Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture.

Alkylphenols act as estrogen mimics by binding to and transactivating estrogen receptors (ERs) in fish. In the present study, activation of ER-mediated production of the estrogenic biomarker vitellogenin (vtg) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes was used to construct a structure-activity relationship for this ubiquitous group of aquatic pollutants. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure was assessed. The results showed that most alkylphenols were estrogenic, although with 3-300 thousand times lower affinity than the endogenous estrogen 17beta-estradiol. Mono-substituted tertiary alkylphenols with moderate (C4-C5) and long alkyl chain length (C8-C9) in the para position exhibited the highest estrogenic potency. Substitution with multiple alkyl groups, presence of substituents in the ortho- and meta-position and lack of a hydroxyl group on the benzene ring reduced the estrogenic activity, although several estrogenic alkylated non-phenolics were identified. Co-exposures with the natural estrogen 17beta-estradiol led to identification of additional estrogenic compounds as well as some anti-estrogens. A combination of low affinity for the ER and cytotoxicity was identified as factors rendering some of the alkylphenols non-estrogenic in the bioassay when tested alone.

Use of fish in vitro hepatocyte assays to detect multi-endpoint toxicity in Slovenian river sediments.

There is an increasing demand for rapid, sensitive and robust methods for toxicity testing of single chemicals, complex mixtures and environmental samples. The objective of this work was to validate and use a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes as a multi-endpoint in vitro bioassay for toxicity characterisation of river sediments from four areas of the Sava and Krupa Rivers (Slovenia). The endpoints were chosen to encompass acute toxicity (cytotoxicity) as well as sub-lethal biomarker and effect endpoints such as metabolic inhibition, DNA damage (Fast Micromethod), endocrine disruption (estrogenicity), and 7-ethoxyresorufin O-deethylase (EROD) activity. Results from these studies show that the primary hepatocyte culture was able to successfully detect effects of single model chemicals in all endpoints analysed. Furthermore, the bioassays were also able to discriminate between contaminated and less contaminated sediments for a number of endpoints such as cytotoxicity, metabolic inhibition and induction of EROD activity, although no increase in DNA damage and estrogenicity was observed above background at any site. The present study shows that primary fish hepatocytes may be used to determine multiple mechanisms of toxic action and that a holistic assessment of effects may improve our understanding of cellular toxicity of complex mixtures such as sediments.

Effluents from oil production activities contain chemicals that interfere with normal function of intra- and extra-cellular estrogen binding proteins.

Some environmental pollutants have the ability to alter the endocrine function in fish through interaction with the estrogen receptor (ER). Many of these chemicals are also able to interfere with the endocrine system through other mechanisms of action, however. The plasma sex steroid-binding protein (SBP), which is involved in regulating circulating levels of endogenous sex steroids, has recently been proposed to contribute to pollutant induced disruption of endocrine homeostasis. The objective of the present work was to determine whether industrial effluents contain chemicals that are able to modulate the endocrine system through interference with the function of the ER and SBP using in vitro biological assays (bioassays) from rainbow trout. The results show that solid phase extracts of process water (produced water) from an oil production facility in the North Sea and a land-based oil refinery contain chemicals that are able to induce estrogenic effects as well as displace natural sex steroid 17beta-estradiol from the SBP. The bioactive chemicals were found to be partly resistant to biological degradation, but the identity of the chemicals was not determined. The alkylphenol 4-tert-butylphenol, which is known to occur in effluents from various oil production facilities, was found to be estrogenic and displace 17beta-estradiol from the SBP and may thus contribute to the observed endocrine disrupting activity.

Characterization of a novel Eph receptor tyrosine kinase, EphA10, expressed in testis.

In mammals, 14 members of the Eph receptor tyrosine kinase family have been described so far. Here we present a not yet described member of this family denoted EphA10. We report the identification of three putative EphA10 isoforms: one soluble and two transmembrane isoforms. One of the latter isoforms lacked the sterile alpha motif commonly found in Eph receptors. The gene encoding EphA10 is located on chromosome 1p34 and expression studies show that EphA10 mRNA is mainly expressed in testis. Binding studies to ephrin ligands suggests that this receptor belongs to the EphA subclass of Eph receptors binding mainly to ephrin-A ligands.

