In this study, the ability of various chemicals to be biotransformed into mutagens by plant cells was investigated. Two thiocarbamate herbicides, diallate and triallate, the sulfonylurea herbicide chlorsulfuron, and the aniline derivative m-phenylenediamine were tested for their ability to revert Salmonella typhimurium (strains TA100 and TA98) in the presence and absence of Nicotiana tabacum (TX1) cell cultures in liquid suspension. Chlorsulfuron and m-phenylenediamine were also tested in the presence and absence of Brassica napus cv. 'Topas' cells. Diallate was found to be activated by TX1 cells into a mutagen that induces base-pair substitution mutations. In the presence of the TX1 plant cell line, chlorsulfuron significantly increased the number of mutations on the strain TA98 of Salmonella. Tobacco TX1 cells did not activate triallate into a mutagen. m-Phenylenediamine was activated into a mutagen by TX1 and Brassica cells as detected by Salmonella TA98. This aniline derivative, in the absence of plant cells and at concentrations higher than 20 $ mu$ Moles/plate, was also able to significantly increase the number of TA98 revertants as compared to the control plants. Finally, Brassica napus cells activated chlorsulfuron into a mutagen that induces frameshift mutations.

Quaternary ammonium compounds (QACs) are ubiquitous contaminants found worldwide in both engineered and natural systems. QACs are toxic to aquatic organisms and cause co-selection for antibiotic resistance, thus providing a reservoir of antibiotic-resistant bacteria, as well as antibiotic resistance genes in QAC-polluted environments. The objectives of the research presented here were to: a) systematically assess the fate and toxicity of QACs using quantitative structure-activity relationships (QSAR); b) evaluate the biotransformation potential of QACs under aerobic, anoxic and anaerobic conditions; and c) assess the potential toxicity of QACs biotransformation products. Nine QACs, belonging to three homologous groups -- monoalkonium, dialkonium and benzalkonium chlorides -- were the target QACs. The QACs critical micelle concentration (CMC) was determined. Then, the CMC was used as a descriptor to derive relationships between QAC structure and partitioning to biosolids as well as acute Microtox® toxicity. QACs with low CMCs had a relatively high adsorption affinity for biosolids and a lower toxicity than QACs with higher CMCs, which suggests that QACs that are more mobile and more (bio)available are more toxic. The biotransformation potential of benzalkonium chlorides (BAC) -- the most commonly used QACs found in engineered and natural biological systems -- under aerobic, methanogenic, nitrate reducing, and fermentative conditions was evaluated using bioenergetics and batch bioassays. The aerobic BAC biotransformation involved sequential dealkylation and debenzylation steps resulting in the formation of benzyl dimethyl amine, and dimethyl amine, respectively. The bacterial community involved in the aerobic BAC degradation was mainly composed of species belonging to the Pseudomonas genus. All QACs tested were recalcitrant under methanogenic conditions and inhibited methanogenesis at and above 25 mg QAC/L. Under nitrate reducing and fermentative conditions, BAC was transformed to alkyldimethyl amines via an abiotic reaction known as modified Hofmann degradation and a biotic reaction known as fumarate addition, respectively. Both reactions are based on a mechanism known as nucleophilic substitution. The discovery of BAC transformation by the above mentioned two reactions is the first ever report to document QAC transformation under anoxic/anaerobic conditions and delineate the transformation pathway.

Orientador: Lucia Regina Durrant / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T08:24:57Z (GMT). No. of bitstreams: 1 Martinez_CamilaOrtiz_M.pdf: 1223287 bytes, checksum: 9db97558a8ad8279e123f2a0318f61d5 (MD5) Previous issue date: 2006 / Mestrado / Mestre em Ciência de Alimentos

Bluegill were exposed to 3 and 30 pg/L lindane and 20 and 200 pg/L naphthalene to determine uptake rate constants, K1 depuration rate constants, K2, and bioconcentration factors, BCF. Correlations were determined between lipid normalized and non-lipid normalized BCFs, and between observed Kl, K2 and BCFs and predicted values. The K1 values for both chemicals and concentrations were similar. The K2 values were different (1.04 day~1, 0.46 day 1). Naphthalene was more rapid. BCFs for lindane (315) and naphthalene (98) were different. Lipid normalized BCFs for naphthalene were more variable than non-lipid normalized BCFs. The reverse was observed for lindane BCFs. Predicted K1, K2 , and BCFs were in agreement with observed values.

