Over the recent years, venlafaxine has become the predominant antidepressant drug detected in municipal wastewater effluents (MWWE) and aquatic systems. However, very little is known about the effect of this drug in the aquatic environment on non-target organisms, including fish. Venlafaxine is a pharmaceutical compound designed to inhibit serotonin and norepinephrine reuptake, thereby increasing the synaptic availability of these neurotransmitters. In teleosts, the key aspect of stress adaptation involves the activation of the hypothalamus-pituitary-interrenal (HPI) axis, leading to the production of cortisol. Given that monoamine neurotransmitters (serotonin, norepinephrine, and dopamine) are involved in the regulation of a wide range of neuroendocrine responses, including stress axis function, my primary hypothesis was that venlafaxine acts as a neuroendocrine disruptor impacting the functioning of the corticosteroid stress axis in rainbow trout (Oncorhynchus mykiss). This hypothesis was tested through a series of in vivo exposure studies, as well as in vitro experiments, using environmentally relevant levels of venlafaxine, in order to tease out potential mode of action of this drug on target tissues involved in HPI axis functioning. The results suggest that venlafaxine alters monoamine neurotransmitter levels and their turnover rates in a region-specific manner in trout brain, and that the midbrain is the prime target. The monoamine changes may be responsible for the downstream effects on neuroendocrine responses coordinated in the hypothalamus, as this region receives monoaminergic inputs from the midbrain. The functional relevance of the above finding was confirmed by showing that venlafaxine exposure disrupted the neuroendocrine responses associated with social stress and appetite regulation. Functional downstream effects of HPI axis dysfunction were further confirmed by subjecting the fish to a handling disturbance, which revealed that the highly conserved cortisol and glucose responses to stressors were disrupted by venlafaxine. Also, there were tissue-specific effects of venlafaxine exposure on metabolic capacities, including enhanced gluconeogenesis and amino acid catabolism in the liver (a key glucose producing tissue), and alterations in the glycolytic capacity and sodium potassium ATPase activity in the gill (a key glucose utilizing tissue). The results suggest that the mode of action of venlafaxine may involve disruption of each target tissue involved in the HPI axis functioning. In vitro mechanistic studies indicated that hypothalamus functioning is disrupted by venlafaxine and this may involve effects mediated by serotonergic pathways. The reduced phosphorylation of cAMP response element binding protein (CREB) suggests that venlafaxine may impact downstream signalling cascades that are CREB-dependent. The transcript changes observed with venlafaxine in the hypothalamus include changes in mRNA levels of key genes involved in appetite regulation and stress response, including corticotropin releasing factor (CRF) and neuropeptide Y (NPY). At the pituitary level, venlafaxine impaired adrenocorticotropic hormone (ACTH) production, and this involved disruption of corticotropin releasing factor-receptor type 1 (CRF-R1), which is a key sensor for CRF stimulation. At the interrenal tissue level, the responsiveness of steroidogenic cells to ACTH stimulation was altered by venlafaxine and the mode of action appears to involve pathways upstream of the intracellular cAMP production. Also, cortisol biosynthetic capacity was disrupted by venlafaxine and this was accompanied by changes in transcript abundances of steroidogenic acute regulatory protein and cytochrome P450 side chain cleavage in the interrenal tissue. Taken together, the results demonstrate for the first time that the antidepressant venlafaxine, a human pharmaceutical contaminating aquatic systems, disrupts neuroendocrine responses and affects stress, feeding and metabolic responses in rainbow trout. The mode of action may include disruptions in brain monoamine levels and pathways involved in CREB signalling, while the exact mechanism of action remains to be elucidated. Exposure of fish to this pharmaceutical drug adversely affects the highly conserved adaptive responses that are essential to cope with subsequent stressors, and may translate into reduced fitness over the long-term. The findings underscore the necessity to understand the mechanisms of action of chemicals present in MWWE, and develop and utilize effective risk management strategies aimed at minimizing discharge of pharmaceuticals into the aquatic environment.

