Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-12T12:12:32Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Mestrado_JulianaScanoni_Corrigido_19.11.pdf: 2056204 bytes, checksum: 8a5314fcf4ab820bdedd049620355c53 (MD5) / Made available in DSpace on 2016-04-12T12:12:32Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Mestrado_JulianaScanoni_Corrigido_19.11.pdf: 2056204 bytes, checksum: 8a5314fcf4ab820bdedd049620355c53 (MD5) Previous issue date: 2015-07-28 / CAPEs / Hidrocarbonetos policíclicos aromáticos (HPAs) são compostos derivados de petróleo conhecidos por seu potencial de toxicidade a organismos aquáticos. Regiões estuarinas são frequentemente contaminadas por HPAs em decorrência de processos de urbanização e atividades industriais, incluindo a cadeia produtiva do petróleo. O trabalho teve o objetivo de avaliar a bioconcentração e efeitos bioquímicos de HPAs em Eugerres brasilianus coletados em quatro estuários no litoral sul de Pernambuco, a fim de obter um diagnóstico da poluição por estes contaminantes nestas regiões. As coletas foram realizadas no rio Ariquindá, sistema estuarino do rio Formoso (AR-SERF), no rio Massangana, no complexo estuarino de Suape (MA-CES), no sistema estuarino de Barra de Jangada (SEBJ), e no complexo estuarino da bacia do Pina (CEBP). A bile dos peixes foi analisada por Fluorescência Fixa para estimar a concentração em equivalentes dos HPAs naftaleno, fenantreno e criseno. Amostras de fígado foram utilizadas para análise de biomarcadores enzimáticos de fase I (etoxiresorufina orto-deetilase, EROD), fase II (glutationa S-transferase, GST), e defesa antioxidante (catalase, CAT, e glutationa redutase, GR). A análise de HPAs na bile e atividades enzimáticas em peixes coletados num ciclo anual em MA-CES e AR-SERF indicou uma semelhança na contaminação por HPAs na bile e nos níveis de atividades enzimáticas dos biomarcadores avaliados entre estes estuários, apesar da diferença do padrão de atividades antrópicas destas regiões. O CES comporta um complexo portuário com alta atividade industrial enquanto o SERF possui baixa densidade populacional e atividades turísticas. Os peixes coletados em SEBJ e CEBP apresentaram bile com concentrações do HPA criseno entre 13 x e 19 x maiores que AR-SERF, respectivamente. Foi detectado aumento das atividades da EROD, GST e CAT das regiões do SEBJ e CEBP, chegando a 30 x para a EROD, e aproximadamente 2 x para GST e CAT em comparação com AR-SERF. A maior bioconcentração de HPAs na bile e maior indução enzimática nos peixes E. brasilianus do SEBJ e CEBP indicam que estes estão dispendendo energia para tentar se adaptar a esta contaminação, o que pode ter consequências para o seu crescimento e sobrevivência nestas regiões. Os resultados indicam que SEBJ e CEBP recebem um maior aporte de HPAs, associado a maior densidade populacional e atividades antrópicas nas bacias desses rios. Os parâmetros utilizados serão úteis para o monitoramento destes sistemas estuarinos, em especial do CES, em franco processo de urbanização e industrialização. / Polycyclic aromatic hydrocarbons (PAHs) are oil derived compounds known for their toxicity potential towards aquatic organisms. Estuarine regions are frequently contaminated with PAHs as a result of urbanization processes and industrial activities, including the oil productive chain. The work aimed to evaluate the bioconcentration and the biochemical effects of PAHs in Eugerres brasilianus sampled from four estuaries in the south coast of Pernambuco, in order to obtain a pollution diagnosis by these contaminants in such regions. The data collection was made in the Aquirindá river, Rio Formoso estuarine system (AR-RFES), in the