Dehydroepiandrosterone 3-sulfate Research Articles

Cortisol, cortisone and DHEAS in epidermis and scales of fish Aphanius fasciatus: HPLC-MS/MS measurement of stress indicators as proxies for natural and human-induced factors

Fish health can be affected by a multitude of stressors. Acute and chronic stress assessment via specific hormones monitoring has become a trending research topic. Common investigated matrices are blood and plasma, but recently less invasive substrates have been identified. As chemical composition of skin mucus/epidermis has been demonstrated to link with acute stress, and of scales with chronic stress in fish, the aim of the study was firstly to improve the determination of three stress hormones, namely cortisol (COL), cortisone (CON), and dehydroepiandrosterone-3-sulfate (DHEAS), in skin mucus/epidermis and scales of Aphanius fasciatus. Secondly, an evaluation of the impact of different environments on hormones concentrations was carried out. A liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) and a preanalytical procedure were validated to determine COL, CON and DHEAS. This methodology was applied to compare a pull of field-collected fish with a pull of fish housed in the laboratory for one year. Our results highlighted a significant presence of cortisol and cortisone in epidermis of the latter pull (averagely 0.10 and 0.14 ng mg−1, respectively), while in the first pull both hormones were much less concentrated (averagely 0.006 and 0.008 ng mg−1, respectively). Scales of both pulls showed presence of hormones, with a higher concentration for fish housed in the laboratory, although a relevant difference in concentration was found only for cortisone. DHEAS was always below the limit of detection.

Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

Per- and polyfluoroalkyl substances (PFAS) inhibit cytochrome P450 CYP3A7 through direct coordination to the heme iron and water displacement

Per- and polyfluoroalkyl substances (PFAS) are a chemical class of highly stable, fluorinated compounds popular for use in a variety of consumer products. PFAS environmental persistence in drinking water contributes to acute exposure in humans and subsequent bioaccumulation of the compounds in the liver and lung tissue. Prenatal PFAS exposure has been associated with lowered birth weight, premature birth, and developmental defects including cranio-facial abnormalities. The cytochrome P450 enzyme CYP3A7 is responsible for facilitating a variety of reactions essential for proper fetal development in humans. In addition to drug metabolism, CYP3A7 is responsible for metabolizing endogenous ligands in the developing human liver, including the steroid precursor dehydroepiandrosterone 3-sulfate (DHEA-S), essential for estriol synthesis during pregnancy, along with the morphogen all-trans-retinoic acid (atRA). Interference with estriol synthesis during pregnancy, as well as atRA clearance, is known to result in similar effects associated with prenatal PFAS exposure including lowered birth weight, premature birth, and developmental defects. We hypothesized that PFAS compounds bind to the CYP3A7 enzyme resulting in its inhibition. We implemented a series of binding studies using spectral characterization of six PFAS compounds (PFOA, PFOS, GenX, PFNA, PFNS, and PFHxS), and evaluated their interactions with recombinant CYP3A7. In addition, we screened PFAS for their ability to inhibit CYP3A7 oxidative activity using dibenzylfluorescein, a fluorescent probe, and DHEA-S, an endogenous substrate of CYP3A7. Our data demonstrate that of the six PFAS tested, PFOA, PFOS, PFNA, and PFHxS bind to and inhibit CYP3A7.

Bilirubin Reduces the Uptake of Estrogen Precursors and the Followed Synthesis of Estradiol in Human Placental Syncytiotrophoblasts via Inhibition and Downregulation of Organic Anion Transporter 4.

