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Oestrogen is one of the most important hormonal regulators and is known to play a key role in the development and growth of breast cancer. The majority of tumours have a hormone dependent growth, and this is indicated by the presence of oestrogen receptors (ERs). About two thirds of breast cancers occur after the menopause when the ovaries have ceased to produce oestrogen and despite the low levels of circulating oestrogen’s the tumour concentrations of oestrone, oestradiol and their sulfates have been shown to be significant. Patients with hormone dependent tumours are candidates for treatment with the anti-oestrogen tamoxifen, which acts by competing with oestrogen for binding to the ER thereby, diminish the transcription of oestrogen regulated genes. The drug is mainly metabolised by cytochrome P450 enzymes in the liver and to a lesser extent locally in the breast, where upon several produced metabolites have higher affinity for the ER than the mother substance. Patients treated with tamoxifen have in general a prolonged disease-free survival. Even if most patients respond well to tamoxifen about 30-50 % either fail to respond or become resistant by incompletely understood mechanisms. Therefore, the aim of this thesis was to investigate possible mechanisms responsible for tamoxifen resistance. In paper I and II we studied genetic variants of enzymes participating in the metabolism of tamoxifen and assessed whether these variants correlated to breast cancer prognosis and/or to the benefit of tamoxifen. The results indicate an influence of CYP2D6, CYP3A5, and SULT1A1 genotypes in tamoxifen response. Further, tamoxifen has shown to compete with oestrogen for the binding to ER. In paper III we measured the expression levels of enzymes involved in the local synthesis of oestrogens in order to see if they correlated to clinical outcome. The protein expression of stromal aromatase was shown to have a prognostic significance, especially in ER-positive patients. Finally, tamoxifen and its ER-active metabolites have shown to induce both cell cycle arrest and apoptosis and one central mediator in these processes is the tumour suppressor protein p53. The proapoptotic activity of p53 is dependent on a proline rich domain containing a common Pro-to-Arg polymorphism. In paper IV we examined the value of this genetic variant as a predictive marker for anti-cancer therapy and found that patients carrying the Pro-allele might be good responders of tamoxifen therapy. The present thesis further indicates the complexity of the mechanisms underlying tamoxifen resistance. In summary, genetic variants of metabolic enzymes, genetic variants in p53, as well as expression levels of enzymes involved in local oestrogen synthesis, may have influence on breast cancer prognosis and may be useful markers in the prediction of tamoxifen response.

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

Currently, many patients with early-stage localized prostate cancer (PrCa) (D’Amico: low risk or low-intermediate risk) do not receive immediate therapy but are monitored within systematic AS programs. Prospective trials showed rates of stage reclassification and progression to the treatment of 20–40% over 2–5 years. However, in certain patients, PrCa progresses rapidly to an advanced stage that requires combined modality therapies, which carry increased risk for toxicity and poor outcomes. There is a need to identify biomarkers that can predict the risk for disease progression in this population. Research showed that dysregulation of metabolism is an important hallmark of cancer progression. Here, we pursued a pilot investigation of enzymes of de novo lipogenesis [ATP-citrate lyase (ACLY), Acetyl-CoA Carboxylase (ACC)], lipid oxidation [a-Methylacyl-CoA Racemase (AMACR)], glucose uptake [facilitative glucose transporter 1 (GLUT1)], and folate – glutamate metabolism (PSMA: prostate-specific membrane antigen) as potential biomarkers of PrCa progression in AS patients. With ethics approval from the Hamilton Integrated Research Ethics Board (HiREB), 40 AS patients were accrued prospectively from the Niagara Health System PrCa diagnostic program clinics and were asked to donate their biopsy tissue. 28 patients progressed on repeat biopsies at 12 or 24 months after initial diagnosis and were included in the “Progressed” group, and 12 did not who were included in the “Non-Progressed” group. Baseline diagnostic prostate core biopsy tissues of both groups were evaluated with H&E and immunohistochemistry (IHC) staining for ACLY, ACC, GLUT1, AMACR and PSMA expression (quantified by H-score). H-scores were evaluated in benign and malignant components (epithelial cells) and were compared between the two groups of patients. We observed statistically significant increased GLUT1 expression in malignant epithelial cells of the progressed group compared to the non-progressed group. Also, we found statistically significant increased PSMA expression in the benign epithelial cells of the progressed group compared to the non-progressed group. Further, our results demonstrated a statistically significant increase in ACLY and ACC expression in malignant epithelial cells compared to benign epithelial cells in the progressed group, while AMACR was detected solely in the malignant component. Overall, the results of this pilot study are consistent with the notion of induction of glycolytic metabolism, de novo lipogenesis and increased PSMA expression associated with the risk for PrCa progression. The levels of expression of PSMA within benign epithelial cells and GLUT1 within malignant epithelial cells may have value as predictive markers of risk for PrCa progression in AS patients. Future studies should investigate this concept systematically in larger AS cohorts. / Thesis / Master of Science (MSc) / Currently, many patients with localized prostate cancer do not receive immediate therapy and are monitored within systematic active surveillance (AS) programs. The main aim of AS management is to prevent overtreatment and treatment-related complications in patients who would otherwise have a good quality of life despite dealing with prostate cancer. However, many of these patients, especially those with low intermediate-risk prostate cancer have a significant risk for disease progression and metastasis. Additionally, there is a lack of promising tissue biomarkers to predict the risk for progression in AS patients at the time of initial diagnosis. Research showed that metabolism dysregulation is an essential hallmark of cancer progression, including prostate cancer. In this pilot study, we examined whether the expression of enzymes involved in lipid, glucose and protein metabolism could have value as biomarkers of risk for prostate cancer progression in patients managed with AS. The expression of five metabolic enzymes (ACLY, ACC, GLUT1, AMACR and PSMA) was examined in tumor and benign regions of diagnostic biopsies of the prostate obtained from men managed with AS. Our early results suggest that the expression of enzymes of protein (PSMA) and glucose (GLUT1) metabolism may have value as biomarkers of risk for prostate cancer progression and should be investigated further in systematic studies.

In vitro genetic toxicity tests using cultured bacterial or mammalian cells provide a cost- and time-effective alternative to animal tests. Unfortunately, existing in vitro assays are not always reliable. This is in part due to the limited metabolic capacity of the cells used, which is often critical to accurately assess chemical genotoxicity. This limited metabolic capacity necessitates the use of exogenous sources of mammalian metabolic enzymes that can simulate in vivo mammalian metabolic activation reactions. In response to this, and other limitations, alongside the worldwide trend to reduce animal testing, there is an acute need to consider various strategies to improve in vitro mutagenicity assessment. This thesis first examined the utility of exogenous metabolic activation systems based on human hepatic S9, relative to conventional induced rat liver S9, for routine genetic toxicity assessment. This was accomplished by critically evaluating existing literature, as well as new experimental data. The results revealed the limitations of human liver S9 for assessment of chemical mutagenicity. More specifically, the analyses concluded that, due to the increased risk of false negative results, human liver S9 should not be used as a replacement for induced rat liver S9. To address the limitations of conventional mammalian cell genetic toxicity assays that require exogenous hepatic S9, the thesis next evaluated the utility of an in vitro mutagenicity assay based on metabolically-competent primary hepatocytes (PHs) derived from the transgenic MutaMouse. Cultured MutaMouse PHs were thoroughly characterized, and found to temporarily retain the phenotypic attributes of hepatocytes in vivo; they express hepatocyte-specific proteins, exhibit the karyotype of typical hepatocytes, and maintain metabolic activity for at least the first 24 hours after isolation. Preliminary validation of the in vitro MutaMouse PH gene mutation assay, using a panel of thirteen mutagenic and non-mutagenic chemicals, demonstrated excellent sensitivity and specificity. Moreover, inclusion of substances requiring a diverse array of metabolic activation pathways revealed comprehensive metabolic competence. Finally, the thesis further investigated the applicability domain of the in vitro MutaMouse PH assay by challenging the assay with selected azo compounds. Comparison of these results with those obtained using the in vivo MutaMouse TGR (transgenic rodent) assay revealed that MutaMouse PHs can carry out some forms of reductive metabolism. Overall, this thesis demonstrated that a gene mutation assay based on MutaMouse PHs holds great promise for routine assessments of chemical mutagenicity.