Massangana river, in the Suape’s estuarine complex (MA-SEC), in the Barra de Jangada estuarine system (BJES) and in the Bacia do Pina estuarine complex (BPEC). The fish’s bile were analyzed using fixed fluorescence to estimate the equivalent concentrations of the PAHs naphthalene, phenanthrene and chrysene. Liver samples were used for phase 1 enzymatic biomarkers analysis (Ethoxyresorufin-O-deethylase, EROD), phase II (glutathione S-transferase, GST) and antioxidant defense (catalase, CAT, and glutathione reductase, GR). The analyzes of PAHs in bile and enzymatic activity in fish that were sampled in an annual cycle in MA-SEC and AR-RFES indicated a similarity between PAHs contamination in bile and the enzymatic activity levels of the biomarkers evaluated among these estuaries, despite the different anthropogenic activity patterns. The SEC comprises a port complex with high industrial activity, while the RFES has low population density and touristic activities. The bile of the fish sampled in BJES and BPEC showed chrysene concentration between 13 x and 19 x higher than AR-RFEC, respectively. An increase in EROD, GST and CAT activities was detected in the regions of BJES and BPEC, reaching 30 x for EROD, and approximately 2 x for GST and CAT. The high PAHs bioconcentration in the bile and the high enzymatic induction in E. brasilianus fish from BJES and BPEC indicate that these fish are wasting energy to try to become adapted to this contamination, which may have consequences to their growth and survival in such regions. The results indicate that BJES and BPEC receive a greater input of PAHs, associated with the higher population density and anthropogenic activities in these rivers’ basins. The parameters used will be useful for the monitoring of these estuarine systems, especially in SEC, which suffers with a rapid urbanization and industrialization process.

Recent studies have shown that dehydroabietic acid (DHAA), a resin acid present in pulp and paper mills, may have anti-estrogenic effects in fish. A chronic-exposure toxicity experiment using immature rainbow trout (Oncorhynchus mykiss) was conducted in order to assess the endocrine disrupting and liver metabolic effects of the wood extractives DHAA and β- sitosterol (BS) regularly present in pulp and paper mills and the model estrogen 17β-estradiol (E2). It was found that exposure to 5 ppm of E2 significantly increased hepatosomatic index (HSI), vitellogenin (VTG) and plasma sorbitol dehydrogenase (SDH). This effect was reduced by mixing E2 with DHAA, indicating that DHAA does not cause its anti-estrogenic effects indirectly due to liver damage. Exposure to 5 ppm of DHAA caused a significant increase in liver citrate synthase (CS), and liver ethoxyresorufin-O-deethylase (EROD) activity after 7 days, however, the fish recovered by 28 days. This study also determined the effect of 14 different pulp and paper mill effluent extracts on liver enzyme metabolism through alterations in the activity of liver lactate dehydrogenase activity (LDH) and CS. This activity varied greatly between mills but most showed an induction of CS after 28 days exposure through i.p. injection. The results of the study indicate that DHAA may alter energy metabolism as well as cause anti-estrogenic effects in female juvenile rainbow trout. / UOIT

