Spironolactone and XPB: An Old Drug with a New Molecular Target.

Spironolactone (SP) is commonly used for the treatment of heart failure, hypertension, and complications of cirrhosis by antagonizing the mineralocorticoid receptor. However, SP also antagonizes the androgen receptor, and thus SP has also been shown to be effective in the treatment of acne, hair loss, and hirsutism in women. Interestingly, recent drug repurposing screens have identified new and diverse functions for SP as a simulator of tumor immunosurveillance and as an inhibitor of DNA repair and viral infection. These novel pharmacological effects of SP have all been linked to the ability of SP to induce the rapid proteolytic degradation of the xeroderma pigmentosum group B (XPB) protein. XPB is a critical enzymatic component of the multi-subunit complex known as transcription factor II-H (TFIIH), which plays essential roles in both DNA repair and the initiation of transcription. Given the critical functions for XPB and TFIIH in these processes, the loss of XPB by SP could lead to mutagenesis. However, the ability of SP to promote cancer stem cell death and facilitate immune recognition may counteract the negative consequences of SP to mitigate carcinogenic risk. Thus, SP appears to have new and interesting pharmacological effects that may extend its potential uses.

Ocular Biodistribution of Spironolactone after a Single Intravitreal Injection of a Biodegradable Sustained-Release Polymer in Rats.

Sustained-release formulations for ocular delivery are of increasing interest given their potential to significantly improve treatment efficacy and patient adherence. The objectives of this study were (i) to develop a sustained-release formulation of spironolactone (SPL) using a biodegradable and injectable polymer, hexyl-substituted poly-lactic acid (hexPLA) and (ii) to investigate the ocular biodistribution and tolerability of SPL and its metabolites in rats in vivo over 1 month following a single intravitreal injection (IVT inj). The concentrations of SPL and its two principal active metabolites, 7α-thiomethylspironolactone and canrenone (CAN), in the different ocular compartments were determined at different time points (3, 7, and 31 days after IVT inj) using a validated ultra-high-performance liquid chromatography-mass spectrometry method. Systemic exposure following a single IVT inj of 5% SPL-hexPLA formulation was evaluated by quantifying SPL and its metabolites in the plasma. Ocular tolerability of the formulation was evaluated using in vivo retinal imaging and histology. In vitro release studies revealed a sustained release of SPL from 5% SPL-hexPLA for up to 65 days. In vivo studies showed that SPL and its metabolites were detected in all ocular tissues at 3 and 7 days post-IVT inj. At 31 days post-IVT inj, SPL and CAN were mainly detected in the retina. These results also highlighted the clearance pathway of SPL and its metabolite involving the anterior and posterior routes in the first week (days 3 and 7), then mainly the posterior segment in the last week (day 31). This study showed that a single IVT inj of 5% SPL-hexPLA in rats enabled sustained delivery of therapeutic amounts of SPL for up to 1 month to the retina without systemic exposure. This formulation may be of interest for the local treatment of diseases involving overactivation of the mineralocorticoid receptor in the chorioretina such as chronic central serous chorioretinopathy.

Synthesis, bioactivity and mode of action of 5A 5B 6C tricyclic spirolactones as novel antiviral lead compounds.

BACKGROUND: Plant viral diseases cause tremendous decreases in yield and quality. Natural polycyclic compounds such as those containing carbocycles are often very important lead compounds for drug and pesticide development. Tricyclic spiranoid lactones with 5A 5B 6C -ring fusion topologies possess various bioactivities. In this study, 33 new 5A 5B 6C tricyclic spirolactones were rationally designed, synthesized, characterized and evaluated for antiviral activities. RESULT: These compounds showed no apparent toxicity against Italian honeybees up to 2.73 µg bee-1 . Spirolactones 14, 16, 19, 23 and 28 at a concentration of 100 µg mL-1 inactivated 90% of tobacco mosaic virus (TMV) infection, making these compounds much more potent than the positive controls. Significantly, compound 19 displayed the best inactivation activity causing inhibition of up to 98%. CONCLUSION: The results of the bioassays and QSAR studies indicated that the carbon-containing cyclic moiety was the antiviral pharmacophore, and derivative 19, which showed the best inactivation activity, could emerge as a potential antiviral agent against TMV. In vitro capsid protein (CP) assembly and TMV assembly inhibition determinations indicated that these compounds induced crosslinking in the TMV and prevented its uncoating, which was a putative new mode of action for TMV inactivation. © 2018 Society of Chemical Industry.