Orientador: Margarida Maria Barros / Resumo: O objetivo do presente estudo foi avaliar os efeitos do concentrado de melão rico em SOD (superóxido dismutase) sobre o desempenho produtivo, perfil hematológico e atividade das enzimas antioxidantes da tilápia-do-Nilo submetidas a desafio por estresse térmico e hipóxia. Um grupo de 462 tilápias revertidas sexualmente (8,87 g ± 0,12) foi distribuído aleatoriamente em 42 aquários de 250 L (11 peixes/aquário) e alimentado com sete dietas práticas contendo níveis de concentrado de melão 0; 0,1; 0,2; 0,3; 0,4; 0,5 e 1,0% por 60 dias. As dietas foram formuladas para conter 29% de proteína digestível e 18 MJ de energia digestível kg-1. Após 60 dias de alimentação, determinou-se o desempenho produtivo, parâmetros hematológicos e atividade das enzimas do sistema antioxidante. Em seguida, os peixes foram submetidos a desafio térmico e hipóxia (34°C/1,87 mg/L-1 de oxigênio dissolvido) por dois dias e os mesmos parâmetros hematológicos e atividade de enzimas do sistema antioxidantes foram determinados após esse período. No presente estudo, o concentrado de melão foi capaz de manter a eritropoiese e a capacidade antioxidante sob estresse térmico e hipóxia, porém não houve efeito no desempenho produtivo. A concentração de malonaldeído presente no filé de peixes alimentados com dietas suplementadas com concentrado de melão diminuiu 1,5 vezes após o estresse. Os resultados do presente estudo sugerem o nível de suplementação entre 0,4 e 0,5% de concentrado de melão capaz de manter a saúde do... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluated the effects of SOD-rich melon pulp concentrate (SOD-rich MPC) on growth performance, hematological profile and antioxidant enzyme activity of Nile tilapia subjected heat/dissolved oxygen-induced stress (HDOIS). A group of 462 male Nile tilapia (8.87 g ± 0.12) was randomly distributed in 42 250-L aquaria (11 fish/tank) and fed seven practical diets with graded levels of SOD-rich MPC at 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0% for 60 days. The diets were formulated to contain 30% crude protein and 18 MJ kg-1 crude energy. After the feeding period, growth performance was evaluated and six fish per treatment were sampled for hematological profile and antioxidant enzyme activity. Then, fish were subjected to HDOIS (34°C/1.87 mg/L-1 dissolved oxygen) for two days and the same hematological profile and antioxidant enzyme activity were determined. In the present study, SOD-rich MPC was able to keep erythropoiesis and antioxidant capacity under HDOIS, but did not affect growth performance. The concentration of MDA in fish fed diets supplemented with SOD-rich MPC decreased by 1.5 fold after HDOIS. The results of the present study suggest that 0.5% SOD-rich MPC dietary supplementation was appropriate to maintain Nile tilapia health and improve their antioxidant capacity under HDOIS. / Mestre

Pyrethroids are ones of the most used pesticides worldwides. The widespread use and high stability of pyrethroids lead to the assumption of that their occurrence in the environment could be quite frequent. They can reach water ecosystem as pollutants. Residues of pyrethroids are not only detected in the water column, but also in sediments and in fish tissues. The first study was devoted to the product Nexide containing 60 g.l-1 of active substance gamma-cyhalothrin. Tested Nexide concentrations were 5, 25, 50, 100, and 250 &microg.l-1. Early life stage test was used.Common carp (Cyprinus carpio) was tested. There were significant mortalities in all concentrations except the lowest concentration during the trial. The lowest concentration tested 5 &microg.l-1 only caused a slightly increased mortality. This lowest concentration influenced the growth in length and weight negatively, decelerated ontogenetic development, and made the body surface of the individuals darker. Histopathology of individuals from this concentration revealed dystrophy in liver. Examination of kidney, intestine and gills did not show significant histopathological differences compared with control. The evaluation of selected parameters of oxidative stress demonstrated a significantly higher activity of detoxification enzyme glutathione-S.transferase (GST) and a significantly lower activity of defensive enzyme glutathione peroxidase (GPx) compared with the control group. The other examined parameters of oxidative stress such as catalase (CAT), glutation reductase (GR), and lipid peroxidation determined by using the thiobarbituric acid-reactive substances (TBARs) were comparable to the control group. Changes in oxidative stress parameters suggest that exposure of the organism to the product Nexide in the given concentration leads to dysbalance of defensive enzymes. The second study was devoted to the product Cyperkill 25 EC containing 250 g.l-1. Tested Cyperkill 25 EC concentrations were 7.2, 36, 72, 144, and 360 &microg.l-1. The procedure of the trial was the same as the preceded one. There were 100% mortalities in all concentrations except the lowest concentration during the trial. The lowest tested concentration 7.2 &microg.l-1 allowed 90% of individuals to stay alive till the end of experiment. The lowest concentration influenced the growth in length and weight negatively and decelerated ontogenetic development compared with the control. Any individual exposed to this concentration did not reach juvenile stage until the end of the trial. Dark pigmentation was visible in 68% of these exposed individuals on the last day. Similar darkening was visible in individuals from higher concentrations shortly before death too. Histological examination did not revealed significant changes in intestine, liver, kidney, and gills compared with the control group. Evaluation of selected parameters of oxidative stress demonstrated significantly lower activities of GST, GR, and GPx. Activities of CAT and TBARS were comparable with the control group. Changes in oxidative stress parameters suggest that exposure of the organism to the product Cyperkill 25 EC in the given concentration could induce oxidative stress and interfere with the activities of antioxidant enzymes. The presented thesis summarises actual data about pyrethroids and their influence on fish. The demonstrated effects confirm high susceptibility of early developmental stages of fish to tested pesticides. When interpreting the results, we have to take into account the fact that studies showed this risk even on single pyrethroid substances. But water organisms are exposed to many other more or less toxic products and substances in a real environment. These xenobiotics could react with each other and their mixture could even potentiate negative effects. The performed studies also clearly show the significant differences in the sensitivity of embryonic and embryolarval tests.