Hormonal activity of combinations of genistein, bisphenol A and 17β-estradiol in the female Wistar rat

Phytoestrogens have been described as weak estrogens,selective estrogen receptor mediators (SERMs) or to exhibit antiestrogenic properties. However, information about their activity in combination with xenoestrogens and 17β-estradiol in vivo, is limited. Therefore, the combinatory activity of the phytoestrogen genistein (Gen), the industrial chemical bisphenol A (BPA), and ethinylestradiol (EE) in ovariectomized Wistar rats was analyzed in this study. All compounds were administered orally on three consecutive days (EE at 30 μg, Gen at 100 mg and BPA at 200 mg per kg body weight per day). The pure antiestrogen fulvestrant (3 mg/kg) served as estrogen receptor (ER) antagonist control. Effects on uterine wet weight, height of the uterine epithelium, uterine clusterin (Clu) and complement C3 expression, and the height of the vaginal epithelium were examined. Treatment with Gen alone resulted in a moderate stimulation of uterine weight; in the vagina the height of the epithelium was strongly stimulated. BPA did not stimulate any of the above-mentioned parameters significantly. In combination with EE, Gen acted on most of the analyzed parameters in an additive manner, whereas BPA significantly antagonized the effects of EE on the uterine epithelium and uterine Clu expression. Given in combination with Gen, BPA was also able to antagonize the stimulatory effect of Gen on the uterine epithelium. In summary, our results demonstrate that Gen, in contrast to BPA, does not exhibit any antiestrogenic properties, even if given at high concentrations. The results of this study characterize BPA as a functional antiestrogen, very likely the result of a lack of ability to activate ER-mediated transactivation after binding to the receptor. This is not the case for Gen. Our results point to the involvement of complex molecular mechanisms in the action of Gen. These mechanisms, especially the role of ERβ have to be characterized in further investigations.     

The xenoestrogens ethinylestradiol and bisphenol A regulate BCRP at the blood–brain barrier of rats

Abstract

1. Breast cancer resistance protein (BCRP) is an ABC-transporter at the blood–brain barrier (BBB) facilitating efflux of xenobiotics into blood. Expression and function are regulated via estrogen-receptors (ERs).

2. 17α-Ethinylestradiol (EE2) and bisphenol A (BPA) represent two prominent xenoestrogens. We studied whether EE2 and BPA regulate BCRP function and expression upon a 6?h treatment in an ER-dependent manner in a rat BBB-ex-vivo-model.

3. Isolated brain capillaries were incubated with EE2 or BPA. BCRP function and expression were analyzed by confocal microscopy and Western-Blot. ERα-antagonist MPP and ER-antagonist ICI182.780 were used to study involvement of ERs.

4. EE2 and BPA down-regulated BCRP transport function and expression. EE2 effects occurred at pharmacologically relevant doses, BPA exhibited only weak influences. Down-regulation by EE2 was reversed by ICI but not MPP. BPA effects were not reversed by either antagonist.

5. EE2 is a potent regulator of BCRP expression and function acting by ERβ-stimulation. Oral contraception could alter uptake of pharmaceuticals to the brain and might thus be considered as an origin of central nervous system (CNS) side-effects. EE2 could also present a novel co-treatment to improve CNS-pharmacotherapy. BPA is a weak modulator of BCRP expression. Its effects appear not to be caused by ERs.     

Subacute oral toxicity study of bisphenol F based on the draft protocol for the “Enhanced OECD Test Guideline no. 407”

Since bisphenol F (4,4’-dihydroxydiphenylmethane) has been reported to exhibit estrogen agonistic properties in the uterotrophic assay, we performed a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) on bisphenol F based on the OECD draft protocols to determine whether it has endocrine-mediated properties. Bisphenol F was orally administered at doses 0, 20, 100 and 500 mg/kg per day for at least 28 days, but no clear endocrine-mediated changes were detected, and it was concluded to have no endocrine-mediated effects in young adult rats. On the other hand, the main effect of bisphenol F was concluded to be liver toxicity based on clinical biochemical parameters and liver weight, but without histopathological changes. The no-observed-effect level for bisphenol F is concluded to be under 20 mg/kg per day since decreased body weight accompanied by decreased serum total cholesterol, glucose, and albumin values were observed in the female rats given 20 mg/kg per day or higher doses of bisphenol F.     

