. The role of sterol and estradiol were investigated by administering 4-aminopyrazolo-[3,4d]pyrimidine (4-APP), aminoglutethimide and/or estradiol to day 12 hypophysectomized-hysterectomized pregnant rats. Estradiol treatment markedly increased mRNA levels of HMG-CoA reductase in the corpus luteum. This stimulatory effect of estradiol was specific for the reductase since the mRNA for luteal P-450scc did not increase following estradiol treatment. To determine whether estradiol's action on HMG-CoA reductase mRNA was mediated by changes in cellular free cholesterol, the separate and combined action of estradiol and either 4-APP or aminoglutethimide on both sterol content and HMG-CoA reductase expression was examined. The estradiol induced rise in HMG-CoA reductase was not accompanied by a decrease in luteal cholesterol. 4-APP treatment depleted cholesterol in the corpus luteum and induced a greater increase in HMG-CoA reductase mRNA than estradiol. Combined treatment with estradiol and 4-APP resulted in a synergistic increase in HMG-CoA reductase mRNA despite the fact that estradiol did not further reduce the 4-APP induced decrease in luteal sterol content. This indicates that luteal cells possess an estradiol-mediated mechanism for up-regulation of HMG-CoA reductase gene expression which is distinct from the cholesterol negative feedback regulation. However, estradiol stimulation of HMG-CoA reductase was totally inhibited when sterol content was elevated by aminoglutethimide, suggesting that estradiol stimulation of HMG-CoA reductase expression is not sufficient to overcome the negative regulatory effect of cholesterol. In contrast to HMG-CoA reductase mRNA which appears to be controlled by sterol and estradiol, P-450scc mRNA levels remained constant in corpora lutea despite wide changes in sterol and steroid content induced by either 4-APP, aminoglutethimide or estradiol treatment. However, interestingly, aminoglutethimide increased substantially the content of P-450scc protein. This increase in P-450scc enzyme was not due to a greater amount of translatable message since similar levels of immunoprecipitable 35S-P-450scc were translated in vitro from luteal mRNA of rats treated with or without aminoglutethimide. In summary, results of this investigation have revealed that the expression of HMG-CoA reductase and cytochrome P-450scc are controlled by totally different mechanisms in the rat corpus luteum.(ABSTRACT TRUNCATED AT 400 WORDS)

Alterations in the urine excretion of estrogen metabolites in breast cancer women treated with aminoglutethimide

The effect of aminoglutethimide treatment on urine estrogen glucuronide excretion was investigated using injections of [4- 14C]estradiol (4 women) or [4- 14Cestrone (2 women). Each patient received 25 μCi of either [4- 14C]estradiol or [4- 14C]estrone as a bolus injection before initiation of aminoglutethimide treatment, and an equal injection following 3–20 weeks on treatment with aminoglutethimide 250 mg q.i.d. with hydrocortisone (50 mg b.i.d. for 2 weeks, then 25 mg b.i.d.). Urine was collected for 24–72 h following each injection. Aminoglutethimide treatment caused significant alterations in the metabolite profiles of estradiol and estrone but with large interindividual variations. [ 14C]Estriol glucuronide excretion was increased by a median value of 48.6%. [ 14C]16α-Hydroxyestrone glucuronide and [ 14C]16-epi-estriol glucuronide excretion was increased by median values of 16.3 and 37.7% respectively, and [ 14C]2-hydroxyestriol glucuronide excretion was increased by a median value of 115.9%. Contrary, excretion of the catechol estrogen glucuronides (2- and 4-hydroxylated metabolites) were reduced (mean reduction of 14.8 and 67.3% respectively). The amount of urine radioactivity excreted as [ 14C]estradiol and [ 14C]estrone glucuronide were consistently reduced by aminoglutethimide treatment (median reduction of 36.8 and 38.2% respectively). These findings suggest aminoglutethimide to stimulate the estrone 16α-hydroxylase and possibly the estrone 16β-hydroxylase located in the hepatic endoplasmic reticulum.

