Changes in estrogen receptors alpha and beta expression in the brain of mice exposed prenatally to bisphenol A

The expression of ERs alpha and beta and serotonergic neurons were evaluated in the brains of mice prenatally exposed to Bisphenol A, a known endocrine disrupting chemical (EDc). Bisphenol A was administered orally at a dose of 2ng/g body weight on gestinational days 11-17 to pregnant ICR mice. Newborn male offspring (Bis-A mice) were evaluated for the immunoreactivity of ERs alpha and beta, serotonin, and serotonin transporter positive cells in the dorsal raphe nucleus (DRN). The serum testosterone level was also evaluated. In the Bis-A mice, the expression of ERs alpha and beta at 5 and 13 weeks was increased compared with the controls (P<0.04), but this difference disappeared by the 9th week. The serotonin, serotonin transporter, and testosterone level differences between two groups did not reach significance. Exposure to bisphenol A may have changed the expression of ERs in the brain, but did not directly affect serotonin neurons in the DRN.     

Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro.

Estrogenic isoflavones, such as genistein and daidzein, are present in virtually all natural-ingredient rodent diets that use soy as a source of protein. Since these compounds are endocrine-active, it is important to determine whether the amounts present in rodent diets are sufficient to affect sexual development. The present study consisted of in vitro and in vivo parts. In the in vitro portion, human hepatoma cells were transfected with either rat estrogen receptor (ER) alpha or beta plus an estrogen-responsive luciferase reporter gene. Genistein and daidzein were complete agonists at both ERs, genistein being more potent than daidzein, and both compounds were more potent at ER beta than ER alpha. In combined studies with estradiol, genistein exerted additive effects with estradiol in vitro. In the in vivo portion of the study, groups of six pregnant Sprague-Dawley females were fed one of the following four diets, and the pups were maintained on the same diets until puberty: (1) a natural-ingredient, open-formula rodent diet (NIH-07) containing 16 mg genistein and 14 mg daidzein per 100 g of feed; (2) a soy- and alfalfa-free diet (SAFD) in which casein and corn oil were substituted for soy and alfalfa meal and soy oil, respectively, that contained no detectable isoflavones; (3) SAFD containing 0.02% genistein (GE.02); or (4) SAFD containing 0.1% genistein (GE.1). In the GE.1 group, effects of dietary genistein included a decreased rate of body-weight gain, a markedly increased (2.3-fold) uterine/body weight (U/BW) ratio on postnatal day (pnd) 21, a significant acceleration of puberty among females, and a marginal decrease in the ventral prostate weight on postnatal day (pnd) 56. However, developmental differences among the groups fed SAFD, GE.02, or NIH-07 were small and suggested minimal effects of phytoestrogens at normal dietary levels. In particular, on pnd 21, the U/BW ratio of the GE.02 and NIH-07 groups did not differ significantly from that of the SAFD group. Only one statistically significant difference was detected between groups fed SAFD and NIH-07: the anogenital distance (AGD) of female neonates on pnd 1 whose dams were fed NIH-07 was 12% larger than that of neonates whose dams were fed SAFD. The results suggest that normal amounts of phytoestrogens in natural-ingredient rodent diets may affect one developmental parameter, the female AGD, and that higher doses can affect several other parameters in both males and females. Based on these findings, we do not suggest replacing soy- and alfalfa-based rodent diets with phytoestrogen-free diets in most developmental toxicology studies. However, phytoestrogen-free diets are recommended for endocrine toxicology studies at low doses, to determine whether interactive effects may occur between dietary phytoestrogens and man-made chemicals.     

A quaternary ammonium glucuronide is the major metabolite of lamotrigine in guinea pigs. In vitro and in vivo studies.