Inhibition of human and rat 11β-hydroxysteroid dehydrogenases activities by bisphenol A

Bisphenol A (BPA) is a potential endocrine disruptor. It has been shown that it can interfere with steroid biosynthesis and metabolism. However, the mechanism is unclear. The objective of the present study is to investigate the effects of BPA on two isoforms of 11β-hydroxysteroid dehydrogenases (11β-HSD1 and 11β-HSD2) in human and rat tissues. Human liver, rat testis microsomes as well as rat adult Leydig cells were used for measurement of 11β-HSD1 activity, and human and rat kidney microsomes for 11β-HSD2 activity. BPA inhibited human and rat 11β-HSD1 activities with the half maximal inhibitory concentrations (IC₅₀s) of 14.81 ± 0.06 μM (mean ± SEM) for human and 19.39 ± 0.09 μM for rat enzyme, respectively. BPA inhibited rat 11β-HSD1 activity in intact rat Leydig cells. BPA also weakly inhibited both human and rat 11β-HSD2 activities. At 100 μM, BPA inhibited human and rat enzymes by 51.16% and 41.61%, respectively. In conclusion, BPA is an inhibitor for both 11β-HSD1 and 11β-HSD2, with selectivity against the type I enzyme.     

Low-dose effects of bisphenol A: a serious threat to human health?

The author tried to review and summarize low-dose effects of endocrine disrupting chemicals (EDCs) through an extensive literature survey of toxicological studies with bisphenol A (BPA), taking BPA as an example for which many studies were published. Data on low-dose effects with BPA, especially on neurobehavioral effects after fetal or early postnatal exposures, suggested that there would be new aspects to be considered. Specific mention for future tasks was made. Firstly, toxicity tests should be designed with more elaboration to ensure a sufficient number of animals with careful handling of litters to allow adequate statistical analysis and appropriate selection of dosages to obtain insight in dose-response relationship. Secondly, precise measurement of plasma levels in both humans and rodents and construction of relevant physiologically-based pharmacokinetic models would help obtain quantitative estimates of intake and target-organ exposure relationship. Thirdly, biological backgrounds, particularly differences and similarities in endocrinological, neurological and immunological aspects among species, should be revisited. Fourthly, mechanistic deliberations on the possibilities of epigenetic mechanism and examinations of putative neurobehavioral effects or a presumptive link of miscarriage with BPA exposures are requested. Finally, general public concerns must be addressed in a thoughtful way so that a simple precautionary approach is not pursued, but uncertainties of the new toxicological aspects should be carefully explained. Further researches and internationally concerted efforts on elucidating risk of low-dose effects by integrating knowledge will contribute to setting new directions in toxicology and improving chemical risk assessments.     

Strain specific induction of pyometra and differences in immune responsiveness in mice exposed to 17α-ethinyl estradiol or the endocrine disrupting chemical bisphenol A

Pyometra is an inflammatory disease of the uterus that can be caused by chronic exposure to estrogens. It is unknown whether weakly estrogenic endocrine disruptors can cause pyometra. We investigated whether dietary exposures to the estrogenic endocrine disruptor bisphenol A (BPA) induced pyometra. Pyometra did not occur in CD1 mice exposed to different dietary doses of BPA ranging from 4.1 to >4000μg/kg-d or 17α-ethinyl estradiol (EE; 1.2 to >150μg/kg-d). In the C57BL/6 strain, pyometra occurred in the 15μg/kg-d EE and 33μg/kg-d BPA treatment groups. At the effective concentration of BPA, histological analysis revealed pathological alterations of uterine morphology associated with a >5.3-fold increase in macrophage numbers in non-pyometra uteri of C57BL/6 mice exposed to BPA. These results suggest that BPA enhances immune responsiveness of the uterus and that heightened responsiveness in C57BL/6 females is related to increased susceptibility to pyometra.     