Effects of aminoglutethimide on plasma estrone sulfate not caused by aromatase inhibition

Plasma levels of estrone and estrone sulfate were measured in 16 postmenopausal women with advanced breast cancer before and during chronic aminoglutethimide therapy. Aminoglutethimide caused a significant reduction in plasma estrone (mean 36%, P < 0.025) and estrone sulfate (mean 65%, P < 0.001). The estrone/estrone sulfate ratio was increased by a mean of 84% ( P < 0.0025). These results suggest that aminoglutethimide influences plasma estrone sulfate by mechanisms unrelated to aromatase inhibition. The findings in this study are consistent with previous results suggesting that aminoglutethimide treatment enhances the rate of estrone sulfate metabolism. This biochemical effect of aminoglutethimide treatment could be a contributory factor to the drugs mechanism of action.

Alterations in the production rate and the metabolism of oestrone and oestrone sulphate in breast cancer patients treated with aminoglutethimide

Plasma level, plasma clearance, production rate and interconversions of oestrone and oestrone sulphate were measured in six breast cancer patients receiving aminoglutethimide therapy. Three additional patients had the production rate of oestrone sulphate investigated. Plasma oestrone and oestrone sulphate levels were reduced by a mean of 46% (P less than 0.05) and 71% (P less than 0.005) respectively. These alterations were due to a combined action of aminoglutethimide inhibiting oestrogen production but also increasing oestrogen metabolism. While oestrone and oestrone sulphate production rate was reduced by a mean of 31% (P less than 0.05) and 41% (P less than 0.005) respectively, the plasma clearance rate of oestrone was found to be increased by a mean of 30% (P less than 0.05), and the plasma clearance rate of oestrone sulphate was increased by a mean of 112% during aminoglutethimide treatment. The fraction of oestrone sulphate converted into plasma oestrone was reduced by 52% (P less than 0.05), the transfer of circulating oestrone into sulphate was non-significantly reduced by a mean of 16%. The findings in this investigation show that aminoglutethimide treatment influences oestrogen disposition by mechanisms unrelated to aromatase inhibition. The possibility that such effects might be partly responsible for the mechanism of action of aminoglutethimide in advanced breast cancer should be considered.

In vivo influence of androgens on the cell kinetics and chromatin pattern of the MXT mouse mammary tumor treated or not by aminogluthetimide

We characterized the influence of androstenedione, 5-alpha-dihydrotestosterone and 17-beta-estradiol on the chromatin organization and the cell kinetics (distribution of the cells into the G0-G1, S and G2+ M fractions) of MXT mouse mammary tumors grafted onto animals that were left intact or that were oophorectomized and/or treated with aminoglutethimide. The cell kinetics and chromatin pattern were monitored by analyzing Feulgen-stained MXT imprint smears through a cell image processor, the SAMBA 200 system. We have also assayed the estrogen and progesterone receptors of these MXT tumors, which appeared to possess both of these biological markers. Our results show that ovariectomy or aminoglutethimide treatment slowed down the MXT tumor growth without any additive effect between them. Androstenedione exerted a stimulating influence on the cell kinetics, which were very similar to those observed after estradiol administration; the treatment of castrated animals with aminoglutethimide completely abolished this androstenedione-induced stimulating influence, however. This androgen lacked any apparent efficiency to transform cell nuclei from a state of castration-induced chromatin condensation into a thinly textured and decondensed state, as did estradiol. Dihydrotestosterone was able to activate the cell kinetics of MXT tumor grafted onto castrated animals as well as those of MXT neoplasms grafted on oophorectomized mice treated with aminoglutethimide. Dihydrotestosterone also possesses the property to transform condensed chromatin into decondensed chromatin. It thus appeared that androstenedione and dihydrotestosterone could activate MXT cell proliferation, as did estradiol, although it appeared that their mechanisms of action were quite distinct from each other.

Relationship between blood flow and steroidogenesis in the rabbit corpus luteum.