Flavonoids, present in fruits, vegetables and beverages derived from plants, have been described as health-promoting, disease-preventing dietary supplements, and have activity as cancer preventive agents. The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xenobiotics. Cytochrome P4501A1 (CYP1A1) is a Phase I enzyme which is known to be involved in the activation of procarcinogens and Glutathione S-Transferase (GST) is a Phase II enzyme which is largely responsible for the detoxification of carcinogens. In this study, it was aimed to investigate the mechanisms of inhibition of CYP1A1 and GST activities of fish by phenolic compounds/flavonoids. Leaping mullet (Liza saliens), captured from highly polluted sites of izmir Bay, expressing high levels of CYP1A, were used in order to investigate these effects. It was demonstrated that all of the phenolic compounds/flavonoids used, exert an inhibitory effect on both CYP1A1 associated 7-Ethoxyresorufin-O-deethylase (EROD) activity and GST activities of fish, although the degree of inhibition was varied with the flavonoid used. Of the flavonoids tested, the most potent inhibitor of CYP1A1 associated EROD activity was found to be quercetin. The potency of the phenolic compounds/flavonoids to inhibit CYP1A1 associated EROD activity follow the sequence of quercetin &gt / resveratrol &gt / naringenin &gt / hesperidin &gt / rutin with IC50 values of 1.32 &micro / M, 3.59 &micro / M, 9.78 &micro / M, 98.5 &micro / M and 0.64 mM respectively. Quercetin, resveratrol, hesperidin and rutin were found to inhibit EROD activity in a competitive manner, on the other hand, naringenin was found to inhibit EROD activity in a non-competitive manner. Inhibition constant (Ki) values of quercetin, resveratrol, naringenin, hesperidin and rutin were calculated from Dixon plots as 0.12 &micro / M, 0.67 &micro / M, 2.63 &micro / M, 18 &micro / M and 0.1 mM, respectively. In the case of GST enzyme, it was demonstrated that all of the phenolic compounds/flavonoids used, exert an inhibitory effect on both total GST and GST-Mu activities of fish. Of the flavonoids tested, the most effective inhibitor of total GST activity was found to be resveratrol. The potency of the phenolic compounds/flavonoids to inhibit total GST activity follow the sequence of resveratrol &gt / quercetin &gt / rutin &gt / naringenin &gt / hesperidin with IC50 values of 7.1 &micro / M, 24.5 &micro / M, 89 &micro / M, 116 &micro / M and 118 &micro / M respectively. Resveratrol, quercetin and hesperidin were found to inhibit total GST activity in a competitive manner, on the other hand, rutin and naringenin were found to inhibit GST activity in a mixed type manner. Ki values of resveratrol, quercetin, hesperidin, naringenin and rutin were calculated from Dixon plots as 3.2 &micro / M, 12.5 &micro / M, 45 &micro / M, 128 &micro / M and 150 &micro / M respectively. In the case of GST-Mu activity, the most potent inhibitor was found to be rutin. The potency of the phenolic compounds/flavonoids to inhibit GST-Mu activity follow the sequence of rutin &gt / resveratrol &gt / quercetin &gt / naringenin &gt / hesperidin with IC50 values of 66.5 &micro / M, 72.3 &micro / M, 113.5 &micro / M, 135.5 &micro / M and 196 &micro / M, respectively. In conclusion, this study indicated that flavonoids were the strong inhibitors of CYP1A1 associated EROD activity and GST activities of mullet liver. The modulation of drug-metabolizing enzymes by flavonoids is important in terms of human health, since these enzymes can activate or inactivate carcinogens. The potential role of xenobiotic metabolizers CYP1 family in the activation of carcinogens and inactivation of chemotherapeutics suggests a potential therapeutic benefits in inhibiting these enzymes. The results of the present study support the hypothesis that flavonoids may be involved in the prevention of malignant transformation, by reducing the formation of carcinogens through inhibition of enzymes such as CYP1A1 which is known to be involved in carcinogen activation.

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants (POP) issus d’une combustion incomplète de matière organique, d’origine naturelle (incendies de forêt) et anthropique (chauffage au gaz, trafic routier, industrie…). La consommation par les animaux d’élevage de couverts végétaux et sols contaminés en HAP, couplée à une forte lipophilie de ces derniers, constitue un risque potentiel en terme de contamination des produits animaux (lait, viande, œufs…). Dans ce travail, nous nous sommes intéressés à l’évaluation de l’exposition du ruminant laitier aux HAP au travers de l’utilisation de biomarqueurs métaboliques d’exposition orale aux HAP. Nous avons tout d’abord testé l’aptitude du 1-hydroxypyrène dans l’urine et/ou le lait à être utilisé comme biomarqueur métabolique spécifique d’exposition orale et subchronique (7 jours, 40 jours) de la chèvre au mélange : phénanthrene, pyrène, benzo(a)pyrene. Ceci en utilisant de l’huile comme vecteur de contamination sur la plage 0.04-50 mg/jour de chacun des 3 HAP. Les résultats démontrent entre autres que (i) le 1-hydroxypyrène est excrété de manière proportionnelle au niveau d’exposition sur toute la plage d’exposition testée dans le lait (taux de transfert stable de l’ordre de 1% dans le lait et 10% dans l’urine) ; (ii) qu’un plateau d’excrétion est obtenu au plus tard 10 jours après le début de l’exposition. La seconde partie de ce travail a permis de démontrer le potentiel de l’activité ethoxyresorufin-o-deethylase (EROD) dans les lymphocyte périphérique sanguins (LPS) à être utilisé comme un biomarqueur d’exposition orale aspécifique du ruminant laitier aux POP CYP450 inducteurs, dont certains HAP. Des cinétiques d’induction EROD lymphocytaire par exposition orale aux HAP ont ainsi pu être modélisées par un modèle de type logistique sur 40 jours d’exposition suivis de 10 jours post-exposition. Une ébauche de courbe dose-réponse de type Michaelis-Menten a par ailleurs pu être déterminée, autorisant plusieurs commentaires relatifs au métabolisme des HAP par le ruminant laitier. Une dernière étude cinétique, menée chez le rat en mode subchronique sur 32 jours, a finalement permis, entre autres, de mettre en évidence une bonne corrélation entre les activités EROD dans les lymphocytes sanguins périphériques, le foie et le tissu cérébral. L’ensemble des résultats obtenus démontre la pertinence de l’usage combiné de l’activité EROD lymphocytaire et du 1-OH pyrène dans le lait ou l’urine comme outils d’évaluation pratiques et accessibles du risque lié à l’ingestion de POP par les ruminants laitiers / Polycyclic aromatic hydrocarbons (PAH) are persistent organic pollutants (POP) produced during the incomplete burning of organic materials. Their production can be of natural origin (forest fire) or anthropic origin (gaz heating, vehicular traffic, industry). The ingestion of PAH contaminated vegetal covers or soils by farm animals, coupled with the high lipophily of these PAH, therefore represents a potential hazard in terms of contamination of animal products (milk, meat, eggs…). In the present work, we focused on the evaluation of the dairy ruminant exposure to PAH through the use of metabolic biomarkers of exposure. At first we tested the potential of 1-hydroxypyrene excreted in urine or milk to be used as a metabolic and specific biomarker of subchronic (7 to 40 days) and oral exposure of the goat to a ternary mixture consisting of phenanthrene, pyrene and benzo(a)pyrene. Each PAH was solubilized in oil to reach contamination levels in the range 0.04-50 mg/day. Results demonstrate that (i) 1-hydroxypyrene excretion in milk and urine is proportional to the level of exposure all along the tested exposure range (stable transfer rates of 1-OH pyrene: about 1% in milk and 10 % in urine); (ii) excretion of 1-OH pyrene reached a plateau at the latest 10 days after the beginning of exposure. In the second part of this work, it was demonstrated that the ethoxyresorufin-o-deethylase (EROD) activity, when measured in peripheral blood lymphocytes (PBL), can be used as a convenient and non-specific biomarker of oral and chronic exposure of dairy ruminant to CYP 450 inducting POP, such as many PAH. Induction kinetic of EROD activity PBL could be fitted with a logistic-like model over 40 days of exposure followed by 10 days post-exposure. An approximate dose/response curve could be fitted using a Michaelis-Menten-like model, allowing for several comments about the metabolism of PAH in dairy ruminant. A final kinetic study, which was run on rats under subchronic conditions (32 days), next to other results, showed a good correlation between EROD activities in PBL, liver and brain. Achieved results demonstrate the relevance of the combined use of the EROD activity in PBL and of the 1-OH pyrene in milk or urine as convenient and cost-limited tools for risk assessment in terms of PAH and more generally POP ingestion by dairy ruminants

L’évaluation de l’impact des polluants dans l’environnement est une des préoccupations majeures qui s’inscrit dans la Directive Européenne Cadre Eau 2000. Les réglementations préconisées ont notamment pour objectif de parvenir au bon état chimique et écologique des masses d’eau. Dans ce contexte, notre étude a consisté à développer une approche multibiomarqueur sur des juvéniles de poisson afin d’évaluer les effets biologiques de la pollution chimique en milieu littoral. Des paramètres moléculaires de détoxification (EROD, GST) et une enzyme antioxydante (CAT) ont été utilisés en tant que «système d’alarme » susceptibles de détecter une perturbation avant l’apparition de signes pathologiques irréversibles. En parallèle, différents biomarqueurs physiologiques (croissance somatique et récente, rapport ARN/ADN, indices morphométrique et lipidique) ont été analysés en considérant que ceux-ci pourraient révéler les dommages induit par les polluants sur l’état de santé des juvéniles. La sensibilité et la pertinence des biomarqueurs moléculaires et physiologiques ont été testés expérimentalement sur des juvéniles : i) de bar exposés à une pollution aigüe de pétrole, ii) de bar et de turbot soumis à des mélanges de contaminants en concentrations environnementales en conditions contrôlée et semi-contrôlée (« caging »). Nos résultats montrent la capacité de l’EROD, et à un degré moindre de la GST, à détecter une exposition courte (2 et 4 jours) des organismes au pétrole et à refléter ses effets délétères sur leur état de santé. Cette relation entre biomarqueurs moléculaires et physiologiques a par contre été plus difficilement établie dans un contexte de pollution multiple. Les indices de croissance et de condition utilisés se sont avérés plus sensibles aux différents niveaux de contamination analysés (métaux et HAPs). Leur utilisation a permis d’évaluer la condition affaiblie des organismes mis en cage en milieu portuaire pendant 38 jours. Cette expérience de « caging » s’est révélée concluante, notamment pour les juvéniles de bar, sur lesquels aucun stress physiologique de la mise en cage n’a été détecté dans la station de référence. Les effets délétères des contaminants chimiques sur l’état de santé des juvéniles de turbot ont également été observés en condition contrôlée après exposition de 21 jours aux mêmes sédiments portuaires et à un sédiment estuarien. En complément de ces expériences, une étude de terrain a été réalisée sur des juvéniles de flet prélevés dans des estuaires le long de la côte française et belge. Une diminution des indices morphométrique et lipidique des juvéniles de flet, issu des trois estuaires anthropisés, a été observée en relation avec des bioconcentrations en métaux plus élevées que l’estuaire de référence. Les résultats issus de ces différentes études montrent la potentialité des indices de croissance et de condition à révéler les effets biologiques des contaminants chimiques sur les juvéniles de poissons marins. Cependant, leur spécificité vis à vis des polluants étant plus faible que les paramètres de détoxification, leur utilisation peut être limitée. Ces travaux montrent ainsi le besoin d’utiliser des biomarqueurs à différents niveaux d’organisation biologique dans les programmes de biosurveillance. / Evaluation of pollutant effects in environment is one of the major issues of the European Water Framework Directive 2000. Regulations have particularly the objective to reach to a good chemical and ecological status of water bodies. In this context, the aim of our study was to develop a multibiomarker approach on juvenile marine fish in order to evaluate the biological effects of chemical pollution in coastal areas. Molecular detoxification parameters (EROD, GST) and an antioxidant enzyme (CAT) were used as early warning tools of toxicity allowing the prevention of irreversible damages. In parallel, different physiological biomarkers (somatic and recent growth, RNA:DNA ratio, morphometric and