Maria Vittoria Cangialosi

Tissue bioaccumulation patterns, xenobiotic biotransformation and steroid hormone levels in Atlantic salmon (Salmo salar) fed a diet containing perfluoroactane sulfonic or perfluorooctane carboxylic acids

In the present study, groups of juvenile Atlantic salmon (Salmo salar) were fed gelatine capsules containing fish-food spiked with PFOA or PFOS (0.2 mg kg(-1) fish) and solvent (methanol). The capsules were given at days 0, 3 and 6. Blood, liver and whole kidney samples were collected prior to exposure (no solvent control), and at days 2, 5, 8 and 14 after exposure (Note: that day 14 after exposure is equal to 7d recovery period). We report on the differences in the tissue bioaccumulation patterns of PFOS and PFOA, in addition to tissue and compound differences in modulation pattern of biotransformation enzyme genes. We observed that the level of PFOS and PFOA increased in the blood, liver and kidney during the exposure period. Different PFOS and PFOA bioaccumulation patterns were observed in the kidney and liver during exposure- and after the recovery periods. Particularly, after the recovery period, PFOA levels in the kidney and liver tissues were almost at the control level. On the contrary, PFOS maintained an increase with tissue-specific differences, showing a higher bioaccumulation potential (also in the blood), compared with PFOA. While PFOS and PFOA produced an apparent time-dependent increase in kidney CYP3A, CYP1A1 and GST expression, similar effects were only temporary in the liver, significantly increasing at sampling day 2. PFOA and PFOS exposure resulted in significant decreases in plasma estrone, testosterone and cortisol levels at sampling day 2, and their effects differed with 17α-methyltestostrerone showing significant decrease by PFOA (also for cholesterol) and increase by PFOS. PFOA significantly increased estrone and testosterone, and no effects were observed for cortisol, 17α-methyltestosterone and cholesterol at sampling day 5. Overall, the changes in plasma steroid hormone levels parallel changes in CYP3A mRNA levels. Given that there are no known studies that have demonstrated such tissue differences in bioaccumulation patterns with associated differences in toxicological responses in any fish species or lower vertebrate, the present findings provide some potential insights and basis for a better understanding of the possible mechanisms of PFCs toxicity that need to be studied in more detail.

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.

Perfluorooctane sulfonamide-mediated modulation of hepatocellular lipid homeostasis and oxidative stress responses in Atlantic salmon hepatocytes.

We have investigated the effects of perfluorooctane sulfonamide (PFOSA) on cellular functions and lipid homeostasis (including β-oxidation) in salmon primary hepatocytes. Salmon hepatocytes were exposed to PFOSA at 0 (control), 2, 20, and 50 μM for 12 and 24 h. Fatty acids (FAs) and lipids were determined by GC-MS; FA elongase (FAE), Δ5-desaturase (FAD5), Δ6-desaturase (FAD6), peroxisome proliferator-activated receptors (PPARs), acyl coenzyme A (ACOX-1), glutathione peroxidase (GPx), catalase (CAT), and glutathione S-transferase (GST) mRNA were analyzed using qPCR. GST activity was analyzed by biochemical assays using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Our data showed that PFOSA produced significant changes in FA composition that predominantly involved a decrease (at 12 h) and an increase (at 24 h) in FA methyl esters (FAMEs), MUFA, total PUFA, and (n-3 and n-6) PUFA. Particularly, an increase of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid [EPA; 20:5n-3], and arachidonic acid [ARA: 20:4n-6] with associated increase in FAE, FAD5, and FAD6 mRNA were observed after PFOSA exposure, while cis-13,16-docosadienoic acid (22:2) was significantly decreased. PFOSA produced apparent concentration-dependent increase of PPARα and PPARγ. CAT, GPx, and GST mRNA show that PFOSA produced concentration- and time-specific increase of CAT and GST, but no changes in GST activity were observed. In general, these responses indicate that PFOSA evokes deleterious effects on cellular lipid homeostasis and transcriptional responses that regulate cellular oxidative homeostasis in salmon hepatocytes.

