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- Environmental risk assessment and toxicity of pharmaceuticals in coastal tropical and temperate organismsPublication . Fonseca, Tainá; Bebianno, Maria JoãoDemographic attributes of the urban society have prompted a milestone shift towards structural aging of global population and increase of life expectancy, crucial to the prominence of cancer as one of the non-communicable diseases leading mortality. Currently, cancer diseases were accountable for about 9.6 million deaths in 2018, with concerning increasing projections of cancer incidence and mortality, by 2030, at a greater proportion in developing countries. As a result, there is an increasing trend of production and consumption of pharmaceuticals applied in cancer treatments. Once administered and metabolized, such drugs are excreted into waterways following to waste water treatment plants (WWTPs), ending up in freshwater and coastal ecosystems, where they can be found at a sub ng to ng L-1 range. Anticancer drugs are designed to damage DNA and disrupt mechanisms of its transcription, replication and synthesis, as well as suppress the cell´s defense, ultimately causing cell death. In cancer therapy, drugs are typically administered in a combinatory cocktail, that covers different molecular targets, reducing the risk of clonal selection based upon cell resistance to a single drug. However, these chemicals may bring potential toxicity if discharged into the aquatic environment, both in temperate and tropical zones, particularly in the benthic compartment, where they are expected to accumulate. In this sense, the present thesis aimed to assess the effects of anticancer agents of different classes, namely the cytotoxic platinum-based cisplatin (CisPt), the alkylating agent cyclophosphamide (CP) and the endocrine disruptor tamoxifen (TAM), on non-target benthic marine species under realistic environmental concentrations, at individual and combined exposures. For this purpose, a multi-biomarker approach was applied including the assessment of behavioural responses, oxidative stress, biotransformation, neurotoxicity, lipid peroxidation and genotoxicity in the temperate polychaete Nereis diversicolor, in addition to the evaluation of acute and chronic effects of drugs in tropical and representative benthic organisms: the sea urchin Echinometra lucunter, the polychaete Scolelepis squamata and the amphipod Tiburonella viscana from the Brazilian coast. The main results revealed that single drug exposures caused alteration of AChE activity, oxidative stress and oxidative damage and, ultimately, DNA damage in N. diversicolor. Besides, alterations in its burrowing behaviour also occurred, as an ecological outcome. These effects were more pronounced in organisms under CisPt at 100 ng Pt L-1, CP at 1000 ng L-1 and TAM at 0.5 ng L-1. Findings from bioassays with N. diversicolor conducted with drugs in tertiary mixtures indicated that each biomarker effect respond differently, according to the trends proposed by models of drugs´s interaction. Toxicity did not increase in a dose-response manner but showed different patterns of effects, in a way that the highest DNA damage was observed at the set of lowest concentrations (Mixture A), an absence of oxidative stress at the intermediary drug levels (Mix B and C), with a potential dominance of TAM´s MoA in organisms exposed to Mixture C and D, suggesting an antagonist interaction between the cytotoxic drugs. Therefore, the effects ruled by single-drug MoAs cannot provide estimations and protective measures for a prospective ERA regarding scenarios of their combination in the environment. Although evidence on estrogen receptors expression is still not disclosed for the herein selected biological models, determination of biochemical and genotoxic outcomes from TAM exposure are highlighted, since it is assumed to comprise a targeted therapy on nuclear estrogen receptors. Anticancer drugs´ concentrations that triggered the above-mentioned responses are potentially present in coastal environments, even more prone to be encountered in developing regions, where technologies of WWTPs are not efficient or are lacking. Accordingly, effects regarding the bioassays conducted with the tropical counterparts showed a non-monotonic reduction of viable pluteus larvae of E. lucunter, at CisPt and CP treatment respectively at the range from 0.1 to 10 ng Pt L-1, and from 50 to 500 ng CP L-1, in contrast to the increasing reduction of embryo larval development under TAM exposure, significant over the whole range of concentrations. In polychaetes S. squamata, a significant acute toxicity was exerted at the higher pharmaceutical doses (Mix C and D). In the amphipod T. viscana demonstrated a higher sensitivity to pharmaceuticals, presenting a significant non-linear reduction of survival in Mix B and D, as a potential outstanding effect of TAM when present as mixtures. By virtue of the non-monotonicity addressed in the dose-responses relationships reported in all bioassays, another concern stands out on the urgency to the development of a risk analysis and safety assessment designated to anticancer drugs.
