Leon Brokken

Over the last few decades, there have been numerous reports of adverse effects on the reproductive health of wildlife and laboratory animals caused by exposure to endocrine disrupting chemicals (EDCs). The increasing trends in human male reproductive disorders and the mounting evidence for causative environmental factors have therefore sparked growing interest in the health threat posed to humans by EDCs, which are substances in our food, environment and consumer items that interfere with hormone action, biosynthesis or metabolism, resulting in disrupted tissue homeostasis or reproductive function. The mechanisms of EDCs involve a wide array of actions and pathways. Examples include the estrogenic, androgenic, thyroid and retinoid pathways, in which the EDCs may act directly as agonists or antagonists, or indirectly via other nuclear receptors. Dioxins and dioxin-like EDCs exert their biological and toxicological actions through activation of the aryl hydrocarbon-receptor, which bes...

Biochemical and immunological characteristics of peroxidase activity of the skin epithelium of common carp (Cyprinus carpio) were investigated and compared with peroxidase activity of blood cells. Skin as well as blood-borne peroxidases eluted from the Superdex column as a 135 kDa protein and both probably are tetrameric molecules. Skin peroxidase activity was characterized by a Vmax of 51.5 ± 1.3 U mg-1 min-1 and a KM of 1.64 ± 0.18 mM ortho-phenylenediamine (OPD), whereas blood-borne peroxidase was characterized by a 1,000 fold higher specific activity (Vmax = 30.5 ċ 103 ± 2.5 ċ 103 U mg-1 min-1) and a higher affinity (KM = 0.875 ± 0.003 mM OPD). Polyclonal antibodies were raised against concanavalin-A purified skin peroxidase as well as blood-borne peroxidase. Immunocytochemical labelling showed that peroxidase is present in mucous cells and in mucus covering the skin and gill epithelia, as well as in erythrocytes and leucocytes. We conclude that the mucous cells of the skin produce a biochemically distinct peroxidase that is released in the mucus and may contribute to the antimicrobial properties of the mucous layer covering the skin. After exposure of the fish to cadmium the kinetic characteristics of the enzyme activity, as determined in skin homogenates, changed considerably. The Vmax increased significantly to 61.9 ± 1.1 U mg-1 min-1, and the affinity for OPD increased to the value demonstrated for blood-borne peroxidase (KM = 0.888 ± 0.045 mM OPD). Increased peroxidase levels after cadmium exposure were also demonstrated immunochemically in a dotblot assay. However, no significant changes were observed when the circulatory system of the fish was perfused prior to sampling, indicating that erythrocytes are a major contributor to the increased peroxidase activity in carp skin during cadmium exposure. This likely reflects the increased vascularization of the connective tissue layer underlying the skin epithelium, which takes place when the fish are exposed to chronic stressors including cadmium. In the cadmium-exposed fish this effect prevented the biochemical detection of stressor-related changes in epithelial peroxidase reported earlier with cytochemical methods.

Fibroblast growth factor-8 (FGF-8) is implicated in the development and progression of breast cancer and its levels are frequently elevated in breast tumors. The mechanisms driving FGF-8-mediated tumorigenesis are not well understood. Herein we aimed to identify target genes associated with FGF-8b-mediated breast cancer cell proliferation by carrying out a cDNA microarray analysis of genes expressed in estrogen receptor negative S115 breast cancer cells treated with FGF-8b for various time periods in comparison with those expressed in non-treated cells. Gene and protein expression was validated for selected genes by qPCR and western blotting respectively. Furthermore, using TRANSBIG data, the expression of human orthologs of FGF-8-regulated genes was correlated to the Nottingham prognostic index and estrogen receptor status. The analysis revealed a number of significantly up- and down-regulated genes in response to FGF-8b at all treatment times. The most differentially expressed genes were genes related to cell cycle regulation, mitosis, cancer, and cell death. Several key regulators of early cell cycle progression such as Btg2 and cyclin D1, as well as regulators of mitosis, including cyclin B, Plk1, survivin, and aurora kinase A, were identified as novel targets for FGF-8b, some of which were additionally shown to correlate with prognosis and ER status in human breast cancer. The results suggest that in stimulation of proliferation FGF-8b not only promotes cell cycle progression through the G1 restriction point but also regulates key proteins involved in chromosomal segregation during mitosis and cytokinesis of breast cancer cells.► FGF-8b is expressed in human breast tumors and it promotes growth of breast cancer cells. ► Using microarray technique we identified FGF-8b-regulated genes in breast cancer cells. ► We show that FGF-8b modulates expression of genes regulating proliferation, cell death, and cancer growth. ► FGF-8b regulates both early and late cell cycle events in breast cancer cell proliferation. ► Expression of FGF-8b-regulated genes correlates with ER status and breast cancer prognosis.

Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) is an important regulator of cell signaling because of its ability to dephosphorylate receptors of growth factors as well as the cytokines and tyrosine-phosphorylated proteins associated with these receptors. In the current study, we used four different prostate cancer cell lines: PC3, DU145, LNCaP and LNCaP-IL6+. Tumor specimens from 122 patients with prostate cancer were analyzed using a tissue microarray. Our data demonstrate that all four prostate cancer cell lines express the SHP-2 protein. Additionally, low staining intensity and SHP-2 expression in the cytoplasm of cancer cells in prostate tumor specimens was inversely correlated with prostate volume (p = 0.041 and p = 0.042, respectively) whereas nuclear staining was positively correlated with extracapsular extension (p = 0.039). In our post-prostatectomy specimens, we found that patients with low SHP-2 expression had less favorable outcomes with respect to biochemical recurrence and clinical progression (p = 0.005 and p = 0.018, respectively). The loss of cytoplasmic SHP-2 expression is associated with increased growth and prostatic cancer progression.

Endocrine disrupting chemicals can induce malformations and impairment of reproductive function in experimental animals and may have similar effects in humans. Recently, the environmental obesogen hypothesis was proposed, suggesting that environmental chemicals contribute to the development of obesity and insulin resistance. These effects could be related to chemical interaction with nuclear receptors such as the peroxisome proliferator activated receptors (PPARs). As several testosterone-reducing drugs are PPAR activators, we aimed to examine whether four PPAR agonists were able to affect fetal testosterone production and masculinization of rats. Additionally, we wished to examine whether these chemicals affected fetal plasma levels of insulin and leptin, which play important roles in the developmental programming of the metabolic system. Pregnant Wistar rats were exposed from gestation day (GD) 7-21 to diisobutyl phthalate (DiBP), butylparaben, perfluorooctanoate, or rosiglitazone (600, 100, 20, or 1 mg/kg bw/day, respectively). Endocrine endpoints were studied in offspring at GD 19 or 21. DiBP, butylparaben and rosiglitazone reduced plasma leptin levels in male and female offspring. DiBP and rosiglitazone additionally reduced fetal plasma insulin levels. In males, DiBP reduced anogenital distance, testosterone production and testicular expression of Insl-3 and genes related to steroidogenesis. PPARalpha mRNA levels were reduced by DiBP at GD 19 in testis and liver. In females, DiBP increased anogenital distance and increased ovarian aromatase mRNA levels. This study reveals new targets for phthalates and parabens in fetal male and female rats and contributes to the increasing concern about adverse effects of human exposure to these compounds.

Adenohypophyseal-hormone production is regulated by hypothalamic peptides and target-gland hormones. Additionally, paracrine regulation by folliculo-stellate cells within the pituitary has been suggested. We recently showed TSH receptor expression in human folliculo-stellate cells and speculated that receptors for other adenohypophyseal hormones might also be expressed by folliculo-stellate cells. Using RT-PCR, we evaluated the expression of receptors for TSH, GH, ACTH, LH, FSH and PRL in a murine folliculo-stellate cell line, TtT/GF. Transcripts of TSH receptor, GH receptor and ACTH receptor were detected in this cell line. LH receptor, FSH receptor and PRL receptor expression, however, could not be demonstrated. We conclude that the TtT/GF cells express some, but not all, receptors for anterior pituitary hormones. This indicates that folliculo-stellate cells might act as mediators in the paracrine regulation of at least some of the hormones secreted by the anterior pituitary.

Thyrotropin secretion from the anterior pituitary is regulated mainly through TRH and thyroid hormones. Recent findings of a TSH receptor (TSHR) on folliculo-stellate (FS) cells in the human anterior pituitary indicate that TSH secretion might, in addition, be regulated in a paracrine manner via FS cells. In order to elucidate the physiological relevance of TSHR expression in FS cells we evaluated the effects of TSH on a murine FS cell line, TtT/GF. First, Western blot analysis confirmed the expression of TSHR protein in these cells. Second, three potential second messenger pathways were studied. Last, cDNA array hybridization was used to evaluate the effect of TSH on gene expression levels. TSH failed to induce either the adenylate cyclase/cAMP pathway, the phosphatidylinositol/calcium pathway, or the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway. Most of the genes regulated by TSH were related to cell proliferation, cell differentiation, and apoptosis. Moreover, TSH induced STAT5a and TGFbeta2 expression. We report that TtT/GF cells express a functional TSHR that is not coupled to cAMP nor IP (3) but probably signals through the JAK/STAT5a pathway. Functional TSHR expression in this cell line offers an in vitro model to study the role of TSHR in FS cells.