Urinary excretion of a variety of quaternary ammonium glucuronides has been generally reported to be confined to humans and some monkeys. Lower animal species appear to lack or have limited ability to form these unusual metabolites. In this report, the excretion of the quaternary ammonium glucuronide of lamotrigine, an investigational 1,2,4-triazine anticonvulsant, in guinea pigs is described. Lamotrigine 2-N-glucuronide accounted for 60% of an i.v. bolus dose of lamotrigine in guinea pig urine. Less than 6% of the dose was excreted unchanged. The pharmacokinetics of lamotrigine after an iv bolus dose of 10 mg/kg were determined with an ion-pairing, reversed-phase HPLC assay. Lamotrigine is a low clearance drug (Cl = 2.51 +/- 0.063 ml/min/kg) with a large volume of distribution (Vss = 2.23 +/- 0.403 liter/kg). The half-life of lamotrigine was 11.5 +/- 2.0 hr. The elimination of the glucuronide was formation rate-limited and it was excreted by extensive tubular secretion. The glucuronide was also formed in Triton-X-100-activated liver microsomes and isolated guinea pig hepatocytes. The KM was 2.10 +/- 0.44 mM and the Vmax was 0.252 +/- 0.0312 nmol/min/mg protein in untreated microsomes. Pretreatment with beta-naphthoflavone did not induce lamotrigine glucuronidation. In hepatocytes, production of the glucuronide was linear for 60 min after a short lag period and 2 mM lamotrigine was not cytotoxic. Lamotrigine is only the second example of a compound that is primarily metabolized to a quaternary ammonium glucuronide in a lower animal species.     

In vitro and in vivo estrogenic activity of chlorinated derivatives of bisphenol A

The estrogenic activity of bisphenol A (BPA) and its chlorinated derivatives, 2-(3-chloro-4-hydroxyphenyl)-2-(4-hydroxyphenyl)propane (3-ClBPA) and 2,2-bis(3-chloro-4-hydroxyphenyl)propane (3,3'-diClBPA) was assessed by determining their relative binding affinity for the human estrogen receptor-alpha and -beta (ERalpha and ERbeta) and also their uterotrophic activity in ovariectomized female rats. BPA and its chlorinated derivatives were active in competing with [3H]17beta-estradiol for their binding to the human ERalpha and ERbeta proteins. While 3-ClBPA and 3,3'-diClBPA competed more effectively for ERalpha binding than BPA (IC50 values of 2.48x10(-5), 1.28x10(-5), and 1.08x10(-4)M, respectively), they had similar activity as BPA for competing the binding to ERbeta (IC50 values of 1.43x10(-5), 1.87x10(-5), and 2.59x10(-5)M, respectively). To determine the uterotropic activity, three doses (10, 50 and 100 mg/kg/day) of BPA and its derivatives were given to mature ovariectomized Sprague-Dawley rats for 3 consecutive days. Treatment of animals with 50 and 100 mg/kg/day of BPA or its chlorinated derivatives caused a significant increase in the uterine wet weight and the endometrial area. The results of our present study demonstrated that the affinities of 3-ClBPA and 3,3'-diClBPA for ERalpha were higher than the affinity of BPA, although the in vivo estrogenic activity of the two chlorinated BPAs in ovariectomized female Sprague-Dawley rats appeared to be comparable to that of BPA.     

Interaction of human estrogen receptors alpha and beta with the same naturally occurring estrogen response elements

Estrogen receptors are derived from two different gene products referred to as estrogen receptor-alpha (ER-alpha) and ER-beta. Both receptors bind to the consensus estrogen response element (ERE) present in the vitellogenin gene, but their binding to hormone response elements present in other estrogen responsive genes has not been reported yet. Using in vitro expressed human receptors, we now show that ER-beta binds to a panel of six endogenous hormone response elements (vitellogenin, c-fos, c-jun, pS2, cathepsin D, and choline acetyltransferase) already known to bind ER-alpha and confer estrogen inducibility to reporter constructs. Binding of ER-alpha and ER-beta occurred at similar DNA concentrations for some EREs, but different DNA concentrations were required to form complexes of the two receptors with other elements. These results illustrate for the first time by direct receptor-DNA binding studies that both ER-alpha and ER-beta bind to a number of EREs present in endogenous hormone regulated genes, and further suggest that the two forms of the receptor display different patterns of affinities for naturally occurring hormone response elements.     

In vivo metabolism of alpha,alpha,beta,beta-tetradeutero-N, N-dimethyltryptamine in rodent brain

The metabolism of alpha,alpha,beta,beta- tetradeutero -N,N -dimethyltryptamine ( D4DMT ) in rat brain in vivo as a function of time and dose was examined. Quantification of D4DMT and its respective deutero-metabolites was accomplished using gas chromatographic/mass spectrometric/selected ion monitoring/isotope dilution techniques. The results of this study indicate that D4DMT is metabolized to the corresponding deutero-N-methyltryptamine, tryptamine, 1,2,3,4-tetrahydro-beta-carboline, and 2-methyl-1, 2,3,4-tetrahydro-beta-carboline in rat brain. The subcellular distribution of D4DMT and the aforementioned metabolites is also reported.     