- Cytotoxic responses of the anticancer drug cyclophosphamide in the mussel Mytilus galloprovincialis and comparative sensitivity with human cells linesPublication . Fernandes, Elna; Fonseca, Tainá; Carriço, Tania; Mestre, Nélia; Tavares, Alvaro; Bebianno, Mariathe rise of cancer cases worldwide led to an increase in production and consumption of anticancer drugs, that ultimately end up in the marine environment and are accumulated in aquatic organisms. Cyclophosphamide (CP) is a cytotoxic alkylating agent frequently prescribed in cancer treatments. This study assess ecotoxicological effects of CP on mussels Mytilus galloprovincialis, through in vivo and ex vivo approaches and compares the sensitivity of mussel haemocytes with well-established human cell lines (RPE and HeLa). Mussels were exposed in vivo to CP (1000 ng L-1) and several biomarkers analysed in gills and digestive glands namely neurotoxicity (AChE activity), oxidative stress (GPx activity), biotransformation (GST activity), lipid peroxidation (LPO) and apoptosis (caspase activity), whereas genotoxicity was determined in mussels' haemocytes. Cytotoxicity was also assessed in haemocytes (in vivo and ex vivo) and human cell lines (in vitro) exposed to a range of CP concentrations (50, 100, 250, 500 and 1000 ng L-1) over 24 h, via neutral red assay. In in vivo exposure, detoxification of CP did not efficiently occur in the gills while in digestive glands GPx and GST activities were induced, jointly with a decrease in lipid peroxidation, indicating a potential outcome of the protective antioxidant mechanisms, whereas no apoptosis was noted. Moreover, cytotoxicity and DNA damage were detected in haemocytes. the ex vivo exposure haemocytes to CP caused cytotoxicity (from 100 ng L-1), whereas no effects occurred in human cell lines. This suggests that, at relevant environmental concentrations, CP cause subtle and irreversible impacts on M. galloprovincialis. (C) 2020 Elsevier Ltd. All rights reserved.
- Ultrasound-assisted extraction as an easy-to-perform analytical methodology for monitoring ibuprofen and its main metabolites in musselsPublication . Malvar, José Luis; Santos, Juan Luis; Martín, Julia; Aparicio, Irene; Fonseca, Tainá; Bebianno, Maria; Alonso, EstebanNon-steroidal anti-infammatory drugs (NSAIDs) have been reported to be the main pharmaceutical class accumulated in seafood. Among them, ibuprofen (IBU) is of special concern as it is used worldwide to treat common pain, does not require a medical prescription, it is often taken in a high daily dose, and has been reported to cause potential adverse efects on aquatic organisms. IBU is highly transformed into hydroxy- and carboxy-metabolites and/or degradation products generated not only after its administration but also during wastewater treatment or in the environment. These compounds can be present in the environment at higher concentrations than IBU and present higher toxicity. In this work, a low-cost and afordable routine analytical method was developed and validated for the frst-time determination of IBU and its main metabolites in mussels. The method is based on ultrasound-assisted extraction (UAE), clean-up by dispersive solid-phase extraction (d-SPE) and analytical determination by liquid chromatography-tandem mass spectrometry. Box-Behnken experimental design was used for method optimisation to better evaluate the infuence and interactions of UAE and d-SPE variables. Extraction recoveries were in the range from 81 to 115%. Precision, expressed as relative standard deviation, was lower than 7%. Method detection limits were in the range from 0.1 to 1.9 ng g−1 dry weight. The method was successfully applied to wild mussels.