Estrogen biosynthesis in human placental trophoblasts requires the human organic anion transporter 4 (hOAT4)-mediated uptake of fetal derived precursors such as dehydroepiandrosterone-3-sulfate (DHEAS) and 16α-hydroxy-DHEA-S (16α-OH-DHEAS). Scant information is available concerning the contribution of fetal metabolites on the impact of placental estrogen precursor transport and the followed estrogen synthesis. This study substantiated the roles of bilirubin as well as bile acids (taurochenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, chenodeoxycholic acid) on the inhibition of hOAT4-mediated uptake of probe substrate 6-carboxylfluorescein and DHEAS in stably transfected hOAT4-Chinese hamster ovary cells, with the IC50 of 1.53 and 0.98 μM on 6-carboxylfluorescein and DHEAS, respectively, for bilirubin, and 90.2, 129, 16.4, and 12.3 μM on 6-CF for taurochenodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, and chenodeoxycholic acid. Bilirubin (2.5-10 μM) concentration-dependently inhibited the accumulation of estradiol precursor DHEAS in human choriocarcinoma JEG-3 cells (reduced by 60% at 10 μM) and primary human trophoblast cells (reduced by 80% at 10 μM). Further study confirmed that bilirubin (0.625-2.5 μM) concentration-dependently reduced the synthesis and secretion of estradiol in primary human trophoblast cells, among which 2.5 μM of bilirubin reduced the synthesis of estradiol by 30% and secretion by 35%. In addition, immunostaining and Western blot results revealed a distinct downregulation of hOAT4 protein expression in primary human trophoblast cells pretreated with 2.5 μM of bilirubin. In conclusion, this study demonstrated that bilirubin reduced the uptake of estrogen precursors and the followed synthesis of estradiol in human placenta via inhibition and downregulation of organic anion transporter 4. SIGNIFICANCE STATEMENT: Fetal metabolites, especially bilirubin, were first identified with significant inhibitory effects on the hOAT4-mediated uptake of estrogen precursor DHEAS in hOAT4-CHO, JEG-3 and PHTCs. Bilirubin concentration-dependently suppressed the estradiol synthesis and secretion in PHTCs treated with DHEAS, which was synchronized with the decline of hOAT4 protein expression. Additionally, those identified bile acids exhibited a weaker inhibitory effect on the secretion of estradiol.

Inhibition of CYP3A7 DHEA-S Oxidation by Lopinavir and Ritonavir: An Alternative Mechanism for Adrenal Impairment in HIV Antiretroviral-Treated Neonates

Prophylactic antiretroviral therapy (ART) in HIV infected pregnant mothers and their newborns can dramatically reduce mother-to-child viral transmission and seroconversion in the neonate. The ritonavir-boosted lopinavir regimen, known as Kaletra, has been associated with premature birth and transient adrenal insufficiency in newborns, accompanied by increases in plasma dehydroepiandrosterone 3-sulfate (DHEA-S). In the fetus and neonates, cytochrome P450 CYP3A7 is responsible for the metabolism of DHEA-S into 16α-hydroxy DHEA-S, which plays a critical role in growth and development. In order to determine if CYP3A7 inhibition could lead to the adverse outcomes associated with Kaletra therapy, we conducted in vitro metabolic studies to determine the extent and mechanism of CYP3A7 inhibition by both ritonavir and lopinavir and the relative intrinsic clearance of lopinavir with and without ritonavir in both neonatal and adult human liver microsomes (HLMs). We identified ritonavir as a potent inhibitor of CYP3A7 oxidation of DHEA-S (IC50 = 0.0514 μM), while lopinavir is a much weaker inhibitor (IC50 = 5.88 μM). Furthermore, ritonavir is a time-dependent inhibitor of CYP3A7 with a KI of 0.392 μM and a kinact of 0.119 min–1, illustrating the potential for CYP3A mediated drug–drug interactions with Kaletra. The clearance rate of lopinavir in neonatal HLMs was much slower and comparable to the rate observed in adult HLMs in the presence of ritonavir, suggesting that the addition of ritonavir in the cocktail therapy may not be necessary to maintain effective concentrations of lopinavir in neonates. Our results suggest that several of the observed adverse outcomes of Kaletra therapy may be due to the direct inhibition of CYP3A7 by ritonavir and that the necessity for the inclusion of this drug in the therapy may be obviated by the lower rate of lopinavir clearance in the neonatal liver. These results may lead to a reconsideration of the use of ritonavir in neonatal antiretroviral therapy.

