The testosterone metabolism of <i>Neomysis integer</i>: the quest continues… (poster)

Both vertebrate and invertebrate species use enzymatic biotransformations for detoxication and elimination of xenobiotics. Testosterone has been used as a substrate to study the multiplicity of these enzymes. Since many of these enzymes are under hormone control, disruption of the hormone function can lead to potential effects on enzyme function and subsequently steroid homeostasis. The testosterone metabolism has therefore been proposed as a biomarker of exposure to endocrine disruptive compounds.In a previous study, the estuarine crustacean Neomysis integer (Crustacea, Mysidacea) was exposed to both testosterone and [14C]-testosterone. Identification and quantification of testosterone metabolites and endogenous steroids was done using TLC and LC-MSn (Verslycke et al., Gen. Comp. Endocrinol., accepted). The use of liquid chromatography coupled with multiple mass spectrometry allows a unique quantification of both endogenously produced steroids and in vivo produced metabolites in single mysid. Recent research has focused on the potential use of these biotransformations as a predictive biomarker for exposure to known endocrine disruptors. In this context, quantitative changes in the biotransformation profile of testosterone were evaluated after exposure to tributyltin (TBT), a compound used in antifouling paint, which has been suspected to interfere with steroid metabolism. The resulting protocol allows a quantitative and qualitative evaluation of the effect of TBT on the testosterone metabolism of N. integer. The results of these exposures will be presented and a possible mechanism of disruption through interaction with the P450 enzyme system is proposed.Future research on the steroid metabolism of N. integer could result in the development of predictive biomarkers for detection of endocrine disruption in estuarine environments

Testosterone metabolism in Neomysis integer following exposure to benzo(a)pyrene

Author Posting. © Elsevier B.V., 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 144 (2006): 405-412, doi:10.1016/j.cbpb.2006.04.001.Cytochromes P450 (CYPs) are important enzymes involved in the regulation of hormone synthesis and in the detoxification and/or activation of xenobiotics. CYPs are found in virtually all organisms, from archae, and eubacteria to eukaryota. A number of endocrine disruptors are suspected of exerting their effects through disruption of normal CYP function. Consequently, alterations in steroid hormone metabolism through changes in CYP could provide an important tool to evaluate potential effects of endocrine disruptors. The aim of this study was to investigate the potential effects of the known CYP modulator, benzo(a)pyrene (B(a)P), on the testosterone metabolism in the invertebrate Neomysis integer (Crustacea; Mysidacea). N. integer were exposed for 96h to 0.43, 2.39, 28.83, 339.00 and 1682.86μg B(a)P L-1 and a solventcontrol, and subsequently their ability to metabolize testosterone was assessed. Identification and quantification of the produced phase I and phase II testosterone metabolites was performed using liquid chromatography coupled with multiple mass spectrometry (LC-MS2). Significant changes were observed in the overall ability of N. integer to metabolize testosterone when exposed to 2.39, 28.83, 339.00 and 1682.86μg B(a)P L-1 as compared to the control animals.This research was supported by a research grant of the Ghent University Research Fund (BOF, 011.072.02). Dr. Tim Verslycke was supported by a Postdoctoral Fellowship of the Belgian American Educational Foundation

TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells

BACKGROUND: TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication. METHODS: To determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells. RESULTS: Our findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G(2)/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G(2)/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells. CONCLUSION: These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis

Co-expression network of neural-differentiation genes shows specific pattern in schizophrenia

Background: Schizophrenia is a neurodevelopmental disorder with genetic and environmental factors contributing to its pathogenesis, although the mechanism is unknown due to the difficulties in accessing diseased tissue during human neurodevelopment. The aim of this study was to find neuronal differentiation genes disrupted in schizophrenia and to evaluate those genes in post-mortem brain tissues from schizophrenia cases and controls. Methods: We analyzed differentially expressed genes (DEG), copy number variation (CNV) and differential methylation in human induced pluripotent stem cells (hiPSC) derived from fibroblasts from one control and one schizophrenia patient and further differentiated into neuron (NPC). Expression of the DEG were analyzed with microarrays of post-mortem brain tissue (frontal cortex) cohort of 29 schizophrenia cases and 30 controls. A Weighted Gene Co-expression Network Analysis (WGCNA) using the DEG was used to detect clusters of co-expressed genes that werenon-conserved between adult cases and controls brain samples. Results: We identified methylation alterations potentially involved with neuronal differentiation in schizophrenia, which displayed an over-representation of genes related to chromatin remodeling complex (adjP = 0.04). We found 228 DEG associated with neuronal differentiation. These genes were involved with metabolic processes, signal transduction, nervous system development, regulation of neurogenesis and neuronal differentiation. Between adult brain samples from cases and controls there were 233 DEG, with only four genes overlapping with the 228 DEG, probably because we compared single cell to tissue bulks and more importantly, the cells were at different stages of development. The comparison of the co-expressed network of the 228 genes in adult brain samples between cases and controls revealed a less conserved module enriched for genes associated with oxidative stress and negative regulation of cell differentiation. Conclusion: This study supports the relevance of using cellular approaches to dissect molecular aspects of neurogenesis with impact in the schizophrenic brain. We showed that, although generated by different approaches, both sets of DEG associated to schizophrenia were involved with neocortical development. The results add to the hypothesis that critical metabolic changes may be occurring during early neurodevelopment influencing faulty development of the brain and potentially contributing to further vulnerability to the illness.We thank the patients, doctors and nurses involved with sample collection and the Stanley Medical Research Institute. This research was supported by either Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq #17/2008) and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). MM (CNPq 304429/2014-7), ACT (FAPESP 2014/00041-1), LL (CAPES 10682/13-9) HV (CAPES) and BP (PPSUS 137270) were supported by their fellowshipsinfo:eu-repo/semantics/publishedVersio

Multiresidue analysis of anabolics in different matrixes: an analytical challenge?

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The testosterone metabolism of the estuarine invertebrate<i> Neomysis integer</i> (Crustacea: Mysidacea): Identification of testosterone metabolites and endogenous vertebrate-type steroids

Testosterone metabolism by Neomysis integer (Crustacea; Mysidacea) was assessed to obtain initial data on its metabolic capacity. N. integer were exposed to both testosterone and [14]testosterone. Identification of testosterone metabolites and endogenous steroids was performed using thin-layer chromatography and liquid chromatography with multiple mass spectrometry. Endogenous production of testosterone in mysids was detected for the first time. N. integer were exposed to testosterone and metabolized administered testosterone extensively. At least 11 polar testosterone metabolites (Rf,metabolite f,testosterone), androstenedione, dihydrotestosterone, and testosterone were produced in vivo by N. integer. A sex-specific testosterone metabolism was also observed, although this observation requires further confirmation. The anabolic steroid ß-boldenone was also identified for the first time in invertebrates. The metabolic pathway leading to the formation of ß-boldenone remains unknown, since the steroidal precursor androstadienedione could not be detected. These results reveal interesting similarities in enzyme systems in invertebrate and vertebrate species. Alterations in steroid hormone metabolism may be used as a new biomarker for the effects of endocrine disruptors in invertebrates