A relation between the chemical structure of a xenobiotic and its steroidal action has not yet been clearly established. Thus, it is not possible to define the estrogenic potency of different xenobiotics. An assessment may be accomplished by the use of different bioassays. We have previously developed a yeast system highly and stably expressing rainbow trout estrogen receptor (rtER) in order to analyze the biological activity of the receptor. The recombinant yeast system appears to be a reliable, rapid and sensitive bioassay for the screening and determination of the direct interaction between ER and estrogenic compounds. This system was used in parallel with a more elaborate biological system, trout hepatocyte aggregate cultures, to examine the estrogenic potency of a wide spectrum of chemicals commonly found in the environment. In hepatocyte cultures, the vitellogenin gene whose expression is principally dependent upon estradiol was used as a biomarker. Moreover, competitive binding assays were performed to determine direct interaction between rtER and xenobiotics. In our study, 50% of the 49 chemical compounds tested exhibited estrogenic activity in the two bioassays: the herbicide diclofop-methyl; the fungicides biphenyl, dodemorph, and triadimefon; the insecticides lindane, methyl parathion, chlordecone, dieldrin, and endosulfan; polychlorinated biphenyl mixtures; the plasticizers or detergents alkylphenols and phthalates; and phytoestrogens. To investigate further biphenyl estrogenic activity, its principal metabolites were also tested in both bioassays. Among these estrogenic compounds, 70% were able to activate rtER in yeast and hepatocytes with variable induction levels according to the system. Nevertheless, 30% of these estrogenic compounds exhibited estrogenic activity in only one of the bioassays, suggesting the implication of metabolites or different pathways in the activation of gene transcription. This paper shows that it is important to combine in vivo bioassays with in vitro approaches to elucidate the mechanism of xenoestrogen actions.
F Petit, P Le Goff, JP Cravedi, Y Valotaire and F Pakdel
Tara Sabo-Attwood, Jason L Blum, Kevin J Kroll, Vishal Patel, Detlef Birkholz, Nancy J Szabo, Suzanne Z Fisher, Robert McKenna, Martha Campbell-Thompson and Nancy D Denslow
The estrogen receptor (ER) signaling cascade is a vulnerable target of exposure to environmental xenoestrogens, like nonylphenol (NP), which are causally associated with impaired health status. However, the impact of xenoestrogens on the individual receptor isotypes (α, βa, and βb) is not well understood. The goal of these studies was to determine the impact of NP on largemouth bass (Micropterus salmoides) ER isotype expression and activity. Here, we show that hepatic expression levels of three receptors are not equivalent in male largemouth bass exposed to NP by injection. Transcript levels of the ERα subtype were predominantly induced in concert with vitellogenin similarly to fish exposed to 17β-estradiol (E2) as measured by quantitative real-time PCR. NP also induced circulating plasma levels of estrogen, which may contribute to overall activation of the ERs. To measure the activation of each receptor isotype by E2 and NP, we employed reporter assays using an estrogen response element (ERE)–luciferase construct. Results from these studies show that ERα had the greatest activity following exposure to E2 and NP. This activity was inhibited by the antagonists ICI 182 780 and ZM 189 154. Furthermore, both βb and βa subtypes depressed ERα activation, suggesting that the cellular composition of receptor isotypes may contribute to the overall actions of estrogen and estrogenic contaminants via the receptors. Results from these studies collectively reveal the differential response of fish ER isotypes in response to xenoestrogens.
Ewa Harasim-Symbor, Karolina Konstantynowicz-Nowicka and Adrian Chabowski
Synthetic and natural glucocorticoids are able to highly modify liver lipid metabolism, which is possibly associated with nonalcoholic fatty liver disease development. We have assessed the changes in lipid and sphingolipid contents in hepatocytes, lipid composition and saturation status as well as the expression of proteins involved in fatty acid transport after both dexamethasone and palmitate treatments. The experiments were conducted on primary rat hepatocytes, incubated with dexamethasone and/or palmitic acid during short (16 h) and prolonged (40 h) exposure. Intracellular and extracellular lipid and sphingolipid contents were assessed by gas liquid chromatography and high-performance liquid chromatography, respectively. The expression of selected proteins was estimated by Western blotting. Short and prolonged exposure to dexamethasone combined with palmitic acid resulted in increased expression of fatty acid transporters, which was subsequently reflected by excessive intracellular accumulation of triacylglycerols and ceramide. The expression of microsomal transfer protein and cassette transporter was also significantly increased after dexamethasone and palmitate treatment, which was in accordance with elevated extracellular lipid and sphingolipid contents. Our data showed additive effects of dexamethasone and palmitate on protein-dependent fatty acid uptake in primary hepatocytes, resulting in the increased accumulation of triacylglycerols and sphingolipids. Moreover, the combined treatment altered fatty acid composition and diminished triacylglycerols desaturation index. Importantly, we observed that additive effects on both increased microsomal transport protein expression as well as elevated export of triacylglycerols, which may be relevant as a liver protective mechanism.
