The acute phase response (APR) has been shown to alter expression and activity of biotransformation enzymes, such as the phase I cytochromes P450 and phase II glutathione s-transferases (GSTs). The cytochromes P450 and GSTs are expressed abundantly and colocalized to non-neuronal cells of the olfactory mucosa. Previous studies indicate that olfactory cytochromes P450 expression and activity is altered during periods of localized inflammation and infection. Little is understood, however, about the influence of the APR on olfactory GST enzymes. This study investigated effects of the APR on olfactory GST isozymes expression and activity in mouse olfactory mucosa after 24-hr treatment with the acute phase inducer, polyinosinic: polycytidylic acid (polyIC). Western blot analysis using antibodies directed against specific GST isoforms α (A1-1), μ (M1-1), and π (P1-1) demonstrated that their expression was unaltered by polyIC treatment. In contrast, olfactory P450 2E1 expression was significantly decreased. Enzymatic activity of the olfactory GSTs toward the general substrate, 1-chloro-2,4-dinitrobenzene (CDNB) was unchanged during the APR. Analysis of olfactory glutathione content during the APR showed that it was also unaffected by polyIC. The insensitivity of these olfactory GST isoforms during the APR may play a significant role toward limiting the impact of infection and inflammation on the olfactory system.

Secoisolariciresinol (SECO) is the major lignan present in flaxseed, but unlike the structurally related lignan nordihydroguaiaretic acid, it is not associated with toxicity. The major phase I metabolite of SECO is lariciresinol, likely formed as a result of para-quinone methide (p-QM) formation followed by an intramolecular cyclization, thereby minimizing any toxicity associated with the p-QM. Four analogues of SECO were used to investigate substituent effects on lignan metabolism and formation of reactive quinones. HPLC methods were developed for analysis of SECO analogues and their metabolites. The stability of SECO analogues (1 mM) in a 50 mM Na2HPO4 buffer at pH 6.0 and 7.4 were quantified. Enzymatic oxidation experiments using mushroom tyrosinase and microsomes harvested from male Sprague-Dawley rats were performed with and without a GSH trapping system. Mass spectrometry and LC-MS were used to identify metabolites. Life Technologies was contracted to perform IC50 inhibition assays on SECO and the SECO analogues against CYP3A4, CYP3A5, CYP2C9 and CYP2C19 cytochrome P450 isoforms. All SECO analogues were stable at pH 6.0. SECO-2 was stable at pH 7.4 but SECO-1, -3 and -4 were unstable at pH 7.4. Autoxidation of SECO -1, -3 and -4 were 1st order reactions with t1/2 of 9.0 h, 1.7 h and 7.0 h respectively. Mushroom tyrosinase oxidations were performed to generate ortho-quinone standards. SECO-1 -3 and -4 were oxidized by mushroom tyrosinase but SECO-2 was not. Trapping with GSH produces aromatic ring conjugates for SECO-1, -3, -4. Results from microsomal oxidations for SECO-1, -3 and -4 are consistent with these standards. SECO-2 was metabolized by a microsomal system to produce a benzyl GSH adduct. Dealkylation products were also observed. All SECO analogues formed quinones but interestingly, GSH conjugation was competitive with intramolecular cyclization. All cytochrome P450 isoforms were inhibited by every analogue tested to varying degrees, a potential cause of toxicity concerns. Quinones are known to cause toxicity in vivo, including cytotoxicity, immunotoxicity, and carcinogenesis. Our results suggest that since the phenol and catechol lignans form GSH adducts in addition to intramolecular cyclization products, this class of lignans have the potential to cause toxicity.