lipidic indices) were analysed as reflecting damages on juveniles health. Sensitivity and relevance of molecular and physiological biomarkers were tested experimentally on juvenile : i) sea bass exposed to acute petroleum pollution, ii) sea bass and turbot submitted to a mix of contaminants in environmental concentrations during controlled and semi-controlled (caging) conditions. Our results show the ability of EROD, and in lower degree the GST, to detect short exposure (2 and 4 days) of organisms to petroleum and to reflect their deleterious effects on fish health. This relationship between molecular and physiological biomarkers was more difficultly established under multiple pollutions. Growth and condition indices were found to be more sensitive to the different levels of chemical contamination analysed (metal, PAHs). Their analyses allowed us to evaluate the weakened condition of organisms caged in the harbour area during 38 days. This caging experiment was relevant especially for juvenile sea bass in which no physiological stress was detected in the reference station. Deleterious effects of chemical contaminant on turbot juvenile health were also observed in controlled condition after 21 days exposure to the same harbour sediments and to an estuarine sediment. In complement to these experiments, a field study was realized on juvenile flounders sampled in some estuaries along the French and Belgium coast. A decrease of morphometric and lipidic indices were found in juvenile flounders from the three anthropogenic estuaries showing the highest metal bioconcentrations compared to the reference estuary. Results from these different studies showed the potentiality of growth and condition indices to reflect biological effects of chemical contaminants on juvenile marine fish. However, their use could be limited by their lower specificity to pollutant than parameters involved in detoxification. These works show therefore the need to use biomarkers at different level of biological organization in biomonitoring programs.

O presente estudo teve como objetivo principal compreender a dinâmica populacional de Mugil curema em duas regiões distintas, tanto sob o ponto de vista oceanográfico como no de ocupação humana, e os possíveis impactos antrópicos no ciclo de vida da espécie. As amostras foram coletadas mensalmente no período de março/09 a fevereiro/10 no estuário de Santos e no período de maio/08 a abril/2009 no sistema costeiro Cananéia-Iguape, utilizando rede de abalo, rede de tarrafa e cerco fixo. Em laboratório os indivíduos coletados foram medidos (mm), pesados (g) e dissecados, para a retirada das gônadas, fígado, rim e otólitos para futuras analises. Os indivíduos de Mugil curema do estuário de Santos e do sistema costeiro Cananéia-Iguape, litoral do estado de São Paulo, Brasil, foram classificadas como pertencentes ao mesmo estoque, não existindo diferenças na estrutura em tamanho, na estrutura etária e na época de desova desses indivíduos. Também foi constatado altos valores do índice de lesões histológicas e da atividade EROD no tecido do fígado e altos níveis de bioacumulação de metais (bromo, cobre, cromo, ferro e zinco) nos tecidos do fígado e rim da espécie nas duas áreas estudadas. Todas as variáveis estimadas não apresentaram diferenças significativas entre as duas áreas de estudo. / The present study has mainly aimed at understanding the population dynamic of Mugil curema from two distinct areas at coast of the state of São Paulo (Brazil), both from oceanographic and human occupation point of view. Samples were collected monthly between March/09 and February/10 of the Santos estuary and between May/08 and April/09 of the Cananéia-Iguape coastal system using gillnet, seine net and fish trap. Collected individuals were measured (mm), weighted (g) and dissected for removal of gonads, liver, kidney and otoliths for further analysis. Specimens of Mugil curema from Santos estuary and Cananéia-Iguape costal system were classified as belonging to the same stock, since the results showed no difference in structure, size, age structure and spawning season. High values of the histological lesions index and EROD activity in the liver tissue as well as increased levels of bioaccumulation of metals (bromine, copper, chromium, iron and zinc) in the liver and kidney tissues were also found. All of the estimated variables for Mugil curema were not significantly different between the two regions.