P R Shepherd, B T Navé and S O'Rahilly
Muscle and adipose tissue play a central role in the maintenance of glucose homeostasis as they account for the majority of insulin-mediated glucose disposal in the post-prandial state. In obese and diabetic subjects, resistance to the stimulatory effects of insulin on glucose disposal into muscle and fat are instrumental in the development of the chronic hyperglycaemic state associated with these conditions. Studies using a range of techniques including nuclear magnetic resonance studies (Rothman et al. 1992), in vivo forearm perfusion studies (Yki-Jarvinen et al. 1990), indirect calorimetry (Butler et al. 1990) and biochemical assays in isolated muscle strips (Dohm et al. 1988, Andreasson et al. 1991) have demonstrated that the insulin resistance appears to be due to the additive effects of defects in multiple aspects of insulin action in muscle. These investigations have identified defects at the level of insulin stimulation of glucose transport across the plasma membrane as
Bolander FF Jr
Prolactin (PRL) is capable of stimulating both calcium and nitric oxide (NO) accumulation in mammary epithelial cells within 15min. A calcium ionophore was also able to stimulate NO levels to an extent similar to that generated by PRL. Furthermore, maximal concentrations of PRL and the ionophore were not additive, suggesting that they were both using the same pathway, i.e. calcium. Finally, the depletion of intracellular calcium completely abrogated the effect of PRL on NO production. No other pathway known to affect NO synthase (NOS) influenced the action of PRL. Specifically, manipulations of protein phosphatase 2B, protein kinase B (PKB), protein kinase C (PKC), and arginine transport did not alter the activation of NOS by PRL. Therefore, the ability of PRL to stimulate NO production at 15min can be completely explained by its ability to elevate intracellular calcium.
Karen Jesus Oliveira, Maria Isabel Chiamolera, Gisele Giannocco, Carmen Cabanelas Pazos-Moura and Tania Maria Ortiga-Carvalho
The modern concept of thyroid disruptors includes synthetic chemicals and bioactive compounds from food that interfere with any aspect of the hypothalamus–pituitary–thyroid axis, thyroid hormone biosynthesis and secretion, blood and transmembrane transport, metabolism and local actions of thyroid hormones. This review highlights relevant disruptors that affect populations through their diet: directly from food itself (fish oil and polyunsaturated fatty acids, pepper, coffee, cinnamon and resveratrol/grapes), through vegetable cultivation (pesticides) and from containers for food storage and cooking (bisphenol A, phthalates and polybrominated diphenyl ethers). Due to the vital role of thyroid hormones during every stage of life, we review effects from the gestational period to adulthood, including evidence from in vitro studies, rodent models, human trials and epidemiological studies.
P. Enyedi, T. Balla, F. A. Antoni and A. Spät
We have previously shown that arginine vasopressin (AVP) stimulates the production of aldosterone in isolated superfused adrenal glomerulosa cells by a mechanism that involves an increased turnover of phosphoinositides. In the present study we compared the characteristics of AVP- and angiotensin II (AII)-induced changes in phosphoinositide turnover and aldosterone production in the rat. Selected concentrations of the two peptides, which were equipotent in terms of the magnitude of changes induced in phosphoinositide turnover, stimulated aldosterone production to the same extent only in the initial phase of the stimulation. A sustained aldosterone response was only observed in AII-stimulated cells. On the other hand, the AVP-induced increase in incorporation of [32P]phosphate into phosphatidyl-inositol and the stimulation of inositol phosphate production were maintained during incubation. Preincubation of the cells with AVP failed to modify the effects of AII on phosphoinositide breakdown or aldosterone production. These results indicate that desensitization at the level of the receptor or at a post-receptor site is not responsible for the transient character of AVP-induced aldosterone production. Delayed activation of an inhibitory mechanism by AVP can also be excluded. Additivity of the stimulation of the phosphoinositide turnover observed at submaximally, but not maximally, effective concentrations of AII indicates that the two agonists act on the same phosphoinositide pool. We suggest that the sustained steroidogenic effect of AII involves an as yet unidentified mechanism, which is absent when the cells are stimulated with AVP.