In vitro interactions of agonists and antagonists with beta-adrenergic receptors

Neurotransmitters and hormones mediate their effects through interaction with specific receptors. A complete understanding of the effects of these chemical signals requires detailed knowledge, at the molecular level, of agonist/receptor interactions. It is likely that agonists and antagonists interact with the same site on a receptor. Agonists, however, are by definition different from antagonists in that agonists are responsible for transducing information across the cell membrane, ultimately resulting in a biological response, while antagonists appear to act through passive occupancy of receptors. Implicit in this concept is the idea that these fundamental differences between agonists and antagonists arise from the sequelae induced by agonist-specific interactions with receptors.     

Differential response of estrogen receptors alpha and beta to SP500263, a novel potent selective estrogen receptor modulator

We determined the differential response of a novel SERM, SP500263, on estrogen receptor (ER) alpha and the more recently cloned ER-beta. Because of the high homology of amino acid residues in the ligand-binding domain of ER-alpha and ER-beta, we were not surprised to find that SP500263 binds to both ERs equally well. In contrast, SP500263 acts as a strong estrogen agonist in a strictly ER-alpha-specific manner in U2OS osteosarcoma cell lines blocking the production of interleukin (IL) 6 and granulocyte macrophage colony-stimulating factor. SP500263 also blocked IL-6 production in primary bone cells. The mechanism of this inhibition is different from the classic estrogen stimulation involving an estrogen response element (ERE). SP500263 does not activate gene expression through an ERE. In contrast to the results observed in U2OS cells, SP500263 acts as a strong estrogen antagonist in an MCF-7 breast cancer proliferation assay. Therefore, SP500263 is a member of a series of next-generation SERMs with functional selectivity toward ER-alpha and a mixed agonist/antagonist profile in a bone cell assay versus a breast cancer assay. The panel of assays described herein allow for the development of receptor-specific ligands that may be further developed into novel pharmaceuticals with an improved profile for the treatments of osteoporosis and breast cancer.     

In vitro synergy between cefotaxime and its main metabolite, desacetylcefotaxime

In vitro synergistic interaction between cefotaxime (CTX) and its main metabolite, desacetyl-cefotaxime (DCTX), against 7 species of clinical isolates (23-27 strains per species) was examined. Complete or partial synergy was noted with a 1:1 combination of CTX and DCTX against 22-78% of the Bacteroides fragilis, Staphylococcus aureus, Citrobacter freundii, Pseudomonas cepacia and Enterobacter cloacae isolates examined. Antagonistic effects of the drugs appeared against 11% of Proteus vulgaris and 4% of Serratia marcescens. When combined at various ratios by the checkerboard method and tested against B. fragilis, CTX and DCTX were found to act synergistically, and no antagonism occurred. The combined use of CTX and DCTX exhibited strong bactericidal activity against B. fragilis and inhibited bacterial regrowth. An experiment with concentrations of CTX and DCTX simulating human serum levels after intravenous administration also showed that the coexistence of DCTX augmented bactericidal activity of CTX against B. fragilis and brought inhibitory effects on bacterial regrowth. It is presumed from the present results that clinically applied CTX would have more potent effects than expected from in vitro sensitivity test data.     

Effects of mycophenolic acid alone and in combination with its metabolite mycophenolic acid glucuronide on rat embryos in vitro