Ephrin-A1 binding to CD4+ T lymphocytes stimulates migration and induces tyrosine phosphorylation of PYK2.

Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape, and mobility. Here we demonstrate that CD4+ T lymphocytes express the EphA1 and EphA4 receptors and that these cells bind the ligand ephrin-A1. Further we show ephrin-A1 expression in vivo on high endothelial venule (HEV) endothelial cells. Ephrin-A1 binding to CD4+ T cells stimulates both stromal cell-derived factor 1alpha (SDF-1alpha)- and macrophage inflammatory protein 3beta (MIP3beta)-mediated chemotaxis. In line with the increased chemotactic response, increased actin polymerization is observed in particular with the combination of ephrin-A1 and SDF-1alpha. Signaling through EphA receptors induces intracellular tyrosine phosphorylation. In particular, proline-rich tyrosine kinase 2 (PYK2) is phosphorylated on tyrosine residues 402 and 580. Ephrin-A1-induced chemotaxis and intracellular tyrosine phosphorylation, including EphA1 and Pyk2, was inhibited by Tyrphostin-A9. In conclusion, ligand engagement of EphA receptors on CD4+ T cells stimulates chemotaxis, induces intracellular tyrosine phosphorylation, and affects actin polymerization. This, together with our finding that ephrin-A1 is expressed by HEV endothelial cells, suggests a role for Eph receptors in transendothelial migration.

Identification of a novel centrosome/microtubule-associated coiled-coil protein involved in cell-cycle progression and spindle organization.

Here we describe the identification of a novel vertebrate-specific centrosome/spindle pole-associated protein (CSPP) involved in cell-cycle regulation. The protein is predicted to have a tripartite domain structure, where the N- and C-terminal domains are linked through a coiled-coil mid-domain. Experimental analysis of the identified domains revealed that spindle association is dependent on the N-terminal and the coiled-coil mid domain. The expression of CSPP at the mRNA level was detected in all tested cell lines and in testis tissue. Ectopic expression of CSPP in HEK293T cells blocked cell-cycle progression in early G1 phase and in mitosis in a dose-dependent manner. Interestingly, mitosis-arrested cells contained aberrant spindles and showed impairment of chromosome congression. Inhibition of CSPP gene expression by small interfering RNAs induced cell-cycle arrest/delay in S phase. This phenotype was characterized by elevated levels of cyclin A, decreased levels of cyclin E and hyperphosphorylation of the S-phase checkpoint kinase Chk1. The activation of Chk1 may indicate a replication stress response due to an inappropriate G1/S-phase transition. Taken together, we demonstrate that CSPP is associated with centrosomes and microtubules and may play a role in the regulation of G(1)/S-phase progression and spindle assembly.

Expression and functional effects of Eph receptor tyrosine kinase A family members on Langerhans like dendritic cells.

BACKGROUND: The Eph receptors are the largest receptor tyrosine kinase family. Several family members are expressed in hematopoietic cells. Previously, the expression of a member of this family, EphA2, was identified on dendritic like cells in tonsils. We therefore specifically examined the expression of EphA2 on in vitro generated dendritic cells. RESULTS: In this study, expression of the EphA2 receptor was identified on in vitro generated Langerhans like dendritic cells compared to in vitro generated dendritic cells. We show that ligand induced engagement of the EphA2 receptor leads to receptor autophosphorylation indicating a functional receptor signaling pathway in these cells. We also observe phosphorylation and dephosphorylation of distinct proteins following ligand activation of EphA receptors. In co-stimulation assays, receptor-ligand interaction reduces the capacity of the Langerhans like dendritic cells to stimulate resting CD4+ T cells. CONCLUSION: Engagement of EphA receptor tyrosine kinases on Langerhans like dendritic cells induces signaling as shown by tyrosine phosphorylation and dephosphorylation of distinct proteins. Furthermore this engagement renders the cells less capable of stimulating CD4+ T cells.