O presente estudo teve como objetivo principal compreender a dinâmica populacional de Mugil curema em duas regiões distintas, tanto sob o ponto de vista oceanográfico como no de ocupação humana, e os possíveis impactos antrópicos no ciclo de vida da espécie. As amostras foram coletadas mensalmente no período de março/09 a fevereiro/10 no estuário de Santos e no período de maio/08 a abril/2009 no sistema costeiro Cananéia-Iguape, utilizando rede de abalo, rede de tarrafa e cerco fixo. Em laboratório os indivíduos coletados foram medidos (mm), pesados (g) e dissecados, para a retirada das gônadas, fígado, rim e otólitos para futuras analises. Os indivíduos de Mugil curema do estuário de Santos e do sistema costeiro Cananéia-Iguape, litoral do estado de São Paulo, Brasil, foram classificadas como pertencentes ao mesmo estoque, não existindo diferenças na estrutura em tamanho, na estrutura etária e na época de desova desses indivíduos. Também foi constatado altos valores do índice de lesões histológicas e da atividade EROD no tecido do fígado e altos níveis de bioacumulação de metais (bromo, cobre, cromo, ferro e zinco) nos tecidos do fígado e rim da espécie nas duas áreas estudadas. Todas as variáveis estimadas não apresentaram diferenças significativas entre as duas áreas de estudo. / The present study has mainly aimed at understanding the population dynamic of Mugil curema from two distinct areas at coast of the state of São Paulo (Brazil), both from oceanographic and human occupation point of view. Samples were collected monthly between March/09 and February/10 of the Santos estuary and between May/08 and April/09 of the Cananéia-Iguape coastal system using gillnet, seine net and fish trap. Collected individuals were measured (mm), weighted (g) and dissected for removal of gonads, liver, kidney and otoliths for further analysis. Specimens of Mugil curema from Santos estuary and Cananéia-Iguape costal system were classified as belonging to the same stock, since the results showed no difference in structure, size, age structure and spawning season. High values of the histological lesions index and EROD activity in the liver tissue as well as increased levels of bioaccumulation of metals (bromine, copper, chromium, iron and zinc) in the liver and kidney tissues were also found. All of the estimated variables for Mugil curema were not significantly different between the two regions.

No description available.

5 Abstract Cytochrome P450 1A1 (CYP1A1) is one of the major isoforms of the cytochrome P450 superfamily. It is primarily an extrahepatic enzyme which is responsible for oxidation of many polycyclic aromatic hydrocarbons and other xenobiotics. Besides of the role in detoxification metabolism CYP1A1 is the one most important isoform involved in activation of procarcinogens. The main aim of this project was preparation of two modifications of the rat CYP1A1 gene with codon optimization for expression in E. coli by gene synthesis. One was wild type (wt1A1) and the other was with modified N-terminal anchor (mod1A1) - for both modifications with or without His Tag at the C-end of CYP1A1. Furthermore, an aim was to compare their level of expression in different strains of E. coli and try to purify and assess enzymatic activity of the gene's products. From pre-prepared oligonucleotides 2 "syntons" (parts of gene) were synthetized and separately inserted into pUC19. After verified sequence of the "syntons" they were cleaved from pUC19 and inserted together into pET-22b. These vectors were prepared for transformation of 3 strains of E. coli (BL-21 (DE3) GOLD, RIL a RIPL). For production of proteins many conditions were tested: temperature (18, 22, 24, 27 a 37 řC), time of production (untill 48 hours), concentration...

<p>A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin <i>O</i>-deethylase (EROD) was developed in rainbow trout (<i>Oncorhynchus mykiss</i>) and applied to Atlantic salmon (<i>Salmo salar</i>), Arctic charr (<i>Salvelinus alpinus</i>), Atlantic cod (<i>Gadus morhua</i>), saithe (<i>Pollachius virens</i>), and spotted wolffish (<i>Anarhichas minor</i>). Exposure to waterborne β-naphthoflavone (βNF; 10^-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10^-9 M) and the textile dye indigo (10^-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity.</p><p>A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne ³H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10^-7 M) and indigo (10^-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10^-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae.</p><p>My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.</p>