In the first part of this work, magnetized nanoporous silica based microparticles (MMPs) are used for horseradish peroxidase (HRP) immobilization and applied in amperometric peroxidase-based biosensors. A homemade magnetized carbon paste electrode permits the MMPs attraction close to the electrode surface. The resulting original biosensor is applied to the investigation of enzymatic oxidation of model drug compounds namely, clozapine (CLZ) and acetaminophen (APAP) by HRP in the presence of hydrogen peroxide. The biosensor operates at a low applied potential and the signal corresponds to the electro-reduction of electroactive species enzymatically generated. The biosensor allows performing the quantitation of the two drug compounds in the micromolar concentration range. It allows also the study of thiol compounds based on the inhibition of the biosensor response. Interestingly, distinct inhibition results are observed for HRP entrapped in the silica microparticles compared to the soluble HRP.<p>We expect that this type of biosensors holds high promise in quantitative analysis and in biotransformation studies of drug compounds.<p><p>In the second part of this thesis work, HRP immobilized magnetic nanoparticles are injected on-line and magnetically retained, as a microreactor, in the capillary of a CE setup. The purpose of such a configuration is to develop an analytical tool for studying “in vitro” drug biotransformation. The advantages expected are (i) minimum sample (drug compound) and biocomponent (enzyme) consumption, (ii) high analysis throughput, (iii) selectivity and sensitivity. In order to illustrate the potential of such an instrumental configuration, it has been applied to study acetaminophen as model drug compound. The mechanistic information obtained by the HRP/H2O2 system is in agreement with literature data on acetaminophen metabolization. Horseradish peroxidase kinetic studies are realized by this setup and the apparent Michaelis constant is determined. Capillary electrophoresis permitted the identification of APAP off-line biotransformed products such as N-acetyl-p-benzoquinone imine (NAPQI), the APAP dimer and APAP polymers as inferred from literature data. The formation of the APAP dimer was further confirmed by electrospray ionization mass spectrometry.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endogenous, as in the case of inborn errors of metabolism. Biotransformation of both exogenous and endogenous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. Fatty acid ?-oxidation deficiencies are associated with characteristic clinical symptoms as a consequence of the accumulation of specific metabolites. For these accumulated metabolites various nutrients are indispensable for optimal biotransformation and continuous accumulation of metabolites can ultimately result in the depletion of biotransformation substrates and cofactors. In this study, a novel model (the unbalanced biotransformation metabolism model) is proposed that describes the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. The value of this model is illustrated by its application to a clinical case investigated. In addition to the use of nutritional supplementation in treatment, biotransformation substrates and cofactors were also used to develop a ?substrate loading cocktail?. This cocktail ensured sufficient availability of biotransformation substrates and precursors to stimulate coenzyme A biosynthesis. The application of this ?substrate loading cocktail? in subjects with both induced and inborn errors in fatty acid oxidation demonstrated that such a novel approach is a useful tool to give new insight into these kinds of deficiencies and open the possibility for the identification of new deficiencies. Interesting observations made in subjects originally referred for biotransformation and oxidative stress status profiles led to the first in vivo evidence of an inhibitory effect of acetylsalicylic acid on short-chain fatty acid metabolism possibly at the level of isobutyryl-CoA dehydrogenase. Since not all individuals were affected to the same degree, this observation can potentially be used to detect individuals with rate-limiting polymorphisms or mutations in the isobutyryl-CoA dehydrogenase enzyme. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

The concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endogenous, as in the case of inborn errors of metabolism. Biotransformation of both exogenous and endogenous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. Fatty acid ?-oxidation deficiencies are associated with characteristic clinical symptoms as a consequence of the accumulation of specific metabolites. For these accumulated metabolites various nutrients are indispensable for optimal biotransformation and continuous accumulation of metabolites can ultimately result in the depletion of biotransformation substrates and cofactors. In this study, a novel model (the unbalanced biotransformation metabolism model) is proposed that describes the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. The value of this model is illustrated by its application to a clinical case investigated. In addition to the use of nutritional supplementation in treatment, biotransformation substrates and cofactors were also used to develop a ?substrate loading cocktail?. This cocktail ensured sufficient availability of biotransformation substrates and precursors to stimulate coenzyme A biosynthesis. The application of this ?substrate loading cocktail? in subjects with both induced and inborn errors in fatty acid oxidation demonstrated that such a novel approach is a useful tool to give new insight into these kinds of deficiencies and open the possibility for the identification of new deficiencies. Interesting observations made in subjects originally referred for biotransformation and oxidative stress status profiles led to the first in vivo evidence of an inhibitory effect of acetylsalicylic acid on short-chain fatty acid metabolism possibly at the level of isobutyryl-CoA dehydrogenase. Since not all individuals were affected to the same degree, this observation can potentially be used to detect individuals with rate-limiting polymorphisms or mutations in the isobutyryl-CoA dehydrogenase enzyme. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Le thème principal de notre travail consistait en la mise en exergue de l'efficience de la mise en œuvre de techniques hybrides associant l’électrochimie à l’élément biologique (biocapteur) ou l’électrochimie aux performances de la spectrométrie de masse (couplage EC-MS). Les informations fournies, jointes aux résultats des mesures en voltampérométrie sur électrodes solides, permettent une bonne compréhension mécanistique quant au devenir oxydatif de substances médicamenteuses. <p>Notre champ d'investigation s'est plus spécifiquement focalisé sur deux familles de molécules psychotropes (les phénothiazines, et une dibenzoazépine). Celles-ci connaissent un usage thérapeutique intensif et un regain d’intérêt pour des applications nouvelles, mais leur utilisation optimale souffre de l’existence d'effets secondaires physiopathologiques importants et dont l’étiologie est encore mal connue. <p>En premier lieu, les résultats de la voltampérométrie cyclique et les différentes modulations en ligne d'une cellule électrochimique couplée à la détection par spectrométrie de masse, nous ont permis de mettre en évidence des différences essentielles dans le devenir des phénothiazines quant aux produits d'oxydations générés. Plus précisément, un comportement clairement distinct entre les phénothiazines garnies de deux (2C) ou trois carbones (3C) entre les deux azotes au niveau de leur chaîne latérale a pu être mis en évidence. Les phénothiazines 3C s'oxydent de manière classique en leur sulfoxyde correspondant. Par contre, les phenothiazines 2C, conjointement à la formation de leur sulfoxyde, souffrent dans des conditions énergiques d’oxydation (persulfate, potentiel élevé) d'une rupture de la chaîne latérale et libèrent la phénothiazine base aisément oxydable et donc subissant elle-même une oxydation. Au vu des structures moléculaires en trois dimensions, nous émettons l’hypothèse que volume trop important de la chaîne latérale des phénothiazines 2C empêcherait le déploiement aisé des structures aromatiques en un radical cation coplanaire lors du phénomène d'oxydation. Les tensions intrastructurelles apparues conduiraient au bris de la chaîne latérale. Différents modes d'oxydation (chimique, électrochimique, enzymatique) ont été utilisés et laissent chacun apparaître la dépendance directe entre la puissance de l'agent oxydant appliqué et les produits d'oxydation obtenus. Chaque technique de détection, de manière individuelle, a bien confirmé la dualité entre les deux groupes de molécules. La mise en commun des divers résultats nous a permis l'identification irrévocable des espèces intermédiaires instables et des composés finaux. Par corollaire, nous avons pu postuler un schéma général d'oxydation pour les dérivés phénothiaziniques. Il nous paraît intéressant de transposer nos résultats aux biotransformations des phénothiazines car les produits identifiés ne possèdent pas l'activité pharmacologique du composé parent