Spironolactone Depletes the XPB Protein and Inhibits DNA Damage Responses in UVB-Irradiated Human Skin.

UVB wavelengths of light induce the formation of photoproducts in genomic DNA that are potentially mutagenic and detrimental to epidermal cell function. The mineralocorticoid and androgen receptor antagonist spironolactone (SP) was recently identified as an inhibitor of UV photoproduct removal in human cancer cells in vitro via its ability to promote the rapid proteolytic degradation of the DNA repair protein XPB. Using normal human keratinocytes in vitro and skin explants ex vivo, we found that SP rapidly depleted XPB protein in both systems and abrogated two major responses to UVB-induced DNA damage, including the removal of UV photoproducts from genomic DNA and the activation of ATR/ATM DNA damage kinase signaling. These effects were also correlated with both mutagenesis and a predisposition to UVB-induced cell death but were unique to SP, because neither the SP metabolites canrenone and 7α-thiomethylspironolactone nor the more specific mineralocorticoid receptor antagonist eplerenone affected XPB protein levels or the UVB response. Our findings provide an approach for studying XPB and its roles in the UVB DNA damage response in human skin ex vivo and indicate that SP may increase UVB mutagenesis and skin cancer risk in certain individuals.

Colectomy induces an aldosterone-mediated increase in jejunal glucose uptake in rats.

AIMS: The main function of the colon is water and electrolyte absorption. Total colectomy eliminates this colonic function and may alter the absorptive capacity of the small intestine for nutrients. This study examines the effect of total colectomy on jejunal glucose absorption and investigates the potential role of aldosterone in mediating the alterations in glucose uptake post-colectomy using the aldosterone antagonist spironolactone. MAIN METHODS: Total colectomy with ileo-rectal anastomosis was performed on anesthetized rats. Sham rats were identically handled without colon resection. Two days post-surgery, groups of colectomized rats were injected with either a daily subcutaneous dose of spironolactone or sesame oil for 12days. Body weight changes and food and water intake were measured in all experimental groups. Glucose absorption was measured by in-vivo single pass perfusion in the rat jejunum of control, sham, colectomized, colectomized with spironolactone, and colectomized with sesame oil treatment. Na/K ATPase, SGK1, SGLT1 and GLUT2 expressions were determined in jejunal mucosa in control, colectomized and colectomized/spironolactone injected rats by Western blot analysis. Histological assessment was performed on jejunal sections in control and colectomized groups. KEY FINDINGS: Glucose absorption significantly increased in colectomized rats with an observed increase in Na/K ATPase and SGK1 expression. No significant expression change in SGLT1 and GLUT2 was detected in the jejunum in colectomized rats. Spironolactone, however, significantly decreased the glucose uptake post-colectomy and normalized Na/K ATPase and SGK1 expression. SIGNIFICANCE: Our results suggest that jejunal glucose uptake increases post-colectomy as a possible consequence of an aldosterone-mediated function.

Metabolomics for informing adverse outcome pathways: Androgen receptor activation and the pharmaceutical spironolactone.

One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone's activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC-MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50µgL-1) for 21days. Metabolite profiles were affected at the two highest concentrations (5 and 50µgL-1), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17ß-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., l-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected amino acid, tryptophan, and fatty acid metabolism. Thus, by identifying possible key events related to changes in biochemical pathways, this approach built upon an established AOP describing spironolactone's androgenic properties and highlighted broader implications potentially unrelated to androgen receptor activation, which could form a basis for the development of an AOP network.

Effects of early aldosterone antagonism on cardiac remodeling in rats with aortic stenosis-induced pressure overload.