Blood flow in the corpus luteum of the pseudopregnant rabbit was measured with tracer-labelled microspheres before and at 1 and 3 h after saline treatment (N = 8) or after inhibition of progesterone synthesis with aminoglutethimide (N = 10). Before treatment luteal blood flow (29.5 +/- 3.9 ml/min.g-1 (mean +/- s.e.m.] was much higher than blood flow to other tissues (ovarian stroma = 2.9 +/- 0.6; uterus = 0.5 +/- 0.1; adrenal gland = 2.6 +/- 0.2 ml/min.g-1). Aminoglutethimide reduced serum progesterone by 60% within 1 h but luteal blood flow was unchanged (26.2 +/- 3.5 ml/min.g-1). At 3 h after aminoglutethimide, serum progesterone remained low and luteal blood flow was slightly reduced to 22.5 +/- 3.4 ml/min.g-1. This reduction was associated with a significant decline in mean arterial blood pressure which resulted in luteal vascular resistance being unaltered by aminoglutethimide treatment. Further analysis of these data indicated that serum progesterone concentration was not significantly correlated with blood flow to the corpora lutea or with blood flow to other tissues. In contrast, mean arterial blood pressure was highly correlated with blood flow to the corpus luteum (r = 0.80; P less than 0.001) but not to the ovarian stroma (r = 0.04), or adrenal gland (r = 0.06). These results indicate that luteal blood flow is not acutely responsive to changes in luteal progesterone production and suggest that luteal blood flow changes passively with changes in arterial blood pressure.

Aminoglutethimide for advanced prostatic cancer resistant to conventional hormonal therapy.

Aminoglutethimide (AG) and hydrocortisone (HC) were given to 20 patients with advanced prostatic cancer resistant to conventional hormonal therapy. Most patients had painful bone metastases and were heavily pretreated. 12 of 16 patients required narcotic analgetics. 8 of 20 were bedridden. AG + HC produced relief of bone pain in 12 patients (75%) and only 4 required narcotics after treatment. The performance status improved in 8 of 20 patients (40%). However, the number of bone metastases seen in bone scans decreased in only 4 patients (22%). The level of serum alkaline phosphatase decreased in 11 of 18 patients and that of acid phosphatase in 8 of 16 patients. The reduction of bone pain lasted approximately 4 months (range 1-15 months). The median lifespan between the start of AG treatment and death was 8 months (range 2-22 months). There was no difference in survival between responders and nonresponders. 3 patients had skin rash, 1 lethargy and 1 thrombocytopenia.

Alterations in the metabolism of oestrogens during treatment with aminoglutethimide in breast cancer patients. Preliminary findings.

In this small study, the effect of aminoglutethimide on the disposition of oestrogens in women with advanced breast cancer was investigated using bolus injections of 4-[14C]-oestradiol and 6,7-[3H]-oestrone sulphate, alone or in combination. No alterations in oestrogen disposition were seen after short term (6 hours) aminoglutethimide administration. During long term (3 weeks to 8 months) aminoglutethimide treatment mean 4-[14C]-oestradiol clearance was not changed. 14C-Oestrone sulphate AUC was reduced by 43% at a low dose of aminoglutethimide (125 mg twice daily) and by 65% at a high dose (250 mg 4 times daily) with hydrocortisone acetate 25 mg twice daily. The oestrone sulphate terminal elimination rate constant (lambda z) was concurrently increased (mean of 46 and 79%, respectively, with the 2 dosage regimens). A possible increase in oestrone sulphate clearance during long term treatment was tested for by injecting 6,7-[3H]-oestrone sulphate. These studies revealed a marked increase (mean 104%) in oestrone sulphate clearance in patients receiving the high dose aminoglutethimide schedule. Following injection of 4-[14C]-oestradiol plus 6,7-[3H]-oestrone sulphate, the fraction of 4-[14C]-oestradiol metabolised to oestrone sulphate was found to be reduced in all patients (mean 13%). A mean increase of 80% in the urinary excretion of 14C-oestriol was observed after 4-[14C]-oestradiol administration. Our results, although preliminary, suggest that aminoglutethimide is a potent inducer of aminoglutethimide metabolism, thereby producing a significant reduction in plasma bioavailability of oestrone sulphate. These effects may have a role in the action of aminoglutethimide, a finding which warrants further investigation.