Sigma-1 Receptor Agonists Induce Oxidative Stress in Mitochondria and Enhance Complex I Activity in Physiological Condition but Protect Against Pathological Oxidative Stress.

The sigma1 receptor (σ1R) is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it modulates Ca2+ exchange between the ER and mitochondria by interacting with inositol-1,4,5 trisphosphate receptors (IP3Rs). The σ1R is highly expressed in the central nervous system and its activation stimulates neuromodulation and neuroprotection, for instance in Alzheimer's disease (AD) models in vitro and in vivo. σ1R effects on mitochondria pathophysiology and the downstream signaling are still not fully understood. We here evaluated the impacts of σ1R ligands in mouse mitochondria preparations on reactive oxygen species (ROS) production, mitochondrial respiration, and complex activities, in physiological condition and after direct application of amyloid Aβ1-42 peptide. σ1R agonists (2-(4-morpholinethyl)-1-phenylcyclohexanecarboxylate hydrochloride (PRE-084), tetrahydro-N,N-dimethyl-5,5-diphenyl-3-furanmethanamine (ANAVEX1-41, AN1-41), (S)-1-(2,8-dimethyl-1-thia-3,8-diazaspiro[4.5]dec-3-yl)-3-(1H-indol-3-yl)propan-1-one (ANAVEX3-71, AN3-71), dehydroepiandrosterone-3 sulfate (DHEA), donepezil) increased mitochondrial ROS in a σ1R antagonist-sensitive manner but decreased Aβ1-42-induced increase in ROS. σ1R ligands (agonists or antagonists) did not impact respiration but attenuated Aβ1-42-induced alteration. σ1R agonists (PRE-084, AN1-41, tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73, AN2-73), AN3-71) increased complex I activity, in a Ca2+-dependent and σ1R antagonist-sensitive manner. σ1R ligands failed to affect complex II, III, and IV activities. The increase in complex I activity explain the σ1R-induced increase in ROS since ligands failed to affect other sources of ROS accumulation in mitochondria and homogenates, namely NADPH oxidase (NOX) and superoxide dismutase (SOD) activities. Furthermore, Aβ1-42 significantly decreased the activity of complexes I and IV and σ1R agonists attenuated the Aβ1-42-induced complex I and IV dysfunctions. σ1R activity in mitochondria therefore results in a Ying-Yang effect, by triggering moderate ROS increase acting as a physiological signal and promoting a marked anti-oxidant effect in pathological (Aβ) conditions.

LC-MS/MS simultaneous analysis of allopregnanolone, epiallopregnanolone, pregnanolone, dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate in human plasma.

Several neuropsychopharmacological properties have been attributed to the 3α-reduced pregnane steroids, allopregnanolone and pregnanolone, as well as to dehydroepiandrosterone sulfate because of their ability to modulate γ-aminobutyric acid (GABAA) receptors in the CNS. In order to understand better their role in several mechanisms in CNS, a new methodology is proposed to monitor these compounds in human plasma. Methodology & results: The analytes were first derivatized with 2-hydrazinopyridine and extracted from plasma using SPE. Then, the compounds were separated and detected by LC-MS/MS. A mobile phase of formic acid (0.1%) in water and methanol through a gradient of composition and a flow rate of 0.3 ml min-1 resulted in good separations of the analytes. Linear responses in wide range of concentrations and LOQs ranging from 10 (dehydroepiandrosterone 3-sulfate) to 40 pg ml-1 (dehydroepiandrosterone) were obtained in <9 min. The method proposed has been validated and then applied to monitor these neurosteroids in plasma samples from ten volunteers. For the first time, a straightforward and reliable method for the chromatographic separation of allopregnanolone, epiallopregnanolone and pregnanolone, as well as of dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate was carried out, with optimal accuracy, sensitivity and specificity.

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (Km and Vmax) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation.

