6 research outputs found
Vitellogenin profiling in liver, plasma and mucus of carp (Cyprinus carpio) exposed to endocrine disrupting chemicals: new facets of an old biomarker gem.
No full textProteome studies of carp and frog exposed to endocrine disrupting compounds
No full textVitellogenin (Vtg) is a well established plasma biomarker for endocrine disrupting compounds with estrogenic properties in fish. In the EU-project EASYRING carp were exposed to estrogens (17-ethynyl-estradiol, EE2), anti-estrogens (tamoxifen), androgens (17-methyldihydrotestosterone), and anti-androgens (flutamide), as well as to caging in polluted stretches of the Rivers Po and Lambro in Northern Italy. Monoclonal antibodies to carp Vtg were used for specific detection of Vtg in 1D- and 2D-western blotting, and in a quantitative sandwich ELISA and a qualitative lateral flow immunoassay (LFIA) dipstick, for measuring Vtg levels in plasma and mucus. Proteomic-based analysis using 2D-gel electrophoresis and mass spectrometry (MALDI-TOF, LC-MS/MS) identified Vtg as a prominent protein in liver, plasma, and mucus after EE2 exposure. At 64 ng EE2/l induction of multiple Vtg forms was observed in all tissues analyzed. In plasma a total of 30 EE2 inducible Vtg spots was identified, a response accompanied by alterations in the levels of other proteins identified as novel biomarker candidates. The induction and presence of numerous Vtg forms was confirmed by 1-D and 2-D western blot analysis. Quantitative Vtg ELISA showed a dose-dependent increase in plasma and mucus-Vtg, significantly different from control after 4 ng EE2/l in mucus, and 16 ng EE2/l in plasma. Regression analysis of plasma vs. mucus Vtg levels gave strong positive correlations for all groups, except for the fish from tamoxifen and field exposure. Analysis of river sediments using YES revealed a mixture of estrogens, and anti-androgens, suggesting that plasma vs. mucus Vtg profiles may be influenced by the nature of endocrine disrupting chemicals present in the environment
Identification of endocrine disrupting chemicals from a polluted river (Lambro) in Italy using bioassays and chemical analysis.
No full textRecently increasing concern evolved about endocrine disrupting chemicals (EDC) in the environment. Some studies showed that these chemicals can influence the reproductive system of animals in the wildlife producing e.g. intersex phenomenon in fishes or in marine snails. In this joint project a polluted river (Lambro) was investigated as a source of EDC to the wildlife of the major Italian watercourse, the river Po. Water and sediment samples from the Lambro river were fractionated and forwarded to bioassays (MVLN cells and yeast screening assay (YES)) in order to identify (anti)estrogenic activities of fractions. All fractions have been chemically analysed qualitatively as well as quantitatively for known EDC and, in addition, an immunoassay for estradiol (E2) was used to quantify E2 amounts. A proteomics analysis of the MVLN cells was conducted from treated cells relative to the control. This combined assay using chemical and biological analyses revealed some chemicals to act estrogenic. Good coincidence was found between the assays and some fractions from the water and the sediment samples were identified as being estrogenic. The chemical analysis showed that E2, E1, E3, Bisphenol A, and teroctylphenol are the most abundant compounds in the fractions with the highest estrogenic activities. The protein pattern of MVLN cells was changed in all treated samples relative to control. We conclude that these chemicals found in the Lambro water are present in concentrations affecting the cell based assays, change the protein patterns in the MVLN cells and may therefore act as EDC in the wildlife. Further investigations will show how fishes and amphibians are affected by the Lambro water conducting morphological studies and the expression of molecular biomarkers
Detection and identification of endocrine disrupting chemicals in water and sediment from the river Lambro, Italy
No full textFlight restriction prevents associative learning deficits but not changes in brain protein-adduct formation during honeybee ageing
No full textSUMMARY
Honeybees (Apis mellifera) senesce within 2 weeks after they discontinue nest tasks in favour of foraging. Foraging involves metabolically demanding flight, which in houseflies (Musca domestica) and fruit flies (Drosophila melanogaster) is associated with markers of ageing such as increased mortality and accumulation of oxidative damage. The role of flight in honeybee ageing is incompletely understood. We assessed relationships between honeybee flight activity and ageing by simulating rain that confined foragers to their colonies most of the day. After 15 days on average, flight-restricted foragers were compared with bees with normal (free) flight: one group that foraged for ∼15 days and two additional control groups, for flight duration and chronological age, that foraged for ∼5 days. Free flight over 15 days on average resulted in impaired associative learning ability. In contrast, flight-restricted foragers did as well in learning as bees that foraged for 5 days on average. This negative effect of flight activity was not influenced by chronological age or gustatory responsiveness, a measure of the bees' motivation to learn. Contrasting their intact learning ability, flight-restricted bees accrued the most oxidative brain damage as indicated by malondialdehyde protein adduct levels in crude cytosolic fractions. Concentrations of mono- and poly-ubiquitinated brain proteins were equal between the groups, whereas differences in total protein amounts suggested changes in brain protein metabolism connected to forager age, but not flight. We propose that intense flight is causal to brain deficits in aged bees, and that oxidative protein damage is unlikely to be the underlying mechanism.</jats:p
