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Trabajo presentado en la Joint 30th Conference of the European Society for Comparative Endocrinology and of the 9th International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022., Fluoxetine (FLX) is a highly prescribed selective inhibitor of serotonin-reuptake and an emerging pollutant affecting fish behaviour, stress and reproduction, but little is known about possible actions and mechanisms in barrier tissues. We combined in vivo and in vitro approaches to demonstrate multi-level impacts of FLX on the sea bass (Dicentrarchus labrax) skin-scale barrier and on the estrogenic system. Juvenile sea bass intraperitoneally injected with FLX had significantly increased levels of FLX and its metabolite nor-FLX. In contrast to the natural estrogen E2, FLX did not increase plasma calcium, phosphorus (P) or vitellogenin, although a slight decrease in scale P content was detected. Quantitative SWATH-MS proteomics of the scales identified 134 proteins that were affected by FLX. Modified proteins were mainly related to extracellular matrix and protein turnover and energy production, 31 of which were also affected by E2. Multiple estrogen receptors and genes related to serotonin activity, transport and degradation were expressed in sea bass scales and transcript abundance of some of them was modulated by E2 and/or FLX. Using a minimally invasive in vitro bioassay with cultured sea bass scales and adhering epithelia we showed direct effects of FLX exposure on enzymatic activity associated with mineral mobilization, while the expression of estrogen receptors was not significantly affected. In in vitro receptor-reporter assays, FLX alone did not activate any of the three sea bass nuclear estrogen receptors but had antiestrogenic effects on Esr1/2b when in co-treatment with E2, and directly activated both plasma membrane Gprotein-coupled estrogen receptors. The combination of in vitro and in vivo assays substantiated the notion that FLX disrupted scale physiology through several different processes, with probable consequences for fish health, and revealed that some of the mechanisms of disruption can result from direct interaction with multiple estrogen ., Projects UIDB/04326/2020, PTDC/AAG-GLO/4003/2012 and DL57/2016/CP1361/CT0015 from FCT (Pt); EU Interreg FR-UK project RedPol; grant AGL2015-67477-C2-1- R (Sp).

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., There are increasing concerns regarding the accumulation of pharmaceuticals in the environment as even low dose exposures can interfere with the endocrine system of fish and other non-target aquatic organisms. In this study, we carried out different in vitro approaches using European sea bass tissues or estrogen receptors to investigate possible estrogenic-disrupting effects of fluoxetine (FLX), a widely used antidepressant and emerging environmental contaminant. Using an in vitro scale bioassay, a slight but significant increase in the enzymatic activity of osteoclast marker TRAP was detected upon exposure to FLX or to the natural estrogen 17b-estradiol (E2) for 24h, while the activity of osteoblast marker ALP was unaffected. The transcript expression of different estrogen receptor subtypes was detected in sea bass scales (nuclear receptors esr2a and esr2b and membrane receptor gperb) but this was not affected by the in vitro exposure to both compounds. Using an in vitro estrogen-response element reporter gene assay, FLX alone did not activate any of the three sea bass nuclear estrogen receptors (Esr1, Esr2a, Esr2b) but when combined with E2 showed antiestrogenic effects on the transactivation through Esr1 and Esr2b. Using an in vitro cAMP response element-luciferase reporter gene assay, significant induction of both membrane estrogen receptors (Gpera and Gperb) was achieved with FLX or E2 alone, but no synergistic effects were detected using the mixture of the two compounds. This combination of in vitro assays suggests different estrogenic and antiestrogenic mechanisms through which FLX may interfere with the scales and other fish tissues and provides promising tools for risk assessment of other potential endocrine disrupting compounds., Work supported by the Portuguese Foundation for Science and Technology (FCT) through projects UIDB/04326/2020 and PTDC/AAG-GLO/4003/2012 and by EU project Interreg France England RedPol. FCT funded the Researcher contract to PP under “Norma Transitória”-DL57/2016/CP1361/ CT0015 and the Spanish Ministry of Science funded grant AGL2015-67477-C2-1-R to AG.

Commercially available, but not yet characterized, the AVG-16 granulosa cell line was established from granulosa cells of medium porcine follicles. To examine the suitability of the AVG-16 cell line for studying the molecular mechanism of action of various environmental oestrogens, we investigated: 1/ cell morphology (by standard haematoxylin and eosin (HE) staining); 2/ basal and follicle-stimulating hormone (FSH) or luteinizing hormone (LH)-stimulated steroid hormone (progesterone; P4 and 17β-oestradiol; E2) secretion (by radioimmunoassay) and 3/ expression of receptors involved in the regulation of granulosa cell function: FSH receptor (FSHR), LH receptor (LHR), oestrogen receptor α (ERα), oestrogen receptor β (ERβ) and aryl hydrocarbon receptor (AhR). mRNA and protein expression was determined by RT-PCR and fluorescence immunocytochemistry, respectively. The secretion of P4 and E2 by AVG-16 cells was in the range of steroid hormone secretion by porcine cultured primary granulosa cells. Neither FSH (100 ng/ml) nor LH (100 ng/ml) affected P4 and E2 secretion by AVG-16 cells. The presence of FSHR and LHR at both mRNA and protein level was not demonstrated in the cells. However, AVG-16 cells were found to express mRNA and protein of ERα, ERβ and AhR. The results of our study showed that AVG-16 cells possess the capability of steroid hormone production, and both oestrogen receptors and AhR are present in these cells. Therefore, AVG-16 cells may serve as an unlimited source of homogenous porcine granulosa cells useful for studying the effects of environmental oestrogens on ovarian physiology.

Objectives To summarize participation and coverage rates in population mammographic screening programmes for breast cancer in Europe. Methods We used the European Network for Information on Cancer (EUNICE), a web-based data warehouse (EUNICE Breast Cancer Screening Monitoring, EBCSM) for breast cancer screening, to obtain information on programme characteristics, coverage and participation from its initial application in 10 national and 16 regional programmes in 18 European countries. Results The total population targeted by the screening programme services covered in the report comprised 26.9 million women predominantly aged 50-69. Most of the collected data relates to 2005, 2006 and/or 2007. The average participation rate across all programmes was 53.4% (range 19.4-88.9% of personally invited); or 66.4% excluding Poland, a large programme that initiated personal invitations in 2007. Thirteen of the 26 programmes achieved the European Union benchmark of acceptable participation (>70%), nine achieved the desirable level (>75%). Despite considerable invitation coverage across all programmes (79.3%, range 50.9-115.2%) only 48.2% (range 28.4-92.1%) of the target population were actually screened. The overall invitation and examination coverage excluding Poland was 70.9% and 50.3%, respectively. Conclusions The results demonstrate the feasibility of European-wide screening monitoring using the EBCSM data warehouse, although further efforts to refine the system and to harmonize standards and data collection practices will be required, to fully integrate all European countries. The more than threefold difference in the examination coverage should be taken into account in the evaluation of service screening programmes.

Objective To estimate the cumulative risk of a false-positive screening result in European mammographic screening programmes, and examine the rates and procedures of further assessment. Methods A literature review was conducted to identify studies of the cumulative risk of a false-positive result in European screening programmes (390,000 women). We then examined aggregate data, cross-sectional information about further assessment procedures among women with positive results in 20 mammographic screening programmes from 17 countries (1.7 million initial screens, 5.9 million subsequent screens), collected by the European Network for Information on Cancer project (EUNICE). Results The estimated cumulative risk of a false-positive screening result in women aged 50-69 undergoing 10 biennial screening tests varied from 8% to 21% in the three studies examined (pooled estimate 19.7%). The cumulative risk of an invasive procedure with benign outcome ranged from 1.8% to 6.3% (pooled estimate 2.9%). The risk of undergoing surgical intervention with benign outcome was 0.9% (one study only). From the EUNICE project, the proportions of all screening examinations in the programmes resulting in needle biopsy were 2.2% and 1.1% for initial and subsequent screens, respectively, though the rates differed between countries; the corresponding rates of surgical interventions among women without breast cancer were 0.19% and 0.07%. Conclusion The specific investigative procedures following a recall should be considered when examining the cumulative risk of a false-positive screening result. Most women with a positive screening test undergo a non-invasive assessment procedure. Only a small proportion of recalled women undergo needle biopsy, and even fewer undergo surgical intervention.