UNLABELLED: Aldosterone plays a pivotal role in the pathophysiology of systolic heart failure. However, whether early aldosterone antagonism improves cardiac remodeling during persistent pressure overload is unsettled. We evaluated the effects of aldosterone antagonist spironolactone on cardiac remodeling in rats with ascending aortic stenosis (AS). METHODS: Three days after inducing AS, weaning rats were randomized to receive spironolactone (AS-SPR, 20mg/kg/day) or no drug (AS) for 18weeks, and compared with sham-operated rats. Myocardial function was studied in isolated left ventricular (LV) papillary muscles. STATISTICAL ANALYSES: ANOVA or Kruskal-Wallis tests. RESULTS: Echocardiogram showed that LV diastolic (Sham 8.73±0.57; AS 8.30±1.10; AS-SPR 9.19±1.15mm) and systolic (Sham 4.57±0.67; AS 3.61±1.49; AS-SPR 4.62±1.48mm) diameters, left atrial diameter (Sham 5.80±0.44; AS 7.15±1.22; AS-SPR 8.02±1.17mm), and LV mass were higher in AS-SPR than AS. Posterior wall shortening velocity (Sham 38.5±3.8; AS 35.6±5.6; AS-SPR 31.1±3.8mm/s) was lower in AS-SPR than Sham and AS; E/A ratio was higher in AS-SPR than Sham. Developed tension was lower in AS and AS-SPR than Sham. Time to peak tension was higher in AS-SPR than Sham and AS after post-rest contraction. Right ventricle weight was higher in AS-SPR than AS, suggesting more severe heart failure in AS-SPR than AS. Interstitial collagen fractional area and myocardial hydroxyproline concentration were higher in AS than Sham. Metalloproteinase-2 and -9 activity, evaluated by zymography, did not differ between groups. CONCLUSION: Early spironolactone administration causes further hypertrophy in cardiac chambers, and left ventricular dilation and dysfunction in rats with AS-induced chronic pressure overload.

Cross-species sensitivity to a novel androgen receptor agonist of potential environmental concern, spironolactone.

Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 µg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 µg/L. Phenotypic masculinization of females of both fish species was observed at 5 µg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing.

Prostate effect in dogs with the aldosterone receptor blocker eplerenone.

Eplerenone (Inspra) is an aldosterone receptor antagonist approved for the treatment of hypertension and heart failure after a myocardial infarction. In vitro receptor binding and transactivation studies showed eplerenone had high selectivity for the mineralocorticoid receptor over other steroid receptors (glucocorticoid, androgen, and progesterone). The most sensitive off-target effect of orally administered eplerenone preclinically was prostate atrophy in dogs. Dose-related prostate atrophy was observed at eplerenone dosages ≥15 mg/kg/day for 13 weeks or longer. The no observed adverse effect level (NOAEL) for the prostate effect in dogs was 5 mg/kg/day. The maximal effect was seen by 13 weeks and the atrophy was reversible even after 1 year of daily treatment. An additional study demonstrated dogs with eplerenone-induced prostate atrophy (confirmed by intrarectal ultrasound) had slightly decreased semen volume but no compound-related effects on libido, semen protein content, sperm motility, daily sperm production, or epididymal sperm transit time. Four possible mechanisms for prostate effect were investigated: (1) inhibition of testosterone synthesis and secretion; (2) inhibition of 5α-reductase, the enzyme within the prostate that converts testosterone into the more active growth factor dihydrotestosterone (DHT); (3) competitive antagonism of the androgen receptor; and (4) inhibition of 5α-reductase or competitive antagonism of the androgen receptor by aldosterone, which increased in dogs treated with eplerenone. Data from these studies supported blockade of androgen receptors at suprapharmacological concentrations of eplerenone. Another mineralocorticoid blocker, spironolactone, had greater antiandrogenic activity than eplerenone both in vivo and in vitro, and it has well known clinically significant antiandrogenic effects in humans, whereas eplerenone does not.

Spironolactone, but not eplerenone, impairs glucose tolerance in a rat model of metabolic syndrome.

Although some clinical studies have suggested that spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, appears to increase the blood glucose levels, experimental studies have not supported this notion. Here, we investigated the effect of SPL on blood glucose levels in SHR/NDmcr-cp(cp/cp) (ND) rats, an animal model of metabolic syndrome, in comparison with that of eplerenone (EPL), another MR antagonist. At the same dose of 100 mg/kg, SPL and EPL increased the urinary sodium-to-potassium ratio to a comparable extent, indicating that both agents have similar renal MR antagonistic efficacy in ND rats. Interestingly, SPL but not EPL significantly increased the level of blood glucose. The oral glucose tolerance test revealed that treatment with SPL led to glucose intolerance. The levels of serum insulin and adiponectin, regulators of the blood glucose level, were virtually unaffected by treatment with SPL. On the other hand, SPL induced a marked increase in the blood level of aldosterone, known to be a risk factor for insulin resistance. These results demonstrate that in comparison with EPL, SPL characteristically impairs glucose tolerance in an animal model of metabolic syndrome, in association with a higher blood level of aldosterone.

Endocrine-disrupting effects of spironolactone in female western mosquitofish, Gambusia affinis.

The discovery of pharmaceuticals in effluent from wastewater treatment plants and drug manufacturing facilities and in receiving waters has raised environmental concern. Because these compounds are ending up in the environment, it is important to investigate the effects of these compounds on wildlife as well as humans. The present study used a fish model to investigate the endocrine-disrupting effects of spironolactone (SPL), an aldosterone antagonist used as a diuretic, but which also exhibits antiandrogenic effects in humans. A dose-response study measured the effects of SPL on anal fin ray elongation, an androgen-dependent secondary sex trait, and expression of the vitellogenin gene, an estrogen-dependent trait, in female western mosquitofish, Gambusia affinis. Fish were exposed to SPL in the water for 35 d at four nominal concentrations: 10, 100, 250, and 500 nM (4.2, 41.7, 104.1, and 208.3 µg/L, respectively) via the static renewal method. Masculinization of females, as evidenced by development of an elongated and modified anal fin, was observed in the fish exposed to the three highest concentrations. Anal fin elongation was observed in the group exposed to the lowest SPL concentration, but without the development of a tip apparatus. These results confirm the results of a preliminary study that, in contrast to antiandrogenic effects seen in humans, SPL has androgenic and/or antiestrogenic activity in a fish.

Hsp72 is an early and sensitive biomarker to detect acute kidney injury.

This study was designed to assess whether heat shock protein Hsp72 is an early and sensitive biomarker of acute kidney injury (AKI) as well as to monitor a renoprotective strategy. Seventy-two Wistar rats were divided into six groups: sham-operated and rats subjected to 10, 20, 30, 45 and 60 min of bilateral ischemia (I) and 24 h of reperfusion (R). Different times of reperfusion (3, 6, 9, 12, 18, 24, 48, 72, 96 and 120 h) were also evaluated in 30 other rats subjected to 30 min of ischemia. Hsp72 messenger RNA (mRNA) and protein levels were determined in both kidney and urine. Hsp72-specificity as a biomarker to assess the success of a renoprotective intervention was evaluated in rats treated with different doses of spironolactone before I/R. Renal Hsp72 mRNA and protein, as well as urinary Hsp72 levels, gradually increased relative to the extent of renal injury induced by different periods of ischemia quantified by histomorphometry as a benchmark of kidney damage. Urinary Hsp72 increased significantly after 3 h and continued rising until 18 h, followed by restoration after 120 h of reperfusion in accord with histopathological findings. Spironolactone renoprotection was associated with normalization of urinary Hsp72 levels. Accordingly, urinary Hsp72 was significantly increased in patients with clinical AKI before serum creatinine elevation. Our results show that urinary Hsp72 is a useful biomarker for early detection and stratification of AKI. In addition, urinary Hsp72 levels are sensitive enough to monitor therapeutic interventions and the degree of tubular recovery following an I/R insult.

The blockade of mineralocorticoid hormone signaling provokes dramatic teratogenesis in cultured rat embryos.

Although the administration of adrenocortical hormones to pregnant rats provokes only limited effect on the growth and development of the fetus, the direct influence of these steroids on cultured embryos has never been studied. The disruption of cell signaling by ZK 91587, which specifically occupies the mineralocorticoid receptor, resulted within 2 days in significant and pronounced adverse effects on the total length, the somite number, the embryo curvature, the communication between vitelline and umbilical blood vessels in the allantoid, and the vascularization of the vitelline sac, in 244-hour Wistar rat embryos in culture. The average score of 16 organs declined in a dose-dependent manner, following exposure to ZK 91587, and this was totally reversed by 10 microM aldosterone which, by itself, did not at all influence the embryonic development. The organogenesis was inhibited in the order: hind limb > fore limb > optic stalk > brain > olfactory pit > otic vesicle. ZK 91587 was completely ineffective in embryos that had attained the age of 260 hours. Similar, but less dramatic, results were obtained with the mineralocorticoid antagonist RU 26752, and with the antiglucocorticoid RU 38486. Sprague-Dawley rat embryos responded in a manner similar to the Wistar conceptuses. Thus, steroid receptor-mediated cell signaling is of critical importance to the growth and development of cultured rat embryos, which form a new model system to unravel adrenocortical hormone action.

Eplerenone: a selective aldosterone receptor antagonist (SARA).

Aldosterone, the final product of the renin-angiotensin-aldosterone system (RAAS), is a mineralocorticoid hormone that classically acts, via the mineralocorticoid (aldosterone) receptor, on epithelia of the kidneys, colon, and sweat glands to maintain electrolyte homeostasis. Aldosterone has also been shown to act at nonepithelial sites where it can contribute to cardiovascular disease such as hypertension, stroke, malignant nephrosclerosis, cardiac fibrosis, ventricular hypertrophy, and myocardial necrosis. Although angiotensin-converting enzyme (ACE) inhibitors and angiotensin type 1 (AT(1)) receptor antagonists act to suppress the RAAS, these agents do not adequately control plasma aldosterone levels--a phenomenon termed "aldosterone synthesis escape." Spironolactone, a nonselective aldosterone receptor antagonist, is an effective agent to suppress the actions of aldosterone; its use is, however, associated with progestational and antiandrogenic side effects due to its promiscuous binding to other steroid receptors. For these reasons, eplerenone--the first agent of a new class of drugs known as the selective aldosterone receptor antagonists (SARAs)--is under development. In rodent models, eplerenone provides marked protection against vascular injury in the kidney and heart. In phase II clinical trials, eplerenone demonstrates 24-h control of blood pressure with once or twice daily dosing, and is safe and well tolerated in patients with heart failure when given with standard of care agents. Pharmacokinetic studies reveal that eplerenone has good bioavailability with low protein binding, good plasma exposure, and is highly metabolized to inactive metabolites and excreted principally in the bile. Eplerenone is well tolerated in acute and chronic safety pharmacology studies. Ongoing phase III trials of eplerenone in the treatment of hypertension and heart failure are underway. These studies will extend our understanding of selective aldosterone receptor antagonism in the treatment of chronic cardiovascular disease.

Effect of spironolactone on cisplatin-induced nephrotoxicity in rabbits.