Mycophenolic acid (MPA) is an immunosuppressive agent that acts as a selective, non-reversible inhibitor of the enzyme inosine-5′-monophosphate dehydrogenase (IMPDH). Malformations have been described in children after maternal exposure to mycophenolate. However, the causal link is unclear in most cases because women had been treated with a combination of drugs and birth defects may have other causes. Therefore, it is important to study the action of this drug and its main metabolite on embryonic tissue. We studied the teratogenic potential of MPA and its major metabolite, the mycophenolic acid glucuronide (MPAG) in the rat whole-embryo culture. A total of 147 day 9.5 embryos were cultivated for 48 h in the standard medium containing 85 % serum. We tested MPA at concentrations of 0.1; 0.25; 0.5; 0.75 mg/l (0.31; 0.78; 1.56; 2.34 μM) and MPA glucuronide at concentrations of 3; 10; 30; 100 mg/l (6.04; 20.14; 60.43; 201.43 μM). Both substances are highly protein bound, and MPA glucuronide might displace MPA from protein binding. Therefore, we examined whether the effects of MPA can be enhanced when studied in combination with the glucuronide. Furthermore, the focus was on additional endpoints to the standard evaluation of cultivated embryos, such as development of cranial nerves [trigeminal nerve (V), facial nerve (VII), glossopharyngeal nerve (IX), vagus nerve (X)] after staining with an antibody against 2H3 neurofilament. Ultrastructural changes were evaluated by electron microscopy. At a concentration of 0.75 mg MPA/l medium, all embryos showed dysmorphic changes. Embryos exposed to 0.25 mg MPA/l medium showed impaired development of nerves, and at 0.1 mg/l, no effects were detectable. Concentration-dependent ultrastructural changes, such as signs of apoptosis, were found by electron microscopy. The examination of the metabolite in this assay showed that at a concentration of 100 mg MPAG/l, the embryos exhibited distinct malformations. This is probably caused by MPA, which was detectable at 0.6 % in the material used for our experiments. The combination of the parent compound (0.03; 0.1; 0.25 mg/l) with its metabolite MPAG (3 mg/l) did not cause enhanced toxicity under our experimental conditions. IMPDH, the target enzyme of MPA, could be detected in rat embryos on day 9.5 of embryonic development as well as at the end of the culture period 48 h later. In summary, MPA impairs embryonic development at low, therapeutically relevant concentrations, but the glucuronide does not exhibit such a potential. Activity of MPA is not enhanced by MPAG.     

In vitro and in vivo evaluations of cytochrome P450 1A2 interactions with duloxetine

OBJECTIVE: To determine whether duloxetine is a substrate, inhibitor or inducer of cytochrome P450 (CYP) 1A2 enzyme, using in vitro and in vivo studies in humans. METHODS: Human liver microsomes or cells with expressed CYP enzymes and specific CYP inhibitors were used to identify which CYP enzymes catalyse the initial oxidation steps in the metabolism of duloxetine. The potential of duloxetine to inhibit CYP1A2 activity was determined using incubations with human liver microsomes and phenacetin, the CYP1A2 substrate. The potential for duloxetine to induce CYP1A2 activity was determined using human primary hepatocytes treated with duloxetine for 72 hours. Studies in humans were conducted using fluvoxamine, a potent CYP1A2 inhibitor, and theophylline, a CYP1A2 substrate, as probes. The subjects were healthy men and women aged 18-65 years. Single-dose duloxetine was administered either intravenously as a 10-mg infusion over 30 minutes or orally as a 60-mg dose in the presence or absence of steady-state fluvoxamine (100 mg orally once daily). Single-dose theophylline was given as 30-minute intravenous infusions of aminophylline 250 mg in the presence or absence of steady-state duloxetine (60 mg orally twice daily). Plasma concentrations of duloxetine, its metabolites and theophylline were determined using liquid chromatography with tandem mass spectrometry. Pharmacokinetic parameters were estimated using noncompartmental methods and evaluated using mixed-effects ANOVA. Safety measurements included vital signs, clinical laboratory tests, a physical examination, ECG readings and adverse event reports. RESULTS: The in vitro results indicated that duloxetine is metabolized by CYP1A2; however, duloxetine was predicted not to be an inhibitor or inducer of CYP1A2 in humans. Following oral administration in the presence of fluvoxamine, the duloxetine area under the plasma concentration-time curve from time zero to infinity (AUC(infinity)) and the maximum plasma drug concentration (C(max)) significantly increased by 460% (90% CI 359, 584) and 141% (90% CI 93, 200), respectively. In the presence of fluvoxamine, the oral bioavailability of duloxetine increased from 42.8% to 81.9%. In the presence of duloxetine, the theophylline AUC(infinity) and C(max) increased by only 13% (90% CI 7, 18) and 7% (90% CI 2, 14), respectively. Coadministration of duloxetine with fluvoxamine or theophylline did not result in any clinically important safety concerns, and these combinations were generally well tolerated. CONCLUSION: Duloxetine is metabolized primarily by CYP1A2; therefore, coadministration of duloxetine with potent CYP1A2 inhibitors should be avoided. Duloxetine does not seem to be a clinically significant inhibitor or inducer of CYP1A2; therefore, dose adjustment of CYP1A2 substrates may not be necessary when they are coadministered with duloxetine.     