Inhibition of the Formation of the Alzheimer's β-Amyloid Peptide

Anorexia and body weight loss are characteristic of many diseases, including cancer and AIDS. Neuropeptides play a pivotal role in the physiological mechanisms regulating food intake and body weight. Neuropeptide Y (NPY) is a key molecule of the orexigenic network for energy intake and for normal adaptive feeding response to energy deficits. Therefore, the NPY receptors (notably, Y1 and Y5) may be one of the most significant target classes for treatment of anorexia and body weight loss. In addition, several anorexigenic peptides including corticotropin-releasing factor (CRF), cholecystokinin (CCK), leptin and melanocortin (MC) are emerging as potential targets for anorexia. Antagonists for CRF 2 receptor, CCK A receptor, MC 4 receptor and leptin receptor may be useful in stimulating food intake. The development of highly specific and selective non-peptide antagonists for these receptors is awaited. Anorexia is a crucial and critical disease. Increasing knowledge of its pathophysiology could lead to innovative new medicines for anorexia-cachexia syndrome.     

Effects of the administration of miconazole by different routes on the biomarkers of the “steroidal module” of the Athlete Biological Passport

This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the “steroidal module” of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC–MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5β-androstane-3α,17β-diol, and 5α-androstane-3α,17β-diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α-androstane-3α,17β-diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1–12.5 μg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 μg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme β-glucuronidase during the sample preparation process. The increase of both incubation time and amount of β-glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results.     

Possible involvement of the opioid receptor gene expression in the mechanisms of the analgesic action of melatonin

In the present study, we attempt to analyse the potential involvement of the opioid receptor gene expression in the mechanisms of the analgesic action of melatonin. A trauma-pain model was established in Wistar rats by combining right - hind limb amputation with 50℃ tail - flick test. Antinociception was determined by tail-flick latency to hot waster at 50℃. Melatonin produced the antinociceptive effect in dose-dependent manner after i. p or i. c.v. administration Injected i. c. v. to rats, naloxone（10μg） obviously antagonized the antinociceptive effect induced by i.p. melatonin.     

Vitellogenic responses of 17β-estradiol and bisphenol A in male Chinese loach (Misgurnus anguillicaudatus)

In this study, male Chinese loaches were exposed to 17β-estradiol (E2), bisphenol A (BPA) and their mixtures, respectively, for 42 days using a semi-static exposure system. Plasma vitellogenin (Vtg) in male Chinese loaches was used as the determining endpoint. The results demonstrated that male Chinese loaches were sensitive to E2, and the vitellogenic responses showed time- and dose-dependent increase. Similarly, BPA induced the estrogenic effects in male Chinese loaches, and the vitellogenic response increased in a time- and dose-dependent manner. The synthesis of Vtg was initiated by the exposure to higher level of BPA (500μg/L) within 7 days, and a relative long-term exposure to lower concentrations of BPA (10-100μg/L) also led to the production of Vtg. The estrogenic effect of the binary mixture of E2 and BPA also showed a time- and dose-dependent response, which was more potent than that of individual compounds, and Vtg contents in the binary mixture group were higher than the summation of Vtg contents in the single-compound groups at the same concentration.     

Proton pump inhibitors: the beginning of the end or the end of the beginning?

Despite the dramatic success of pharmacological acid suppression in healing peptic ulcers (PUs) and managing patients with gastro-esophageal reflux disease (GERD) a number of challenges remain in the management of acid-related disorders. Several new drugs are currently being investigated to provide a significant advance over current treatments. These include new drug formulations, novel proton pump inhibitors (PPIs) as well as potassium-competitive acid blockers (P-CABs), which have already reached clinical testing. Some others (like NO-releasing antisecretory compounds) are still in preclinical development and require proof of concept in humans. While H(2)-receptor antagonists (especially soluble or OTC formulations) will become the 'antacids of the third millennium' and will be particularly useful for on-demand symptom relief, clinicians will continue to rely on PPIs to control acid secretion in GERD and other acid-related diseases. Since an increasing proportion of patients fail to respond to the best PPI treatment, more potent and long-acting drugs and more effective regimens are needed.     

Hydrogen sulfide: from the smell of the past to the mediator of the future?

Gases such as nitric oxide and carbon monoxide play important roles both in normal physiology and in disease. In recent years, interest has been directed towards other naturally occurring gases, notably hydrogen sulfide (H₂S), which is both a potent vasodilator and a mediator of long-term potentiation in the brain. This article focuses on recent work that suggests a role for H₂S, and perhaps other gases, in the CNS and cardiovascular system.     