Toxicogenomics of Aryl Hydrocarbon- and Estrogen Receptor Interactions in Fish: Mechanisms and Profiling of Gene Expression Patterns in Chemical Mixture Exposure Scenarios
No full textAlmost without exception, biological processes such as overt morphological changes, development (both reproductive and growth), toxicological responses and clinical manifestation to disease, have molecular basis. From our perspective (i.e. toxicological perspective), the evidence of receptor-mediated mechanisms of xenobiotic-induced effects is provided if the effect is tissue specific, predictable, if increases in the transactivation of specific genes can be demonstrated, transcriptional responses occur rapidly, compounds bind reversibly to intracellular macromolecules or compounds are stereo-specific. Thus, the primary objective of toxicological in vitro studies on cells and tissues is to characterize cellular and molecular substrates and pathways that contribute to adverse effects in an organism after toxicant exposure. The estrogenic and xenobiotic biotransformation gene expressions are receptor-mediated processes that are ligand structure-dependent interactions with estrogen-receptor (ER) and aryl hydrocarbon receptor (AhR). The anti-estrogenic activities of AhR agonists have been reported in vitro and in vivo studies. In teleost species, exposure to AhR agonists has been associated with reduced vitellogenin (Vtg) synthesis or impaired gonadal development. Recently, several studies have shown that AhR-agonists directly activate ERs and induce estrogenic responses in mammalian in vitro systems. The overall objective of this thesis was to develop diagnostic gene and protein response tools in the study of the molecular mechanisms of gene expression patterns of xenoestrogens and xenobiotic interactions in wildlife species. Contaminants known to be estrogenic (ethynylestradiol; EE2 and nonylphenol; NP) and/or anti-estrogenic (PCBs), either by direct ER or indirect AhR mechanistic pathways, were used as model xenobiotics and evaluated either singly or in combination using in vitro and in vivo test systems.
Suppressive subtractive hybridization (SSH) was used to create a cDNA library of clones containing differentially expressed genes from Atlantic salmon (Salmo salar) separately exposed to ER and AhR agonists. Based on differentially expressed genes from the library, a targeted cDNA array (SalArray) was developed. Cellular in vitro systems, like cell and tissue models, facilitate the investigation of the direct molecular mechanisms accounting for predictable adverse effects of xenobiotic compounds on wildlife and humans. Consequently, in the studies presented primary hepatocyte cultures were isolated from the liver of trout and salmon by the collagenase perfusion method. The targeted SalArray and quantitative real-time PCR (q-PCR) were used to demonstrate that exposure of salmon hepatocytes to the ER-agonist NP singly or in combination with the produced differential gene expression patterns in salmon liver.Exposure of hepatocytes to NP mainly altered genes involved in the estrogenic pathway, including genes involved in steroid hormone synthesis and metabolism. The anti-estrogenic properties of PCB77 were demonstrated in the array analysis as NP induced gene expressions decreased by exposure of hepatocytes to PCB77. Our data showed a reciprocal inhibitory interaction between ER- and AhR-agonists. PCB77 produced anti-estrogenic effects by decreasing the mRNA expression of ER-responsive genes, and NP produced anti-AhR mediated effects as inhibitor of AhRR, Arnt, CYP1A1 and UGT expression. In vivo exposure of salmon to EE2 produced a significant decrease of CYP1A1 expression and these effects paralleled EROD activity and AhRR mRNA, suggesting a direct role of EE2 in controlling the cellular detoxification machinery.
While a clear pattern of negative effects on ER-mediated gene expression was found in hepatocytes exposed to PCB77, exposure of cells to the more potent AhR-agonist and dioxinlike PCB126 induced transcriptional activation of ER signalling demonstrated by increased Vtg and ERα mRNA and ERα protein levels. The decreased levels of ERα and Vtg expression in cells treated with PCB126 in the presence of ICI is novel, indicating a possible, but not conclusive “ER-hijacking” not previously reported in any fish species or lower vertebrate. Different gene expression patterns were obtained at similar time-interval with fish from different seasons, demonstrating the complexity of AhR-ER interactions. Thus, the direct estrogenic actions of PCB126 observed contribute new insight on the complexity of the mechanisms involved in ER-AhR crosstalk, prompting a new wave of discussion on whether AhR-mediated anti-estrogenicity is an exception, rather than a rule of action. This thesis demonstrates a complex mode of interactions between two different classes of ligandactivated receptors and provides novel mechanistic insights on signalling pathways. Therefore, the degree of simultaneous interactions between the ER and AhR gene transcripts demonstrated support the concept of cross-talk between these signalling pathways, in addition to generating new hypotheses that need to be evaluated empirically. AhR-agonist PCB77PhD i biologiPhD in Biolog