C Massart, J Gibassier, C Lucas, F Le Gall, S Giscard-Dartevelle, J Bourdinière, M S Moukhtar and M Nicol
We studied the hormonal secretion of a human mixed follicular and medullary carcinoma. Thyroglobulin (Tg) secretion, especially by large cells and sometimes by small ones, was visualized with immunoenzymatic staining. Calcitonin (CT) was produced by small spindle-shaped cells. Moreover, immunofluorescence double staining performed on the resected thyroid tissue showed the secretion of both Tg and CT in a small number of cells. The cells lost their hormonal secretion after 2 months of culture. Hormonal secretion was modulated by different additives in the medium. Tg secretion was induced when TSH was added to the culture medium; the maximal effect was produced with the addition of 1 mU TSH/ml and 1 μm cortisol, which potentiated the effect of TSH on Tg production. A durable Tg secretion was obtained by embedding the cells in Engelbretch—Hohn—Swarn (EHS) tumour matrix. The CT production was reinduced by the addition of 4 mm Ca2+, 1 μm glucagon and 1 μm cortisol to the culture medium. These findings show that different cells are found in a mixed follicular and medullary carcinoma, some of which can secrete both CT and Tg. They can remain differentiated for a long period after being embedded in EHS tumour matrix with Ca2+ and hormonal components.
B. Quérat, A. Hardy and Y.A. Fontaine
The gonadotrophic function of the European eel (Anguilla anguilla L.) at the silver stage is very weak: gonadotrophin-releasing hormone (GnRH) secretion is deficient and, moreover, dopamine inhibition overrides GnRH action. At the silver stage, œstradiol stimulates the biosynthesis of the type-II gonadotrophin (GTH-II). To study the molecular mechanism of this activation further, we examined the effect of testosterone and œstradiol administration on pituitary levels of mRNA encoding GTH-II α and β subunits. Corresponding eel cDNA probes and Northern blot analysis were used. After 2 weeks, testosterone and œstradiol implantation resulted in a strong increase in mRNA encoding the GTH-II β subunit (7-fold and 25-fold, respectively) and in a slight, but non-significant, rise in the a subunit mRNA level (1.8-fold and 1.5-fold, respectively). Co-implantation of these two steroids suggested a potentiation of their effects on the β subunit (104-fold) while an additive effect was indicated on the α mRNA level (2.9-fold). Effects were detectable within 4 days and were maximal 4 weeks after implantation. These results indicate that in the European eel at the silver stage, gonadal steroids stimulate differentially the expression of GTH-II subunit genes at a pretranslational level.
B Gallwitz, M Witt, U R Fölsch, W Creutzfeldt and W E Schmidt
Glucagon-like peptide-1(7–36)amide (GLP-1(7–36) amide) and gastric inhibitory polypeptide (GIP), peptides of the glucagon family, stimulate insulin secretion in vitro and in vivo. They possess high N-terminal sequence homology. Binding studies with 125I-labelled GIP and 125I-labelled GLP-1(7– 36)amide were performed in RINm5F insulinoma cells to investigate receptor specificity and to compare both receptors directly. Both binding sites were highly ligand-specific: GIP did not bind to the GLP-1(7–36)amide receptor and vice versa. Both peptides increased intracellular cyclic AMP levels; GLP-1(7– 36)amide was 100-fold more potent in stimulating cyclic AMP production when compared with GIP. At ranges of 1–10 nmol GLP-1(7–36)amide/1 and 0·1–10 GIP/1, corresponding to submaximal binding concentrations, the hormones showed an additive effect on cyclic AMP production. The N-terminal portion of GIP was important for binding, as GIP(1–30) showed almost full binding and biological activity. GIP(17–42) bound in a concentration-dependent manner with approximately 500-fold lower potency than GIP. At concentrations of up to 10 μmol GIP(17–42)/1 no stimulation of cyclic AMP was observed.