DHEA-S inhibits human neutrophil and human airway smooth muscle migration

Airway diseases such as asthma, emphysema, and chronic bronchitis are, in part, characterized by reversible airflow obstruction and inflammation. In severe disease, marked decreases in lung function are associated with airway smooth muscle proliferation and airway neutrophilia. Inhaled glucocorticoids attenuate increased airflow obstruction and airway inflammation that occur, in part, due to increased smooth muscle migration and proliferation, as well as the airway neutrophilia. Glucocorticoids, however, have adverse side effects and, in some patients, are ineffective despite high doses. Recent research has explored the effects of non-traditional steroids on attenuation of inflammation associated with airway diseases. These non-traditional steroids have improved side effect profiles in comparison to glucocorticoid therapy. Our studies assessed effects of dehydroepiandrosterone-3-sulfate (DHEA-S) on migration of both human peripheral blood neutrophils (PMN) and human airway smooth muscle cells (HASM). DHEA-S dose-dependently inhibited chemotaxis of PMN and HASM while having no effect on the phosphorylation levels of Akt, ERK1/2, p38 MAPK or PKC, canonical positive regulators of cell migration. These studies demonstrate direct effects of DHEA-S on cell migration, thereby suggesting that DHEA-S may attenuate airway inflammation and cell migration.

Nebulized dehydroepiandrosterone-3-sulfate improves asthma control in the moderate-to-severe asthma results of a 6-week, randomized, double-blind, placebo-controlled study

Inhaled dehydroepiandrosterone-3-sulfate (DHEAS), but not dehydroepiandrosterone (DHEA), possesses anti-inflammatory activity in in vitro assays and in models of allergen and lipopolysaccharide challenges. We postulated whether an inhaled suspension of DHEAS delivered via nebulizer would improve asthma control in moderate-to-severe asthma patients. We also characterized the safety profile of an inhaled suspension of DHEAS. Patients receiving at least 500 μg of fluticasone equivalent plus long-acting beta-agonists (LABA) entered a 5-week run-in where the dose of inhaled corticosteroids was reduced to 200 μg of fluticasone plus LABA per day. Patients were randomized to 70 mg of DHEAS or placebo if their Asthma Control Questionnaire (ACQ) score was ≥2.0 and their FEV(1) ≥ 50%. When compared with control, a statistically significant improvement in ACQ in 6 weeks of treatment with 70 mg of DHEAS was observed. The median improvement in ACQ was -0.72 and -0.43 for the active and placebo groups, respectively (p = 0.0389); the percentage of patients with at least minimally clinically important difference of -0.50 from baseline was significantly greater in the DHEAS group versus the placebo, (59.4% versus 45.7%; p = 0.0236). Asthma symptom scores, the proportion of symptom-free days and symptom nights, although not statistically significant, had positive trends supporting the improvement in ACQ. Fewer patients were withdrawn from the study for respiratory events on DHEAS compared with placebo. There were few adverse events and no changes in sex hormones despite increases in circulating levels of DHEAS. An inhaled suspension of DHEAS delivered via nebulizer improved asthma control scores in subjects with poorly controlled moderate-to-severe asthma. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY ANZCTR.ORG.AU IDENTIFIER: 012607000192482.

Organic Solute Transporter, OSTα-OSTβ: Its Role in Bile Acid Transport and Cholestasis

Organic solute transporter alpha-beta (OSTalpha-OSTbeta) is a unique heteromeric transporter localized to the basolateral membrane of epithelial cells involved in sterol transport. It is believed to be the primary bile acid efflux transporter in the intestine of mammals and is therefore essential to bile acid homeostasis and the enterohepatic circulation. First described in the evolutionarily primitive small skate, LEUCORAJA ERINACEA, this facilitated transporter requires expression of both subunits for its function. It can transport a variety of bile acids, as well as estrone 3-sulfate, dehydroepiandrosterone 3-sulfate, digoxin, and prostaglandin E (2). Expression of both subunits is variable between species and tissues; in humans high expression is noted in the liver, small intestine, kidney, testis, and adrenal gland. OSTalpha-OSTbeta is directly regulated by the bile acid sensing nuclear receptor, farnesoid X receptor (FXR). Furthermore, it is part of the complex regulatory pathway that controls bile acid synthesis and homeostasis. Hepatic OSTalpha-OSTbeta is upregulated in cholestasis in both humans and rodents, where it appears to play a protective role. Additional studies are necessary to determine its role in liver injury, bile acid malabsorption, and lipid and glucose metabolism, as well as a potential protective role for kidney OSTalpha-OSTbeta in cholestasis.

Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16α-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E 1S) uptake by these transporters stably expressed in FlpIn™-HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E 1S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E 1S by OAT4 was partly Na +-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na +-independent. Kinetic analysis of the initial uptake rates of E 1S by OAT4 and OATP2B1 gave very similar values for K m (about 20 μM) and V max (about 600 pmol/(min × mg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower ( K m > 200 μM) then for OAT4 ( K m = 29 μM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation.

Helices F–G Are Important for the Substrate Specificities of CYP3A7

CYP3A7 is a member of the human CYP3A family and a major form of P450 expressed in human fetal livers. Although CYP3A7 shares nearly 90% base sequence with CYP3A4, CYP3A7 shows striking functional differences in the catalytic preference for several substrates, such as dehydroepiandrosterone (DHEA) or dehydroepiandrosterone 3-sulfate (DHEA-3S). First, to clarify the reason for the differences between CYP3A7 and CYP3A4, a homology model of CYP3A7 was constructed using the CYP3A4 crystal structure. Because these two structures were similar, four kinds of chimeric enzymes were constructed to determine which sequences are important for exhibiting the characteristics of CYP3A7. The results of kinetic analysis of DHEA and DHEA-3S 16alpha-hydroxylations by CYP3A7, CYP3A4, and CYP3A chimeras suggested that the amino acid residues from Leu(210) to Glu(279) were important to express the specificity for substrates as CYP3A7. This region was on the F and G helices of the modeled CYP3A7. Furthermore, to assess which amino acid in this sequence is important for the substrate specificity of CYP3A7, a one-point mutation of CYP3A7 to CYP3A4 was made by site-directed mutagenesis. The mutants of K224T and K244E had lost DHEA and DHEA-3S 16alpha-hydroxylation activities. The mutants also greatly decreased the metabolism of testosterone, erythromycin, nevirapine, and triazolam relative to those activities of CYP3A7 wild-type enzyme. From these results, it is expected that CYP3A7 can recognize specific substrates using the lysines in F-G loops.

Rapid column‐switching liquid chromatography/mass spectrometric assay for DHEA‐sulfate in the plasma of patients with Alzheimer's disease

A simple and highly sensitive method for the quantification of dehydroepiandrosterone-3-sulfate (DHEAS) in human plasma was developed. DHEAS was directly determined in plasma using column-switching liquid chromatography/mass spectrometry (LC-MS). The plasma was filtered with a membrane filter. The filtrate was injected onto a pre-column without further sample preparation such as extraction or derivatization. The pre-column was washed with an aqueous solution to remove interference and the analyte was eluted into a reversed-phase C(18) analytical column for separation and detection using a column-switching valve. The calibration range of DHEAS was 0.01-10 micromol/L, and the linearity of the method was 0.999. The limit of detection (LOD) at a signal-to-noise (S/N) ratio of 3 was 5 nmol/L. The accuracy and precision (%CV) were less than 10% in within-day and day-to-day variations. To explore the relationship between Alzheimer's disease and the DHEAS level in human plasma, the concentrations of DHEAS in female patients with Alzheimer's disease (n = 20) and in normal female subjects (n = 20) were measured. The level of DHEAS was significantly decreased in the plasma of patients with Alzheimer's disease (p < 0.0002) compared with that in normal subjects. From the results, we concluded that our method is sufficiently sensitivity and reliability for the quantification of DHEAS in clinical samples. Plasma DHEAS concentration could be an important marker to understand the pathogenesis of Alzheimer's disease.