- Microplastics effects in Scrobicularia planaPublication . Ribeiro, Francisca; Garcia, Ana; Pereira, Beatriz P.; Fonseca, Maria; Mestre, Nélia; Fonseca, Tainá; Ilharco, Laura M.; Bebianno, Maria JoãoOne of the most common plastics in the marine environment is polystyrene (PS) that can be broken down to micro sized particles. Marine organisms are vulnerable to the exposure to microplastics. This study assesses the effects of PS microplastics in tissues of the clam Scrobicularia plana. Clams were exposed to 1mgL-1(20μm) for 14days, followed by 7days of depuration. A qualitative analysis by infrared spectroscopy in diffuse reflectance mode period detected the presence of microplastics in clam tissues upon exposure, which were not eliminated after depuration. The effects of microplastics were assessed by a battery of biomarkers and results revealed that microplastics induce effects on antioxidant capacity, DNA damage, neurotoxicity and oxidative damage. S. plana is a significant target to assess the environmental risk of PS microplastics.
- Ecotoxicological effects of chemical contaminants adsorbed to microplastics in the clam Scrobicularia planaPublication . O'Donovan, Sarit; Mestre, Nélia; Abel, Serena; Fonseca, Tainá; Carteny, Camilla C.; Cormier, Bettie; Keiter, Steffen H.; Bebianno, Maria JoãoAlthough microplastics (MPs) are distributed globally in the marine environment, a great deal of unknowns relating to their ecotoxicological effects on the marine biota remains. Due to their lipophilic nature, microplastics have the potential to adsorb persistent organic pollutants present in contaminated regions, which may increase their detrimental impact once assimilated by organisms. This study investigates the ecotoxicological effects of exposure to low-density polyethylene (LDPE) microplastics (11-13 beta m), with and without adsorbed contaminants (benzo[a]pyrene- BaP and perfluorooctane sulfonic acid-PFOS), in the peppery furrow shell clam, Scrobicularia plana. Environmentally relevant concentrations of contaminants (BaP-16.87 +/- 0.22 mu g g(-1) and PFOS-70.22 +/- 12.41 mu g g(-1)) were adsorbed to microplastics to evaluate the potential role of plastic particles as a source of chemical contamination once ingested. S. plana were exposed to microplastics, at a concentration of 1 mg L-1, in a water-sediment exposure setup for 14 days. Clams were sampled at the beginning of the experiment (day 0) and after 3, 7, and 14 days. BaP accumulation, in whole clam tissues, was analyzed. A multi-biomarker assessment was conducted in the gills, digestive gland, and haemolymph of clams to clarify the effects of exposure. This included the quantification of antioxidant (superoxide dismutase, catalase, glutathione peroxidase) and biotransformation (glutathione-Stransferases) enzyme activities, oxidative damage (lipid peroxidation levels), genotoxicity (single and double strand DNA breaks), and neurotoxicity (acetylcholinesterase activity). Results suggest a potential mechanical injury of gills caused by ingestion of microplastics that may also affect the analyzed biomarkers. The digestive gland seems less affected by mechanical damage caused by virgin microplastic exposure, with the MPs-adsorbed BaP and PFOS exerting a negative influence over the assessed biomarkers in this tissue.
- Metal bioaccumulation by the neotropical clam Anomalocardia flexuosa to estimate the quality of estuarine sedimentsPublication . Cruz, Ana C. F.; Pauly, Guacira F. E.; Araujo, Giuliana S.; Gusso-Choueri, Paloma; FONSECA, TAINÁ; Campos, Bruno G.; Santelli, Ricardo E.; Freire, A. S.; Braz, Bernardo F.; Bosco-Santos, Alice; Luiz-Silva, Wanilson; Machado, Wilson; Abessa, Denis M. S.This investigation evaluated the bioaccumulation potential of the tropical estuarine bivalve Anomalocardia flexuosa for trace metals. To this aim, chemical and sedimentological analyses and bioaccumulation tests were performed. The sediments were mainly composed by fine-sands and mud, with variable levels of organic matter and CaCO3. Muddy sediments from a depositional site (P2) presented the highest concentrations of metals, despite SEM/AVS not indicating bioavailability. Bioaccumulation factors showed high ratios for Cd, Ni, and Zn, while associations between the contents of mud, organic matter, CaCO3 and metals in sediments and tissues of A. flexuosa were indicated by a principal component analysis. The SEM/AVS was not effective to predict the bioavailability through dissolved metals. The results showed that contaminants were bioavailable, while the performed bioaccumulation test proved to be a reliable technique for assessing sediment contamination in estuarine regions. Moreover, A. flexuosa was considered an adequate test organism for bioaccumulation studies.