Xenobiotic Organic Compounds (XOCs) are present in wastewater and wastewater-impacted environmental systems. Pharmaceuticals and personal care products are a broad and varied category of chemicals that are included among these compounds. Although, these compounds have been detected at low levels in surface water, concerns that these compounds may have an impact on human health and aquatic life, have led to increased interest in how XOCs are removed during wastewater treatment. Recognizing specific mechanisms in recent literature and simulating those mechanisms responsible for the removal of XOCs is the main objective of this study. Conventional models, such as the popular activated sludge models (ASM1, ASM2, etc), do not sufficiently address the removal processes; therefore, a fate model is created to provide a means of predicting and simulating removal mechanisms along with experimental analyses. GPS-X is a multi-purpose modeling tool for the simulation of municipal and industrial wastewater treatment plants. This software package includes conventional models as built-in libraries, which can be used as bases on which new models can be created. In this thesis, the removal mechanisms of XOCs are recognized and investigated; a new library for GPS-X is also created to include XOCs. As a first step the uncalibrated fate model, which includes all mechanisms of interest with their process rates and state variables, is developed using in GPS-X software. A modified ASM1 (Mantis model) is used as a basis for developing the fate model. Since only a group of mechanisms is responsible for the removal of each compound the mechanisms are categorized in three different case studies as the next step. Thus, one submodel is associated with each case study. The model developer toolbar in GPS-X software is used to develop the model for these case studies. The first case study involves the removal of antibiotics, such as Sulfamethoxazole. The removal mechanisms used in this case are biodegradation, sorption, and parent compound formation, with co-metabolism and competitive inhibition effects being inserted into the structure of the model. Secondly, the removal of nonylphenol ethoxylates (NPEOs) occurs through abiotic oxidative cleavage, hydrolysis, and biodegradation. The third case study includes removal mechanisms of biodegradation and sorption for neutral and ionized compounds. In the calibration process, model parameters are tuned such that the model can best simulate the experimental data using optimization methods. A common error criterion is Sum of Squared Errors (SSE) between the simulated results and the measured data. By minimizing SSE, optimal values of parameters of interest can be estimated. In each case study different data sets were used for the validation process. To validate the calibrated model, simulated results are compared against experimental data in each case study. The experimental data set used in the validation process is different from that used for calibrating the model, which means the validation process data set was obtained from the different literature. By looking at the validation results, it is concluded that the proposed model successfully simulates removal of XOCs. Since the operating parameters of wastewater treatment plants, such as Solids Retention Time (SRT) and Hydraulic Retention Time (HRT) are crucial for the fate of XOC???s, a sensitivity analysis is carried out to investigate the effect of those parameters. Moreover, the pH effect is studied because it relates to the ionized XOCs. Sensitivity analysis results show that the fate model is more sensitive to model parameters i.e. biodegradation rate constant (kb) than the operational parameters, i.e. SRT and HRT. Furthermore, the responses showed sensitivity to pH, whereby acidic conditions provide a better environment for removing neutral forms and alkaline conditions were suitable for removing ionized forms, according to the ionized compound fate model.

MetaPrint2D, a new software tool implementing a data-mining approach for predicting sites of xenobiotic metabolism has been developed. The algorithm is based on a statistical analysis of the occurrences of atom centred circular fingerprints in both substrates and metabolites. This approach has undergone extensive evaluation and been shown to be of comparable accuracy to current best-in-class tools, but is able to make much faster predictions, for the first time enabling chemists to explore the effects of structural modifications on a compound’s metabolism in a highly responsive and interactive manner. MetaPrint2D is able to assign a confidence score to the predictions it generates, based on the availability of relevant data and the degree to which a compound is modelled by the algorithm. In the course of the evaluation of MetaPrint2D a novel metric for assessing the performance of site of metabolism predictions has been introduced. This overcomes the bias introduced by molecule size and the number of sites of metabolism inherent to the most commonly reported metrics used to evaluate site of metabolism predictions. This data mining approach to site of metabolism prediction has been augmented by a set of reaction type definitions to produce MetaPrint2D-React, enabling prediction of the types of transformations a compound is likely to undergo and the metabolites that are formed. This approach has been evaluated against both historical data and metabolic schemes reported in a number of recently published studies. Results suggest that the ability of this method to predict metabolic transformations is highly dependent on the relevance of the training set data to the query compounds. MetaPrint2D has been released as an open source software library, and both MetaPrint2D and MetaPrint2D-React are available for chemists to use through the Unilever Centre for Molecular Science Informatics website.

Les connaissances sur les risques liés à l'inhalation de mycotoxines restent limitées, alors que ces toxines, présentes au niveau des spores fongiques, peuvent atteindre directement les épithéliums respiratoires. Les enzymes du métabolisme des xéniobiotiques pulmonaires peuvent moduler la toxicité de ces mycotoxines en les détoxifiant ou en formant des métabolites plus réactifs. Notre objectif était d'étudier in vitro le métabolisme pulmonaire de la stérigmatocystine et de la méthoxy-stérigmatocystine, deux mycotoxines présentes dans les environnements contaminés par les moisissures, et de prédire les éventuelles conséquences toxiques. L'étude du métabolisme en présence de cytochromes P450 exprimés dans des systèmes hétérologues, puis dans un modèle de culture primaire de cellules épithéliales trachéales porcines, a montré que ces mycotoxines étaient oxydées, principalement par les cytochromes P450 1A, et conjuguées par glucucoridation et sulfo-conjugaison. Ces deux toxines semblent principalement détoxifiées au niveau pulmonaire. Si une activation métabolique de la stérigmatocystine est possible, celle-ci semble limitée / Human health effects of inhaled mycotoxins remains poorly documented, despite these toxins are present in fungal spores et can directly reach the airway epithelia. Xenobiotic metabolozing enzymes can modulate lung toxicity of these mycotoxins through detoxification or reactive metabolite formation. Our aim was to study in vitro the metabolism and the cellular toxic consequences of two mycotoxins present in mold-contaminated environments, e.g. sterigmatocystin and methoxy-sterigmatocystin. The metabolism studies using recombinant cytochromes P450 enzymes and porcine tracheal epithelial cell primary cultures, showed that these mycotoxins could be oxidized by cytochrome P450 1A and conjugated by glucucoridation and sulfo-conjugation. Sterigmatocystin and methoxysterigmatocystin seem mainly detoxified in respiratory cells. If a metabolic activation of sterigmatocystin is possible, it seems limited