Phencyclidine-Induced Cognitive Deficits in Mice are Improved by Subsequent Subchronic Administration of Fluvoxamine: Role of Sigma-1 Receptors

This study was undertaken to examine the effects of the selective serotonin reuptake inhibitors fluvoxamine and paroxetine on cognitive deficits in mice after repeated administration of the N-methyl-D-aspartate receptor antagonist phencyclidine (PCP). In the novel object recognition test, repeated administration of PCP (10 mg/kg/day, 10 days) significantly decreased the exploratory preference in the retention test session, but not in the training test session. PCP-induced cognitive deficits were significantly improved by subsequent subchronic (2-week) administration of fluvoxamine (20 mg/kg/day), but not paroxetine (10 mg/kg/day). Furthermore, the effect of fluvoxamine on PCP-induced cognitive deficits was antagonized by co-administration of the selective sigma-1 receptor antagonist NE-100 (1 mg/kg/day). Moreover, PCP-induced cognitive deficits were also significantly improved by subsequent subchronic (2-week) administration of the selective sigma-1 receptor agonist SA4503 (1 mg/kg/day) or neurosteroid dehydroepiandrosterone 3-sulfate (DHEA-S; 25 mg/kg/day). The effects of SA4503 or DHEA-S were also antagonized by co-administration of NE-100 (1 mg/kg/day), suggesting the role of sigma-1 receptors in the active mechanisms of these drugs. In contrast, acute single administration of these drugs (fluvoxamine, paroxetine, SA4503) alone or combination with NE-100 did not alter PCP-induced cognitive deficits. The present study suggests that agonistic activity of fluvoxamine at sigma-1 receptors plays a role in the active mechanisms of fluvoxamine on PCP-induced cognitive deficits in mice. Therefore, sigma-1 receptor agonists such as fluvoxamine would be potential therapeutic drugs for the treatment of the cognitive deficits of schizophrenia.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 μM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Biology of a Novel Organic Solute and Steroid Transporter, OSTΑ-OSTβ

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.

Functional characterization of a human organic anion transporter hOAT4 in placental BeWo cells

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. hOAT4 is expressed in the placenta and kidney. In the current study, we stably transfected hOAT4 into human placental BeWo cells and the functional properties of hOAT4 and its regulation were investigated in these cells. hOAT4-mediated uptake of estrone sulfate, a protypical organic anion for hOAT4, was dose- and time-dependent, and saturable ( K m = 4.2 μM). The substrate specificity of hOAT4 includes various steroid sulfates, such as β-estradiol-3,17-disulfate, 17-β-estradiol-3-sulfate, β-estradiol-3-sulfate, and dehydroepiandrosterone-3-sulfate (DHEAS), but does not include p-aminohippuric acid (PAH) and tetraethylammonium (TEA). Pre-incubation of hOAT4-expressing BeWo cells with phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), both of which are protein kinase C (PKC) activators, acutely inhibited the transport activity. The inhibition by PDBu resulted in a decreased V max without significant affecting the K m. Establishment of hOAT4-expressing BeWo cells provided useful tool for further pharmacological and molecular biological studies of placental transport of organic anions mediated by this carrier.

Enzymic conversion of 3β-hydroxy-5-ene-steroids and their sulfates to 3-oxo-4-ene-steroids for increasing sensitivity in LC–APCI-MS

A method of increasing the sensitivity of 3β-hydroxy-5-ene (Δ 5)-steroids in liquid chromatography–atmospheric pressure chemical ionization-mass spectrometry (LC–APCI-MS) based on the structural conversion by cholesterol oxidase (ChO) was demonstrated. The Δ 5-steroids were rapidly converted to their 3-oxo-4-ene (Δ 4)-forms by the treatment with ChO and the obtained Δ 4-forms provided 3–14-fold higher sensitivity compared to intact steroids in the positive-APCI-MS. This enzymic conversion method was also applied to the sulfated conjugates of Δ 5-steroids after solvolysis. The method enabled the detection of trace levels of dehydroepiandrosterone and androstenediol 3-sulfate in human serum, which could not be detected by the usual LC–MS.