mais présentent un profil toxicologique bien répertorié dans la littérature. Nos résultats suggèrent d’approfondir les études de biotransformation afin de déterminer si ‘l’éclatement’ oxydatif des phénothiazines 2C est également observé in vivo. Une relation cause/effet de ces métabolites pourrait ainsi être établie. <p>En deuxième point, au travers de l'association CE/SM ou CE/CL/SM, nous avons étudié l’électroxydation de la clozapine. La génération et l'identification des principaux métabolites de phases I et II, illustre un mimétisme certain avec le CYP450, et nous a permis de confirmer de nombreuses données de la littérature quant à l'oxydation in vivo et in vitro de la clozapine. L'oxydation électrochimique ne génère cependant pas l'ensemble des réactions de métabolisation prises en charge par le système CYP450. Lors de la combinaison CE/SM, par l'absence de séparation chromatographique dans cette configuration, le spectre de masse présente un pic correspondant à un intermédiaire à demi-vie courte, difficilement et rarement mis en évidence: l'ion nitrénium. Cette espèce hautement réactive envers les fonctions thiols des petites molécules et des protéines, se trouve très régulièrement tenue pour responsable majeur de la toxicité avérée de la clozapine. <p>L'apparition plus abondante de dérivés déméthylés démontre l'influence du potentiel appliqué à l'électrode de travail lors de l'oxydation électrochimique. En effet, les processus de déméthylation nécessitent des potentiels élevés pour être observés. En présence de glutathion, aux différents pics antérieurement identifiés, des pics supplémentaires relatifs à la formation d'adduits de GSH sur la CLZ apparaissent. Les courbes voltampérométriques réalisées sur la clozapine suggèrent la distinctement la formation de l'ion nitrénium et d'une nouvelle espèce aisément électroréduite, probablement une structure quinone imine. L'addition de GSH provoque la disparition des pics de réduction de la CLZ. Ces comportements en VC corroborent les interprétations issues des mesures par couplage EC/CL/SM. <p>La dernière partie de notre travail a consisté en la construction d'un biocapteur à pâte de carbone solide avec inclusion au sein de cette matrice de peroxydase de raifort. Basé sur la capacité reconnue de l'HRP à reproduire in vitro des produits d'oxydation similaires à la métabolisation in vivo, nous avons exploité un tel biocapteur pour l'analyse de la clozapine et de composés thiols. Une compréhension fine du mécanisme opérationnel intrinsèque du biocapteur a pu être suggérée. La génération à la surface de l'électrode de l'ion nitrénium par oxydation enzymatique de la clozapine par l'HRP, suivie de sa réduction immédiate fournit un courant ampérométrique substantiel. Sous des conditions de pH optimales, ce courant de réduction autorise la détermination quantitative de la clozapine dans un domaine de linéarité compris entre 1 x 10-5 M et 1 x 10-6 M. L'addition de composés thiols dans le milieu occasionne une chute de courant par action de ceux-ci sur la structure radical cation ou nitrénium par addition nucléophile. La disparition de l'ion nitrénium et la formation d'un adduit GSH-CLZ inhibent tout processus de réduction à l'électrode du biocapteur. Cette diminution de courant proportionnelle aux concentrations en thiols introduits, permet la détermination quantitative de dérivés thiols. Les courbes de calibration exprimées en pourcentage d'inhibition conduisent facilement à l'évaluation de la constante d'inhibition (Ki) et de CI50. L'étude de la réponse ampérométrique de la clozapine à l'EPC/HRP en l'absence ou présence d'un dérivé thiol envisagé permet la détermination de Km et de caractériser le type d'inhibition qui entre en jeu. De tels paramètres cinétiques nous ont habilités à classer les thiols considérés en fonction de leur puissance réactionnelle envers les substances oxydées de la clozapine.<p><p>Au terme de ce travail, nous espérons avoir illustré, par l’étude de quelques molécules modèles, l’intérêt de la mise en œuvre des techniques électrochimiques couplées à l’élément biologique ou à la spectrométrie de masse. Des améliorations au niveau de la cellule électrochimique sont envisageables par l’emploi d’électrodes modifiées, elles laissent entrevoir la possibilité de mimer totalement le système CYP450.<p>Les résultats fournis par ces techniques hybrides et par voltampérométrie cyclique sont complémentaires, ils procurent un éventail d'informations d'une utilité estimable pour une application dans des études prédictives précoces de candidats médicament. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished