In vitro assessment of corticosteroid effects of eight chiral herbicides.

Information regarding the enantioselective endocrine disruption of chiral herbicides is scarce. This study assessed the disrupting effects of eight typical chiral herbicides on corticosteroids (including glucocorticoids and mineralocorticoids). Enantioselectivity of eight chiral herbicides were evaluated for their agonistic/antagonistic effects on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) with CHOK1 cell line using reporter gene assay. Their influence on the production of corticosteroids were further investigated in H295R cell line using enzyme-linked immunosorbent assay (ELISA). None of the racemates or enantiomers of eight chiral herbicides exhibited GR or MR agonistic activity at non-cytotoxic concentrations. However, rac-propisochlor and S-imazamox antagonized cortisol-induced transactivation of GR by 21.79% and 38.73% at the concentration of 1.0 × 10-7 M and 1.0 × 10-6 M, respectively, and R-napropamide remarkably attenuated aldosterone-induced MR transactivation by 68.78% at 1.0 × 10-6 M. The secretion of cortisol was significantly restrained after treated with 1.0 × 10-6 M rac-propisochlor and rac-/R-napropamide at the concentration of 1.0 × 10-6 M by 26.49%, 30.10% and 35.27%, respectively, while this glucocorticoid was remarkably induced by 1.0 × 10-5 M rac-diclofop-methyl and its two enantiomers at the concentration of 1.0 × 10-5 M by 75.60%, 100.1% and 68.78%, respectively. Exposure to rac-propisochlor (1.0 × 10-6 M), S-diclofop-methyl (1.0 × 10-5 M) or rac-/S-/R- acetochlor (1.0 × 10-6 M) and rac-/S-/R-lactofen (1.0 × 10-6 M) inhibited the secretion of aldosterone by approximately 40%. Our findings suggested that chiral herbicides disrupted corticosteroid homeostasis in an enantioselective way. Therefore, more comprehensive screening is required to better understand the ecological and health risks of chiral pesticides.

Potential endocrine-disrupting effects of metals via interference with glucocorticoid and mineralocorticoid receptors.

As a result of human activities, the pollution of metals is becoming ubiquitous in the environment. Among various toxicological mechanisms of action, metals have been considered as endocrine-disrupting chemicals (EDCs) through interference with steroid receptors. However, information regarding the potential endocrine disruption of metals on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is especially scarce. In this study, a total of 16 metals were assessed for their GR/MR activities using luciferase reporter gene assay. None of the tested metals exhibited GR or MR agonistic activity, but a total of 7 and 5 candidate metals showed obvious GR and MR antagonistic properties, respectively. All 7 GR antagonistic metals [ BaCl2, CoCl2, CuCl2, Pb(NO3)2, LiCl, SnCl2 and ZnCl2] inhibited glucocorticoid-responsive gene GILZ expression in J774A.1 cells. Further investigations indicated that the 5 MR antagonistic metals [ CdCl2, Pb(NO3)2, LiCl, MnCl2 and SnCl2] antagonized aldosterone-inhibited hepatocellular carcinoma cell proliferation. Among these metals, Pb(NO3)2, LiCl, and SnCl2 showed both anti-glucocorticoid and anti-mineralocorticoid activities. Comprehensive screening and evaluation of GR and MR antagonists and agonists among metals should be considered to better understand the ecological and health risks of metals.

Novel Pathways of Endocrine Disruption Through Pesticides Interference With Human Mineralocorticoid Receptors.

Mineralocorticoid receptor (MR) is one member of the steroid receptor family. In addition to its important role in Na+/K+ homeostasis, MR is reported as a tumor-suppressor in carcinogenesis. So far, little was known about the ability of pesticides to interfere with MR. In this study, a total of 43 pesticides and/or metabolites were investigated for their potential effects on human MR. None of the tested pesticides exhibited MR agonistic potency, whereas 16 compounds showed antagonistic activities. Further investigations indicated that these 16 chemicals individually antagonized aldosterone-induced alkaline phosphatase expression in vascular smooth muscle cells and aldosterone-inhibited hepatocellular carcinoma cell proliferation at higher concentrations, and the mixture of these 16 pesticides at environmentally relevant concentrations significantly disrupted MR activity. The additional quantitative mixture experiments indicated a good agreement between the combined anti-mineralocorticoidic activities of 16 pesticides and the responses predicted by concentration addition model instead of independent action model. The interruption of nuclear translocation of MR was clarified as a main mechanism for the anti-mineralocorticoidic activities by these pesticides. These data suggest that the health risk may increase when multiple MR antagonists cooperate following concentration addition model and exhibit a combined effect. Our findings emphasize that comprehensive risk assessment of adverse effects of environmental MR ligands on human health should be considered.