- Perfluorooctane sulfonic acid (PFOS) adsorbed to polyethylene microplastics: accumulation and ecotoxicological effects in the clam Scrobicularia planaPublication . Islam, Naimul; FONSECA, TAINÁ; Vilke, Juliano Marcelo; Gonçalves, Joanna M.; Pedro, Paulo; Keiter, Steffen; Cunha, Sara C.; Fernandes, José O.; Bebianno, Maria JoãoMicroplastics are widespread in the marine environment, whereby organisms’ uptake these tiny particles, presenting adverse biological responses. Plastic debris also act as a vector of many contaminants which intensify their effects to marine organisms that depend on type, size, shape and chemical properties. This study aimed to assess the accumulation and potential toxicity of different sizes of microplastics with and without adsorbed perfluorooctane sulfonic acid (PFOS) in the clam Scrobicularia plana. Clams were exposed to 1 mg L-1 of virgin and PFOS (70.2 ± 12.4 μg g−1) contaminated lowdensity polyethylene microplastics of two different sizes (4-6 and 20-25 μm) over 14 days. Microplastic and PFOS accumulation and filtration rate were determined along with a multi biomarker approach to access the biological effects of microplastics accumulation. Biomarkers include oxidative stress (superoxide dismutase, catalase, glutathione peroxidases), biotransformation enzymes (glutathione-S-transferases) activity, neurotoxicity (acetylcholinesterase activity), oxidative damage and apoptosis. Microplastics and PFOS accumulated were microplastic size dependent but not PFOS dependent and filtration rate was reduced at the end of the exposure. Reactive oxygen species in gills and digestive gland were generated as a result of exposure to both types of microplastics, confirming the disturbance of the antioxidant system. Larger microparticles lead to stronger impacts, when compared to smaller ones which was also supported by Integrated Biomarker Responses index calculated for both tissues. An anti-apoptotic response was detected in digestive glands under exposure to any of the MPs treatments as a probable mechanism associated to carcinogenic responses derived from MPs uptake.
- Ecotoxicological assessment of the anticancer drug cisplatin in the the polychaete Nereis diversicolorPublication . Aureliano, M.; Bebianno, Maria João; Abessa, D. M. S.; Rocha, T.; FONSECA, TAINÁAnticancer drugs are designed to inhibit tumor cell proliferation by interacting with DNA and altering cellular growth factors. When released into the waterbodies of municipal and hospital effluents these pharmaceutical compounds may pose a risk to non-target aquatic organisms, due to their mode of action (cytotoxic, genotoxic, mutagenic and teratogenic). The present study aimed to assess the ecotoxicological potential of the alkylating agent cisplatin (CisPt) to the polychaete Nereis diversicolor, at a range of relevant environmental concentrations (i.e. 0.1, 10 and 100 ng Pt L−1 ). Behavioural impairment (burrowing kinetic impairment), ion pump effects (SR Ca2+-ATPase), neurotoxicity (AChE activity), oxidative stress (SOD, CAT and GPXs activities), metal exposure (metallothionein-like proteins - MTLP), biotransformation (GST), oxidative damage (LPO) and genotoxicity (DNA damage), were selected as endpoints to evaluate the sublethal responses of the ragworms after 14-days of exposure in a water-sediment system. Significant burrowing impairment occurred in worms exposed to the highest CisPt concentration (100 ng Pt L−1 ) along with neurotoxic effects. The activity of antioxidant enzymes (SOD, CAT) and second phase biotransformation enzyme (GST) was inhibited but such effects were compensated by MTLP induction. Furthermore, LPO levels also increased. Results showed that the mode of action of cisplatin may pose a risk to this aquatic species even at the range of ng L−1