I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems. By this time (beginning of 2002), I had finished my first semester and a major part of the course work, so we decided to start a new project focusing on some of the unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his Ph. D. from the lab, helped me in literature work and in finding targets for structural studies. Finally, we decided to target enzymes involved in the propionate etabolism. The pathways for propionate metabolism in Escherichia coli as well as Salmonella typhimurium were just established and there were no structural information available for most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle. Most of the enzymes involved in 2-methylcitrate pathway were not characterized biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected a complete diffraction data to a resolution of 2.5 Å which was later on extended to a resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems. Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the plausible rationale for different substrate specificities of these two enzymes. Due to unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we decided to target other enzymes involved in propionate metabolism. We looked into the literature for the metabolic pathways by which propionate is synthesized in the Salmonella typhimurium and finally decided to target enzymes present in the metabolic pathway which converts L-threonine to propionate. Formation of propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments. By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme. The structural studies carried out on these three enzymes have provided structural as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.

Les récepteurs nucléaires des xénobiotiques et les enzymes métaboliques P450 (CYP) constituent les principaux éléments contrôlant la biotransformation des médicaments, ainsi que le maintien de des barrières physiologiques au niveau périphérique, plus particulièrement, dans le foie et dans l’intestin. Plusieurs études ont mis en évidence la présence des CYP ainsi que les récepteurs nucléaires contrôlant leur expression, tels que le Constitutive Androstane Receptor et le Pregnane Xenobiotic Receptor (CAR et PXR). Des résultats précédant indiquent la surexpression des CYP2E1 et CYP3A4 dans des tissus et des cellules isolées du cerveau de patients épileptiques pharmacorésistants. L’importance de ces résultats réside dans le rôle du CYP2E1 et CYP3A4 dans la biotransformation de plusieurs médicaments antiépileptiques (MAE) suggérant ainsi un mécanisme de pharmacorésistance aux médicaments. Contrairement aux autres récepteurs nucléaires, les fonctions physiologiques des récepteurs nucléaires des xénobiotiques au niveau vasculaire sont mal connues. Nos résultats montrent des changements spatio-temporaux de l’expression des CYP2E1 et CYP3A4 dans le cerveau après une crise aigüe et pendant le processus d’épileptogenèse chez la souris. Une exposition in vivo et in vitro au MAE Phénytoïne induit une augmentation du niveau du CYP2E1. Le Phénobarbital et la Carbamazépine n’ont pas eu d’effet. Les souris privées des récepteurs nucléaires des xénobiotiques (PXR KO et CAR KO) ne présentent pas de changement de niveau basal des CYP dans le cerveau. Cependant, les souris CAR KO présentent des dysfonctionnements neuronaux (altération de la mémoire, comportement anxieux et une diminution de l’intensité des rythmes EEG entre 3.5-7 Hz) et des modifications caractérisées par une augmentation de perméabilité vasculaire et une dispersion des neurones hippocampiques. L’ensemble des résultats indique une régulation dynamique des CYP dans le cerveau avec une extension de l’impact des récepteurs des xénobiotiques à des fonctions neurovasculaires. / Xenobiotic nuclear receptors and P450 metabolic enzymes (CYP) are pivotal controllers of drug biotransformation and barrier functions in peripheral organs, including the intestine and the liver. Accumulating evidence suggested that, in human, central nervous system cells express significant levels of P450 enzymes and their upstream regulators e.g. Constitutive Androstane and Pregnane Xenobiotic Receptors (CAR, PXR). We previously showed the increased and ectopic CYP2E1 and CYP3A4 expression in brain specimens or cells obtained from drug resistant epileptic patient. These results are significant as CYP2E1 and CYP3A4 are responsible for the metabolism of several antiepileptic drugs (AED), therefore, suggesting a possible new mechanism of drug resistance. However, the exact determinants of CYP expression in the epileptic brain remain unknown. In addition, the exact role of nuclear xenobiotic receptor