The effects of a single interaperitoneal dose of cisplatin (6.5 mg kg day(-1)), oral doses of spironolactone (20.0 mg kg day(-1)) for 5 days or the combined treatment (spironolactone+cisplatin) on the kidney function and liver function parameters, as well as the serum, liver and kidney cortical lipid contents were studied. The serum urea and creatinine concentrations (measured as kidney function parameters) were not altered by spironolactone treatment, but were significantly (P<0.001) elevated by cisplatin administration. However, animals exposed to both spironolactone+cisplatin revealed drastic increases in the serum creatinine and urea concentrations amounting to about four- and twofold those of cisplatin-alone treated animals, respectively. The histological examination of slides of kidneys from animals exposed to the combined drugs exhibited more extensive necrosis in the tubules compared to those from animals treated with cisplatin alone. Non of the drug treatments had any effects on the serum alanine transaminase (ALT) and aspartate transaminase (AST) levels (measured as liver function parameters) or liver protein content or hepatic alkaline phosphatase (ALP) activity. The histological examination also revealed apparently normal livers in all experimental groups. The cisplatin-induced nephrotoxicity was accompanied by hypercholesterolaemia and hyperphospholipidaemia, whereas spironolactone showed a hypocholesterolaemic effect. The concomitant treatment with both cisplatin and spironolactone significantly (P<0.05) raised the serum triacylglycerol (TAG) concentration compared to the cisplatin-alone-treated group. Both spironolactone and cisplatin administered separately or jointly caused accumulation of cholesterol and TAG in the kidney cortex with significant depletion of the liver cholesterol content. The present results indicated that spironolactone aggravates the cisplatin-induced nephrotoxicity in the rabbit.

Adrenocorticotrophin-induced hypertension: effects of mineralocorticoid and glucocorticoid receptor antagonism.

OBJECTIVE: To examine whether the increase of blood pressure in adrenocorticotrophin-treated rats is mediated through mineralocorticoid or glucocorticoid receptors or corticosterone 6 beta-hydroxylation inhibition. DESIGN: Rats were randomly allocated to 14 treatment groups for 10 days. The treatments included sham injection (n = 35), adrenocorticotrophin (5, 100, 500 micrograms/kg per day, subcutaneously, n = 5, 15 and 15, respectively), spironolactone (100 mg/kg per day, subcutaneously, n = 15), standard-dose or high-dose RU 486 (70 mg/kg every 3 days or 70 mg/kg per day, subcutaneously, n = 5 and 10, respectively), spironolactone + adrenocorticotrophin (100 micrograms/kg per day, n = 5, or 500 micrograms/kg per day, n = 10), standard-dose RU 486 + adrenocorticotrophin (500 micrograms/kg per day, n = 5), high-dose RU 486 + adrenocorticotrophin (100 micrograms/kg per day, n = 10), troleandomycin (40 mg/kg per day, subcutaneously, n = 5) and troleandomycin + adrenocorticotrophin (5 micrograms/kg per day, n = 5). Systolic blood pressure and metabolic parameters were measured every second day. RESULTS: Adrenocorticotrophin treatment increased systolic blood pressure dose-dependently (5 micrograms/kg per day: +14 +/- 2 mmHg; 100 micrograms/kg per day: +20 +/- 2 mmHg; 500 micrograms/kg per day: +28 +/- 2 mmHg, all P < 0.001). Adrenocorticotrophin at 100 and 500 micrograms/kg per day increased plasma sodium and decreased plasma potassium concentrations. Spironolactone did not block adrenocorticotrophin-induced systolic blood pressure changes but did block changes in plasma sodium and potassium levels. Standard-dose RU 486 did not modify the adrenocorticotrophin-induced (500 micrograms/kg per day) systolic blood pressure rise but blocked the effect of adrenocorticotrophin on body weight. High-dose RU 486 partially blocked the adrenocorticotrophin-induced (100 micrograms/kg per day) systolic blood pressure increase (adrenocorticotrophin at 100 micrograms/kg per day: 143 +/- 3 mmHg; high-dose RU 486 + adrenocorticotrophin at 100 micrograms/kg per day: 128 +/- 5 mmHg, P < 0.001) and body-weight loss. Troleandomycin did not alter the development of adrenocorticotrophin-induced hypertension. CONCLUSIONS: Spironolactone and standard-dose RU 486 did not modify adrenocorticotrophin-induced hypertension despite demonstrable antimineralocorticoid and antiglucocorticoid actions. High-dose RU 486 partially blocked adrenocorticotrophin-induced (100 micrograms/kg per day) hypertension, suggesting either a permissive effect of glucocorticoid on blood pressure or other antihypertensive actions of RU 486. Inhibition of glucocorticoid 6 beta-hydroxylation by troleandomycin did not modify adrenocorticotrophin-induced hypertension, suggesting that effects of corticosterone 6 beta-hydroxylation in adrenocorticotrophin-induced hypertension are negligible.