- Ecotoxicological assessment of chemotherapeutic agents using toxicity tests with embryos of Mellita quinquiesperforataPublication . Mello, Luiza Costa; Fonseca, Tainá; de Souza, Abessa Denis MoledodeThe consumption of anticancer agents has increased in the recent decades, and these substances may be present in sewage. Consequently, they may reach the environment when sanitation infrastructure is ineffective. This study evaluated the toxicity of three anticancer agents-Tamoxifen (TAM), Cisplatin (CisPt), and Cyclophosphamide (CP)-on the development of embryos of the sand-dollar Mellita quinquiesperforata. Adult individuals were collected in sandy beaches, and gametes were obtained. Freshly-fertilized eggs were exposed to increasing sets of concentrations of each compound, and the effective concentrations needed to cause a 50% effect in the organisms (EC50) were calculated. The three compounds were toxic, and their EC50 values were 16.78 +/- 2.42 ng.L-1 (TAM), 27.20 +/- 38.26 ng.L-1 (CisPt), and 101.82 +/- 70.96 ng.L-1 (CP). There is no information on the environmental levels of these compounds in Brazil, but as they were already detected in ng.L-1 levels worldwide, it can be expected that these substances pose environmental risks to the marine biota.
- Effects of the UV filter, oxybenzone, adsorbed to microplastics in the clam Scrobicularia planaPublication . O'Donovan, Sarit; Mestre, Nélia; Abel, Serena; FONSECA, TAINÁ; Carteny, Camilla C.; Willems, Tim; Prinsen, Els; Cormier, Bettie; Keiter, Steffen S.; Bebianno, Maria JoãoMicroplastics (MPs) lipophilic nature and widespread distribution raises concerns due to their increasing presence in the marine environment and their ability to adsorb organic contaminants, as being potential vehicles for transport and potential source of accumulation of organic contaminants by marine organisms. The organic UV-filter, oxybenzone (BP-3) is a constituent of sunscreens and personal care products, entering the marine environment either by direct contact with swimmers or by wastewater effluents. In this study the ecotoxicological effects of exposure to low-density polyethylene (LDPE) microplastics with and without adsorbed BP-3 were investigated in the peppery furrow shell clam, Scrobicularia plana. LDPE microplastics with a size range of 11–13 μm were previously contaminated with an environmentally relevant concentration of BP-3 (82 ng g−1 ). S. plana individuals were exposed to a concentration of 1 mg L−1 of microplastics with and without BP-3 adsorbed in a water-sediment exposure system for 14 days. Clams were sampled at the beginning of the experiment and after 3, 7, and 14 days of exposure. Multiple biomarkers were analysed to investigate the effect of exposure in different clam tissues, gills, digestive gland, and haemolymph. Antioxidant (superoxide dismutase, catalase, glutathione peroxidase) and biotransformation (glutathione-S-transferases) enzyme activities, oxidative damage (lipid peroxidation), genotoxicity (single and double strand DNA breaks), and neurotoxicity (acetylcholinesterase activity) were assessed along with two biomarker indexes to assess the overall health status. Results indicate that after 7 days of exposure MPs with adsorbed BP-3 induced oxidative stress and damage, when compared to exposure to virgin MPs and control treatments. Neurotoxic effects were also noted in MPs with adsorbed BP-3 after 14 days exposure, while some evidence points to increased genotoxicity with exposure time. Overall results indicate that gills were more affected by exposure to microplastics than digestive gland and that biomarkers alterations are apparently more related to the toxicity of BP-3 adsorbed than virgin MPs alone.