A review of the fate of inhaled α-quartz in the lungs of rats

The distribution of dust particles within the lungs and their excretion are highly associated with their pulmonary toxicity. Literature was reviewed to discern pulmonary translocation pathways for inhaled α-quartz compared to those for inhaled TiO₂. Accordingly, it was hypothesized α-quartz particles in the alveoli were phagocytized by alveolar macrophages but silica-containing macrophages remained in the alveoli for longer time in contrast to the rapid elimination from the alveoli seen for TiO₂-containing macrophages. In addition, it was presumed that free silica particles are translocated in the interstitium, possibly through the cytoplasm of Type I epithelial cells, as observed with TiO₂. Free silica particles are presumed to be phagocytized by interstitial macrophages soon after the particles penetrate the interstitium; these dust cells are then translocated to the ciliated airway regions in the lumen through bronchus-associated lymphoid tissue (BALT). The pulmonary retention half-time of dust particles in rats exposed to α-quartz is several times longer than that of rats exposed to TiO₂, as long as the lung dust burden is ≈ 3?mg. The reduced pulmonary particle clearance ability in rats exposed to α-quartz aerosol is presumably attributed to the long-term retention of dust cells both in the alveoli and in the interstitium; this retention may be caused by the reduced chemotactic abilities of α-quartz-containing dust cells. However, the accumulation of α-quartz-containing dust cells in the lungs is not associated with the occurrence of pulmonary inflammation.     

Increase in the levels of chaperone proteins by exposure to β-estradiol,bisphenol A and 4-methoxyphenol in human cells transfected with estrogen receptor α cDNA

We examined changes in the levels of chaperone proteins to evaluate the toxic effects of environmental chemicals in human cells in vitro. Some chaperones are up-regulated by estrogenic chemicals, but the effect is not necessarily dependent on the receptor. Thus we also investigated whether a chemical-induced change in chaperone protein expression is human estrogen receptor (hER)-dependent or not, using cultured human cell lines transfected with hERα cDNA or an empty vector. In the hERα-expressed cells, the protein levels of the heat shock protein 27 (HSP27), the glucose-regulated protein 78 (GRP78/BiP), and GRP94 increased after exposure to β-estradiol (E₂) (from 10^?9 M to 10^?6 M) and bisphenol A (BPA) (from 10^?6 M to 10^?5 M). On the other hand, the increase was not observed in the cells without hERα expression. These results suggest that the E₂- and BPA-induced increase in the protein levels were hERα dependent. We next examined the effect of four phenolic chemicals similar in structure to BPA, and found that among them, 4-methoxyphenol (from 10^?6 M to 10^?5 M) increased the levels of the chaperone proteins with hERα dependency. Thus the human cultured cells would be suitable for evaluating whether an increase in chaperone proteins occurs upon exposure to environmental chemicals and whether the effect is ER-dependent.     

Blockade of γ-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen

Summary When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, Choecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mol/l). This release is affected by dopamine and by -aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D₂-receptors and decreases it via D₁-receptors. GABA_A-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABA_B-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D₂-receptors. The GABA_A-receptor antagonist bicuculline (10 mol/l)and the blocker of the GABA_A-receptor ionophore picrotoxin (1 mol/l) did not affect the dopamine (0.1 mol/1)-induced increase in the release of CCK-LI. However, the GABA_A-receptor agonist muscimol (1 mol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mol/l). This effect of muscimol was blocked by bicuculline (10 mol/l). In the presence of -amino-n-valeric acid (0.1 mmol/l), which has been described to block GABA_B-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. -Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mol/l) and 1 mol/l in the presence of the preferential D₁-receptor antagonist SCH 23390. The effect of -amino-n-valeric acid persisted in the presence of bicuculline (10 mol/l and 100 mol/l). (+)-Baclofen, a partial agonist at GABA_B-receptors, and the stereoisomer (–)-baclofen, a full agonist, also prevented the effect of dopamine on the veratridine-induced release of CCK-LI. The effects of both drugs may be due to desensitization of GABA_B-receptors, which has been described to develop quite rapidly. It is concluded that -amino-n-valeric acid blocks GABA_B-receptors and in this way prevents the enhancement of the veratridine-induced release of CCK-LI caused by dopamine via D₂-receptors. These data are interpreted as evidence that dopamine and GABA-neurons can directly or indirectly interact in the rat neostriatum. Send offprint requests to D. K. Meyer at the above address