Pig Leydig cell culture: a useful in vitro test for evaluating the testicular toxicity of compounds.

In vivo studies have shown that the testis is a target organ for drugs and chemicals. In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model. Testosterone and progesterone secretion were measured to evaluate testicular function. Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs. Ketoconazole and aminoglutethimide inhibited both testosterone and progesterone secretion, but without modifying cell viability. Spironolactone specifically blocked testosterone secretion and increased progesterone concentration without inducing cell mortality. Cycloheximide altered testicular steroid secretion by another mechanism of action. Chlorpromazine, which interferes with the secretion of gonadotropins in vivo, produced a significant inhibition of progesterone and testosterone secretion as a result of the cytotoxic effects of the drug. In conclusion, this in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.

Relationship between covalent binding to microsomal protein and the destruction of adrenal cytochrome P-450 by spironolactone.

Previous investigations have demonstrated that guinea pig adrenal microsomes catalyze an NADPH-dependent activation of spironolactone (SL) resulting in the degradation of cytochrome(s) P-450 and decreases in steroidogenic enzyme activities. Studies were done to evaluate the relationship between the destruction of cytochrome P-450 and the covalent binding to microsomal protein by SL and by 7 alpha-thiospironolactone (7 alpha-thio-SL), an obligatory intermediate in the activation pathway. NADPH-dependent irreversible binding to guinea pig adrenal microsomal protein was demonstrable with 22-14C- and with 35S-labelled SL or 7 alpha-thio-SL as substrates. In the absence of NADPH, there was relatively little binding. NADPH-dependent covalent binding was not demonstrable with hepatic microsomal preparations. The amount of covalent binding to adrenal microsomes was far greater with 7 alpha-thio-SL than with SL and also greater with 35S-labelled than with 14C-labelled substrates. The latter results suggest the possibility of more than one reactive metabolite. Time-course experiments revealed a good correlation between covalent binding and P-450 destruction by SL and by 7 alpha-thio-SL. In addition, the 17 alpha-hydroxylase inhibitor, SU-10'603, and the 17 alpha-hydroxylase substrate, progesterone, prevented both the degradation of cytochrome P-450 and the NADPH-dependent covalent binding by 7 alpha-thio-SL. Reduced glutathione also decreased covalent binding but did not diminish P-450 destruction. The latter results indicate that some of the covalent binding is unrelated to the degradation of cytochrome P-450. However, all of the data are consistent with the hypothesis that 7 alpha-thio-SL is a suicide inhibitor of adrenal cytochrome P-450 and that covalent binding to protein is involved in the degradation of cytochrome P-450.

The effects of chronic ingestion of spironolactone on serum thyrotropin and thyroid hormones in the male rat.

Male rats were fed spironolactone admixed with feed at doses of 6, 50, and 200 mg/kg/day for up to 13 weeks. After 13 weeks of treatment, there were dose-related increases in thyroid weight and follicular hypertrophy. Serum thyrotropin (TSH) concentrations were significantly increased throughout the treatment period. Serum thyroxine (T4) and triiodothyronine (T3) were significantly decreased at Weeks 2 and 4, but returned to control concentrations by Week 13. The TSH increase and transient T4 decrease suggested that the thyroid hypertrophy was a compensatory reaction to lowered thyroid hormone levels. To determine the effect of spironolactone ingestion on T4 synthesis and metabolism, male rats were fed spironolactone admixed with feed at 200 mg/kg for 2 weeks. The decrease in T4 was not due to decreased synthesis, since iodide uptake and organification were significantly increased by spironolactone treatment. Since uridine diphosphate glucuronosyl transferase activity was significantly increased by spironolactone treatment, it appears that, by increasing hepatic clearance of T4, spironolactone causes a decrease in the serum concentration of this hormone. The lower T4 level causes a release of feedback inhibition and an increase in TSH resulting in the increase in thyroid gland size and activity.