Valentina Mansueto

Screening of ovarian steroidogenic pathway in Ciona intestinalis and its modulation after tributyltin exposure.

In this study, we have identified several ovarian steroids in Ciona with high similarity to vertebrate steroids and showed that cholesterol, corticosterone, dehydroepiandrosterone, estrone, estradiol-17beta, testosterone, pregnenolone, progesterone, have identical molecular spectra with vertebrate steroids. In addition, we have studied the effects of an endocrine disruptor (tributyltin: TBT) on these sex hormones and their precursors, ovarian morphology, and gene expression of some key enzymes in steroidogenic pathway in the ovary of Ciona. Ovarian specimens were cultured in vitro using different concentrations of TBT (10(-5), 10(-4) and 10(-3)M). Ethanol was used as solvent control. Gene expression analysis was performed for adrenodoxin (ADREN) and adrenodoxin reductase (ADOX) (mediators of acute steroidogenesis) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). These transcripts were detected and measured by quantitative (real-time) polymerase chain reaction (qPCR). Sex steroids and their precursors were identified and quantified by a gas chromatography-mass spectroscopy (GC-MS) method. Exposure of Ciona ovaries to TBT produced modulations (either increased or decreased) of sterols and sex steroid levels, whereas no significant differences in ADREN, ADOX or 17beta-HSD mRNA expression patterns were observed. Histological analysis shows that TBT produced several modifications on Ciona ovarian morphology that includes irregular outline of nuclear membrane, less compacted cytoplasm, in addition to test and granulosa cells that were detached from the oocyte membrane. Given that the ascidians represent very simple experimental models for the study of endocrine disruption by environmental contaminants, our findings provide excellent models for multiple identification and quantification of sex steroid and their precursors in biological samples exposed to endocrine-disrupting chemicals and for direct extrapolation of such effects across taxonomic groups and phyla. In addition, these results suggest that Cionaintestinalis may be a suitable species for molecular ecotoxicological studies and biomarker model for endocrine-disrupting effects in marine invertebrates.

Acetylcholinesterase activity in juvenile Ciona intestinalis (Ascidiacea, Urochordata) after exposure to tributyltin

The acetylcholinesterase (AChE) activity has been studied in 4-day post-fertilization juveniles of Ciona intestinalis exposed to tributyltin (TBT) at 10-5 M using the histochemical method of Karnovsky and Roots. Relative to vertebrate tissues and organs, the development of adult form of ascidians is interesting, because the analysis of many nuclear genes indicated that the ascidians are the closest living relatives of the vertebrates. Therefore, toxicity research using different approaches could provide data for comparative studies with vertebrates. AChE is over-expressed under chemical stress and in some diseases of vertebrates. Therefore, AChE is considered a biomarker of environmental contamination. Recently, we showed that eggs and embryos of ascidians over-express AChE activity after TBT exposure. Thus, the aim of the present study is to extend our previous findings by evaluating the effects of TBT on AChE activity in the embryos of this chordate and their suitability as good materials for comparative ecotoxicological studies. Our results demonstrate an increased expression of AChE activity in nervous-, blood progenitors- and tunic cells. The specific AChE inhibitor, BW284C51, inhibited this enzymatic activity. The presence of AChE activity in these cells has no obvious relations to their classical functions and seems to show a behavior similar to that of other chordates under changing stimuli. These preliminary descriptions provide a basis for further studies on cellular and molecular mechanism underlying the development of adult organs and tissues of this chordate, under chemical and physiological stress conditions.

Post-embryonic development effect of Bisphenol A and Tributyltin effects in Ciona intestinalis

Abstract In the present study we have determined the effects of endocrine disrupting chemicals such as Tributyltin (TBT) and Bisphenol-A (BPA) in juvenile Ciona intestinalis (Ascidiacea, Urochordata). The interest in Urochordata is due to its close phylogenetic relation to vertebrates. Moreover, in the juvenile the organs of a form similar to the adult form are present and they can easily be studied for the eventual morphological alterations that can be induced under stress conditions. Juvenile Ciona intestinalis of 4 days post fertilization were incubated for 1 h at increasing concentrations of either TBT or BPA solutions (0.1, 1 and 10 μM). The morphology of several organs was altered in a concentration-dependent manner in both TBT and BPA treated animals. BPA seems to be more toxic than TBT, destroying the tunic, the gonad cells and inhibiting the rhythmic body contractions. The TBT and BPA induced toxicity on the gonads is in agreement with previous data demonstrating that many chemicals can endanger the reproductive system leading to reproductive failure and consequently a population decline. These results suggest that the juvenile Ciona intestinalis can be used as an alternative or supplemental model for toxicological studies regarding the effects of toxicants not only on organs but also on metamorphosis and on reproductive, defense and nervous systems.

Bisphenol A (BPA) and atrazine inhibit the embryonic development of Ciona intestinalis (Ascidiacea, Urochordata)

The toxicities of bisphenol A (BPA) and atrazine in Ciona intestinalis development were examined in two separate series of experiments. The fertilized eggs were exposed to BPA or atrazine at concentrations of 0.1, 1 and 10 μM. Pasteurized filtered sea water (PFSW) and lipid solvent (ethanol 0.0005%) solutions were used in the control groups. The embryos of PFSW and lipid solvent groups developed up to swimming larvae after 22 h post-fertilization. Both BPA and atrazine induced concentration-dependent effects on Ciona embryo development. In BPA 1 and 10 μM groups the fertilized eggs were blocked at morula and at 2–4-cell stages, respectively. Changes noted in these groups include abnormal distribution of blastomeres that resulted in fused or absent blastomeres and lack of follicular and test cells. In all atrazine treated groups the development of fertilized eggs was blocked at 2–4 cells; no further development was observed beyond this stage. Anomalies noted include the unsegmented fertilized eggs and the fusion of blastomeres. The lowest observed effect concentration in BPA and atrazine was 1 and 0.1 μM, with embryos blocked at morula and at 2–4-cell stage, respectively.