Modulation of Membrane Lipid Composition and Homeostasis in Salmon Hepatocytes Exposed to Hypoxia and Perfluorooctane Sulfonamide, Given Singly or in Combination

The relative importance of environmental hypoxia due to global climate change on organismal ability to adapt to chemical insult and/or mechanisms of these responses is not well understood. Therefore, we have studied the effects of combined exposure to perfluorooctane sulfonamide (PFOSA) and chemically induced hypoxia on membrane lipid profile and homeostasis. Primary salmon hepatocytes were exposed to PFOSA at 0, 25 and 50 µM singly or in combination with either cobalt chloride (CoCl2: 0 and 150 µM) or deferroxamine (DFO: 0 and 100 µM) for 24 and 48 h. CoCl2 and DFO were used to induce cellular hypoxia because these two chemicals have been commonly used in animal experiments for this purpose and have been shown to increase hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) levels. Fatty acid (FA) profiles were determined by GC-MS, while gene expression patterns were determined by quantitative PCR. Hypoxic condition was confirmed with time-related increases of HIF-1α mRNA levels in CoCl2 and DFO exposed cells. In general, significant alterations of genes involved in lipid homeostasis were predominantly observed after 48 h exposure. Gene expression analysis showed that biological responses related to peroxisome proliferation (peroxisome proliferator-activated receptors (PPARs) and acyl coenzyme A (ACOX)) and FA desaturation (Δ5- and Δ6-desaturases: FAD5 and FAD6, respectively) and elongation (FAE) were elevated slightly by single exposure (i.e. either PFOSA, CoCl2 or DFO exposure alone), and these responses were potentiated in combined exposure conditions. Principal component analysis (PCA) showed a clustering of peroxisome proliferation responses at transcript levels and FA desaturation against membrane FAs levels whose changes were explained by PFOSA and chemically induced hypoxia exposures. Overall, our data show that most of the observed responses were stronger in combined stressor exposure conditions, compared to individual stressor exposure. In general, our data show that hypoxia may, singly or in combination with PFOSA produce deleterious health, physiological and developmental consequences through the alteration of membrane lipid profile in organisms.

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.

Seasonal screening of AChE, GSH and gonad histology, in European sea bass Dicentrarchus labrax L. reared in three different fish farms

The aim of this work was to do a preliminary seasonal screening of ecotoxicological biomarkers in European sea bass Dicentrarchus labrax in three different fish farms, to know if the different location and typology can discriminate them. A set of selected biomarkers of xenobiotic exposure, such as acetylcholinesterase (AChE) activity, Glutathione (GSH) and gonad morphology were investigated seasonally in male European sea bass D. labrax (L.) reared in three different intensive farms: a land-based farm of cement tanks (T), an in-shore sea cages farm (C1) and an off-shore sea cages farm (C2). The results showed that both location and typology can discriminate AChE activity, GSH content and gonad morphology. Further investigation is needed to propose these biomarkers in the protocol of fish farm quality control.

Screening of ecotoxicological, qualitative and reproductive variables in male European sea bass Dicentrarchus labrax (L.) reared in three different fish farms: Facility location and typology

The aim of this study was to investigate the influence of both facility location and typology of fish farm on some ecotoxicological, qualitative and reproductive variables in European sea bass Dicentrarchus labrax L. Several variables were investigated: gonado-somatic index (GSI), liver-somatic index (LSI); 7-ethoxyresorufin-O-deethylase (EROD), benzo(a)pyrene monooxygenase and acetylcholinesterase activities; glutathione (GSH), testosterone, 17β-estradiol, total lipid, phospholipid (PL) and triglyceride contents. In addition, the histological sections of gonads were examined. Results suggest that LSI, EROD activity, GSI, GSH, PL, hormone levels and gonad morphology were influenced by different facility locations and typologies of fish farm.

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.

Preliminary identification and quantification of steroid hormones in the red palm weevil, Rhynchophorus ferrugineus

In the present preliminary study, we used a gas chromatography–mass spectroscopy (GC-MS) method to identify and quantify steroid hormones, their precursors and metabolites in whole body of red palm weevil (RPW) R. ferrugineus adults. We identified a total of seven steroids by single ion monitoring mode (SIM) analysis and compared them to the National Institute of Standards and Technology (NIST) library. The steroids include: dehydroepiandrosterone (DHEA), estrone, estradiol-17β, testosterone, progesterone, cortisol and cholesterol, whereas pregnenolone, pregnan-20-one-17-hydroxy and corticosterone were not detected. This study shows that some invertebrate groups seem to use partially or totally comparable hormones to vertebrates and produce vertebrate-type steroids with functional roles. However, some steroids of the endocrine system of invertebrates are still lacking or yet to be identified in most phyla.