in the brain is understudied. The latter represents a significant knowledge gap as nuclear receptors other than the xenobiotic ones were shown to contribute to physiological neurovascular functions. Our results show spatio-temporal changes of CYP2E1 and CYP3A4 brain expression occuring after status epilepticus and during epileptogenesis in mice. Exposure to the AED phenytoin, phenobarbital, but not carbamazepine, increased brain CYP2E1 expression in vivo and in vitro. Lack of the specific xenobiotic receptors CAR and PXR did not impact basal CYP brain levels. However, we found an unexpected contribution of CAR to neuronal dysfunctions (memory impairment, anxiety like-behavior and decrease 3.5-7 Hz EEG waves) and neurovascular development, as indicated by increase vascular permeability and hippocampal neuronal dispersion. These results depict a dynamic regulation of P450 enzymes in the brain also expanding the impact of xenobiotic receptors to previously unexplored neurovascular functions.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Environmental contamination caused by frequent use of herbicides in agriculture has increased in last decades. The quinclorac and clomazone herbicides are widely used in agriculture, which can cause contamination to organisms, such as fish. However, it is necessary studies in order to minimize the toxic effects of these compounds on aquatic ecosystems. Micronutrients such as selenium, which is essential in the nutrition of fish, may have antioxidant functions against oxidative damage caused by herbicides. The objective of this study was to verify whether a diet supplemented with diphenyl diselenide [(PhSe)2], has a protective effect against damage induced by herbicides quinclorac and clomazone in fish species Cyprinus carpio (carp) and Rhamdia sp. (silver catfish). In the first experiment, carp were fed a diet without (PhSe)2 or a diet containing 3.0 mg/Kg of (PhSe)2 for 60 days and after were exposed to 1 mg/L of quinclorac for 192 h. Thiobarbituric acid reactive substances (TBARS), protein carbonyl, non-protein thiols (SHNP), ascorbic acid (AA), and the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) were determined in liver, gills, brain and muscle of carp. The quinclorac increased TBARS levels in liver and gills, decreased NPSH levels in brain and muscle, AA in muscle, inhibited enzymes SOD in liver and GST in liver and brain. The (PhSe)2 reversed these effects by preventing the increase of TBARS in liver and gills and recovering GST activity in liver and NPSH in brain and muscle. In the second experiment, silver catfish were fed a diet without (PhSe)2 or a diet containing 3.0 mg/Kg of (PhSe)2 for 60 days and after were exposed to 1 mg/L of quinclorac for 192 h. We investigated the effect of (PhSe)2 on plasma metabolic changes, enzymes of intermediary metabolism, pro-oxidants parameters and antioxidant defense in the liver of silver catfish. The weight, length and hepatic somatic index (HSI) were also determined. Animals exposed to quinclorac showed a decrease at HSI, an increase lactate levels in plasma and in the enzymes fructose bifosfatase (FBPase), glucose-6-phosphatase (G6Pase), glycogen phosphorylase (GPase) and aspartate aminotransferase (AST) in liver. Furthermore, were observed increased of TBARS, decreased NPSH and AA levels and inhibition of SOD in liver of silver catfish. The (PhSe)2 was effective in protecting the liver of silver catfish by decreased TBARS, increasing NPSH levels, AA and the activity of SOD. However, the (PhSe)2 was not effective in recovery the effects caused by the increase of hepatic enzyme AST. In the third experiment investigated the ability of (PhSe)2 in reducing oxidative damage in the liver, gills and muscle of carp and silver catfish exposed to clomazone. Silver catfish and carp were fed with a diet without (PhSe)2 or a diet containing 3.0 mg/Kg of (PhSe)2 for 60 days and after were exposed to 1 mg/L of clomazone (192 h). At the end of the exposure period, oxidative parameters and antioxidant defenses were determined. Silver catfish exposed to clomazone showed increased TBARS in liver and muscle and protein carbonyl in liver and gills. In addition, decrease in levels of NPSH in liver and gills, AA in the liver and of enzyme glutathione peroxidase (GPx) in liver was observed. The (PhSe)2 reversed some effects caused by clomazone in silver catfish, preventing the increase in TBARS and protein carbonyl and by increasing NPSH and AA levels. Moreover, clomazone no caused apparent situation of oxidative stress in carp and thus cannot assess the role of (PhSe)2 in this species exposed to that herbicide. However, the (PhSe)2 in both species reduced per