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.

Drospirenone intake alters plasmatic steroid levels and cyp17a1 expression in gonads of juvenile sea bass.

Drospirenone (DRO) is one of the most widely used progestins in contraceptive treatments and hormone replacement therapies. The pharmacokinetics and potential toxicological effects of DRO were investigated in juvenile sea bass (Dicentrarchus labrax) exposed through the diet (0.01-10 µg DRO/g) for up to 31 days. DRO was detected in the blood (4-27 ng/mL) of fish exposed to the highest concentration, with no significant bioaccumulation over time and no alteration of hepatic metabolizing enzymes, namely, CYP1A and CYP3A-catalysed activities and UDP-glucuronyltransferase (UGT). Pregnenolone (P5), progesterone (P4), 17α-hydroxyprogesterone (17P4), 17α-hydroxypregnenolone (17P5), androstenedione (AD) and testosterone (T) were determined in plasma and gene expression of cyp17a1, cyp19a1a and cyp11ß analysed by qRT-PCR in gonads. The significant increase in plasmatic levels of 17P5, 17P4 and AD detected after 31 days exposure to 10 ng DRO/g together with the increased expression of cyp17a1 in females evidence the ability of DRO to alter steroid synthesis at low intake concentrations (7 ng DRO/day). However, the potential consequences of this steroid shift for female reproduction remain to be investigated.

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.

DNA damage in tissues of rat treated with potassium canrenoate.

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463-472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.

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.

Genotoxicity testing of potassium canrenoate in cultured rat and human cells.

Potassium canrenoate (PC), a competitive aldosterone antagonist used as a diuretic and in the treatment of hypertension, was examined for its capacity to produce genotoxic effects in cultured rat and human cells. At subtoxic concentrations (10-90 microM) PC was found to induce a dose-dependent degree of DNA fragmentation, as detected by the Comet assay, and of DNA repair synthesis, as measured by quantitative autoradiography, in primary cultures of hepatocytes from rat and human donors of both genders. In rat hepatocytes both DNA fragmentation and DNA repair were more marked after 3 h than after 20 h exposure and in cultures from females than from males. In human hepatocytes from one male and two female donors, PC caused a similar effect in terms of DNA fragmentation, whereas DNA repair was detected in cultures from only two of the same three donors and was less marked than in rat hepatocytes. A modest but statistically significant increase in micronucleated cells was present in primary cultures of replicating rat hepatocytes exposed to 10 or 30 microM PC for 48 h, the response being, in this case also, more evident in females than in males. In contrast, PC did not induce micronucleus formation in human hepatocytes from two female donors. Any evidence of DNA fragmentation and micronucleus formation was absent in cultured human lymphocytes. Taken as a whole these findings support the hypothesis that hepatocytes activate PC to DNA-damaging reactive species. PC induced the observed genotoxic effects at concentrations close to those produced in humans by the administration of therapeutic doses, but these effects were as a whole more marked in rat than in human hepatocytes. Since PC shares the 17-hydroxy-3-oxopregna-4,6-diene structure with cyproterone acetate, chlormadinone acetate and megestrol acetate, previously found to be genotoxic to both rat and human hepatocytes, the potential carcinogenic hazard of this type of steroids cannot be neglected.

Inhibition of sodium transport by hyperbaric oxygen in the toad urinary bladder.

In the isolated toad bladder, 2--10 ATA of oxygen reversibly inhibited the active transport of sodium ions across the bladder, measured as the short-circuit current. Extent of inhibition was dependent on the oxygen pressure and length of exposure. Oxygen at 1 ATA had no effect on sodium transport for up to 2 days of exposure. However, bladders exposed for 18 h to oxygen at 1 ATA were more sensitive to a subsequent exposure to hyperbaric oxygen. The hormonal stimulation of sodium transport by pitressin or aldosterone was inhibited by hyperbaric oxygen but was unaffected by prolonged exposure to oxygen at 1 ATA. Incubation of bladders with aldosterone or sodium pyruvate increased the sensitivity of sodium transport to the inhibitory effects of hyperbaric oxygen. The mechanism of the inhibition of sodium transport in the toad bladder by hyperbaric oxygen was not known.