In vitro molecular mechanisms of bisphenol A action

Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS# 80-05-7) is a chemical used primarily in the manufacture of polycarbonate plastic, epoxy resins and as a non-polymer additive to other plastics. Recent evidence has demonstrated that human and wildlife populations are exposed to levels of BPA which cause adverse reproductive and developmental effects in a number of different wildlife species and laboratory animal models. However, there are major uncertainties surrounding the spectrum of BPA's mechanisms of action, the tissue-specific impacts of exposures, and the critical windows of susceptibility during which target tissues are sensitive to BPA exposures. As a foundation to address some of those uncertainties, this review was prepared by the "In vitro" expert sub-panel assembled during the "Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health" workshop held in Chapel Hill, NC, Nov 28-29, 2006. The specific charge of this expert panel was to review and assess the strength of the published literature pertaining to the mechanisms of BPA action. The resulting document is a detailed review of published studies that have focused on the mechanistic basis of BPA action in diverse experimental models and an assessment of the strength of the evidence regarding the published BPA research.     

In vivo effects of bisphenol A in laboratory rodent studies

Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/(kg day), in laboratory exposures with mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol and ethinylestradiol. For most effects, the potency of BPA is approximately 10-1000-fold less than that of diethylstilbestrol or ethinylestradiol. Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long lasting, organizational effects in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely, but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that developmental effects occur in the female reproductive system.     

In vitro and in vivo metabolism of a potent and selective integrin alpha v beta 3 antagonist in rats, dogs, and monkeys.

Compound A (3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyrindin-2-yl)propyl]-imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)-propionic acid), a potent and selective antagonist of integrin alpha(v)beta(3) receptor, is under development for treatment of osteoporosis. This study describes metabolism and excretion of A in vivo in rats, dogs, and monkeys, and metabolism of A in vitro in primary hepatocytes from rats, dogs, monkeys, and humans. In all three animal species studied, A was primarily excreted as unchanged drug and, to a lesser degree, as phase I and phase II metabolites. Major biotransformation pathways of A included glucuronidation/glucosylation on the carboxylic group to form acyl-linked glucuronides/glucosides; and oxidation on the tetrahydronaphthyridine moiety to generate a carbinolamine and its further metabolized products. Minor pathways involved O-demethylation and hydroxylations on the alkyl chain. Only in rats, a glutathione adduct of A was also observed, and its formation is proposed to be via an iminium intermediate on the tetrahydronaphthyridine ring. Similar metabolic pathways were observed in the incubates of hepatocytes from the corresponding animals as well as from humans. CYP 3A and 2D subfamilies were capable of metabolizing A to its oxidative products. Overall, these in vitro and in vivo findings should provide useful insight on possible biotransformation pathways of A in humans.     

A32390A, a new biologically active metabolite. III. In vitro and in vivo antifungal activity.

A32390A, an isonitrile-containing derivative of mannitol, represents a new class of antifungal antibiotics. In vitro antifungal activity of A32390A was found against Candida albicans, Cryptococcus neoformans and Histoplasma capsulatum. In vivo antifungal activity of A32390A was demonstrated in mice infected with C. albicans. Accumulative doses of 37.5 approximately 600 mg/kg, administered subcutaneously over a 24-hour period, showed significant activity without demonstrating toxicity. A32390A was effective, but not as effective as amphotericin B, in reducing the number of Candida cells isolated from the kidney of infected mice. Urinary excretion of A32390A accounted for only 10% of the administered dose. Improved bioavailability of A32390A was accomplished when the antibiotic was combined with polyvinyl pyrrolidone (PVP) in a solid dispersion. Administration of A32390A as a 10% dispersion in PVP resulted in increased urinary excretion of the drug and reduced the amount of drug required for in vivo activity.     

Estrogenic effects of fluorotelomer alcohols for human estrogen receptor isoforms alpha and beta in vitro

The present study demonstrates the estrogenic effects of fluorotelomer alcohols (FTOHs). In a yeast two-hybrid assay, treatment with 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH), 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH) and 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1-decanol (NFDH) showed a dose-dependent interaction between the human estrogen receptor (hER) isoforms hERalpha or hERbeta ligand-binding domain and coactivator TIF2, whereas there were no estrogenic effects of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) for these hERs. The estrogenic effects of FTOHs on hERalpha were higher than those on hERbeta, indicating a differential responsiveness of hERs to FTOHs. The relative ranks of tested chemicals on the estrogenic effects for hERalpha and hERbeta descended in the order of estradiol-17beta>6:2 FTOH>NFDH>8:2 FTOH. These results suggest that certain FTOHs including 6:2 FTOH, 8:2 FTOH and NFDH interact with hER isoforms alpha and beta in vitro. Further studies are necessary to investigate contamination levels, potential biological effects and the risks of these compounds on human health.