se the TBARS levels in liver and muscle, increased NPSH and AA levels and the activity of GPx in liver. Considering that exposure to herbicides is more frequent and that is the cause of several changes in fish, the results of these studies are of great importance, since the (PhSe)2 may represent an alternative to prevent or mitigate toxicity caused by herbicides in different fish species of commercial importance. / A contaminação ambiental causada pelo uso frequente de herbicidas na agricultura tem aumentado nas últimas décadas. O quinclorac e o clomazone são herbicidas amplamente utilizados na agricultura, os quais podem causar contaminação a organismos, como os peixes. Entretanto é necessário estudos no sentido de minimizar os efeitos tóxicos desses compostos em ecossistemas aquáticos. Micronutrientes como o selênio, o qual é essencial na nutrição de peixes, podem ter funções antioxidantes contra danos oxidativos causados por herbicidas. Assim, o objetivo deste trabalho foi verificar se uma dieta suplementada com o disseleneto de difenila [(PhSe)2], possui efeito protetor contra danos induzidos pelos herbicidas quinclorac e clomazone em peixes das espécies Cyprinus carpio (carpas) e Rhamdia sp. (jundiás). No primeiro experimento carpas foram alimentadas com uma dieta sem (PhSe)2 ou uma dieta contendo 3.0 mg/Kg de (PhSe)2 por 60 dias e após foram expostas a 1 mg/L do quinclorac por 192 horas. Substâncias reativas ao ácido tiobarbitúrico (TBARS), carbonilação de proteínas, tióis não proteicos (SHNP), ácido ascórbico (AA), bem como a atividade das enzimas catalase (CAT), superóxido dismutase (SOD) e glutationa S-transferase (GST) foram determinados em fígado, brânquias, cérebro e músculo de carpas. O quinclorac aumentou os níveis de TBARS em fígado e brânquias, diminuiu os níveis de SHNP em cérebro e músculo e de AA no músculo e inibiu as enzimas SOD em fígado e GST em fígado e cérebro. O (PhSe)2 reverteu esses efeitos prevenindo o aumento do TBARS em fígado e brânquias e recuperando a atividade da GST em fígado e SHNP em cérebro e músculo. No segundo experimento jundiás foram alimentados com uma dieta sem (PhSe)2 ou uma dieta contendo 3.0 mg/Kg de (PhSe)2 por 60 dias e após expostos a 1 mg/L do quinclorac por 192 horas. Investigou-se o efeito do (PhSe)2 sobre as alterações metabólicas no plasma, enzimas do metabolismo intermediário, bem como parâmetros pró-oxidantes e de defesa antioxidante em fígado dos jundiás. O peso, comprimento e índice hepatossomático (IHS) também foram determinados. Animais expostos ao quinclorac apresentaram diminuição no IHS, aumento nos níveis de lactato em plasma e das enzimas frutose bifosfatase (FBPase), glicose 6-fosfatase (G6Pase), glicogênio fosforilase (GPase) e da asparato aminotransferase (AST) em fígado. Além disso, foram observadas aumento do TBARS, diminuição nos níveis de SHNP e AA e inibição da enzima SOD no fígado dos jundiás. O (PhSe)2 foi efetivo em proteger o tecido hepático dos jundiás por diminuir o TBARS, aumentar os níveis de SHNP, AA, e a atividade da SOD. Porém o (PhSe)2 não foi eficaz na recuperação dos efeitos causados pelo aumento da enzima hepática AST. No terceiro experimento investigou-se a capacidade do (PhSe)2 em reduzir o dano oxidativo em fígado, brânquias e músculo de carpas e jundiás expostos ao clomazone. Jundiás e carpas foram alimentados com uma dieta sem (PhSe)2 ou uma dieta contendo 3.0 mg/Kg de (PhSe)2 por 60 dias e após foram expostos a 1 mg/L do clomazone (192 horas). No final do período de exposição, parâmetros oxidativos e defesas antioxidantes foram determinados. Jundiás expostos ao clomazone apresentaram aumento do TBARS em fígado e músculo e da proteína carbonil em fígado e brânquias. Além da diminuição nos níveis de SHNP em fígado e brânquias, AA em fígado e inibição da enzima glutationa peroxidase (GPx) em fígado. O (PhSe)2 reverteu alguns efeitos causados pelo clomazone em jundiás, prevenindo o aumento do TBARS e da proteína carbonil e aumentando os níveis de SHNP e AA. Por outro lado, o clomazone não causou uma aparente situação de estresse oxidativo em carpa e assim não podemos avaliar o papel do (PhSe)2 nesta espécie exposto a esse herbicida. Porém o (PhSe)2 em ambas as espécies diminuiu per se os níveis de TBARS em fígado e músculo, aumentou os níveis de SHNP e AA e atividade da enzima GPx em fígado. Considerando-se que a exposição aos herbicidas é cada vez mais frequente e que é a causa de diversas alterações em peixes, os resultados desses trabalhos são de grande importância, uma vez que o (PhSe)2 pode representar uma alternativa para prevenir ou atenuar a toxicidade causada por herbicidas em diferentes espécies de peixes de importância comercial.