É grande a preocupação na identificação de compostos envolvidos no desenvolvimento de processos deletérios, como o câncer. O tetraidrofurano (THF) é um solvente amplamente utilizado em indústria e pesquisa e estudos recentes indicaram uma atividade carcinogênica em animais experimentais, incentivando a investigação da sua possível interação com biomoléculas. Adutos de DNA servem como marcadores para a identificação da atividade genotóxica de substâncias e para o monitoramento da exposição humana aos agentes genotóxicos. Estudos realizados in vitro, em células em cultura, bem como em animais experimentais, permitem que mecanismos sejam desvendados e metodologias sejam desenvolvidas para uma posterior avaliação da exposição humana. O contínuo desenvolvimento de técnicas de análise de adutos em DNA para monitoramento biológico constitui importante preocupação para pesquisadores envolvidos com a saúde ocupacional e a medicina preventiva. No presente trabalho foi investigada a reação de produtos de oxidação do THF com os nucleosídeos 2\'-desoxiadenosina (dAdo) e 2\'-desoxicitidina (dCyd). As estruturas dos adutos formados foram elucidadas por análises de espectros de massas (ESI/MS-MS) e ressonância magnética nuclear (RMN) após purificação de quantidade suficiente dos produtos por cromatografia líquida de alta eficiência (HPLC). Com isso, foi aberta a possibilidade de validação de novos marcadores de exposição a THF. Tais lesões foram detectadas em DNA incubado com THF oxidado in vitro, após o desenvolvimento de uma metodologia sensível e seletiva baseada em HPLC/ESI/MS-MS. Além disso, camundongos foram expostos a vapor de THF e foi feita análise de dano oxidativo (8-oxo-2-desoxiguanosina) no DNA de fígado e rim. Um aumento de aproximadamente oito vezes no nível de 8-oxo-2-desoxiguanosina foi observado no DNA dos órgãos dos camundongos expostos ao THF. Nossos estudos in vitro apontam para a possibilidade de ação genotóxica do THF oxidado (formação de adutos com o DNA). A ocorrência dessa via in vivo será investigada em estudos posteriores. Adicionalmente, há aumento de dano oxidativo em órgãos alvos de carcinogênese induzida por esse solvente. / There is a huge concern to identify compounds involved in the development of harmful processes, like cancer. Tetrahydrofuran (THF) is a solvent widely used in industries and recent researches and studies indicated a carcinogenic activity in experimental animals, instigating the investigation of its possible interaction with biomolecules. DNA adducts are used as markers for the identification of genotoxic activity of substances and monitoring the human exposition to genotoxic agents. Studies carried out in vitro, in culture cells and in experimental animals allow to disclose mechanisms and develop methodologies for a posterior evaluation of the human exposition. The continuous development of techniques for the analysis of DNA adducts for biological monitoring constitutes a remarkable concern for the researchers involved with occupational health and preventive medicine. In the present paper, the reaction of THF oxidation products with the nucleosides 2\'-deoxyadenosine (dAdo) and 2\'-deoxycitidine (dCyd) were investigated. The structures of the formed adducts were elucidated by mass spectra analysis (ESI/MS-MS) and nuclear magnetic resonance (RMN) after purification of sufficient quantities of the products by high-performance liquid chromatography (HPLC). Thus, the validation of new markers of exposition to THF was made possible. Such damages were detected in DNA incubated with oxidized THF in vitro, after the development of a sensible and selective methodology based on HPLC/ESI/MS-MS. Moreover, mice were exposed to THF vapors and an oxidative damage analysis (8-oxo-2\'-deoxyguanosine) was carried out on DNA from liver and kidney. An approximate 8-fold increase in the level of 8-oxo-2\'-deoxyguanosine was observed on DNA from the mices organs exposed to THF. Our in vitro studies indicate the possibility of oxidized THF genotoxic action (formation of adducts with DNA). The occurrence of this path in vivo will be investigated in future studies. In addition, the oxidative damage in target organs of carcinogenesis induced by this solvent was increased.

Epitélios confluentes de queratinócitos autólogos, cultivados segundo metodologia padronizada por Rheinwald e Green em 1975, têm sido usados como enxertos em diferentes situações clínicas. Contudo, a presença de componentes xenobióticos empregados nesta metodologia implica na possibilidade de transmissão de zoonoses, príons e viroses aos pacientes, além de envolver questões éticas relacionadas à utilização de animais. Tais preocupações têm direcionado pesquisadores a buscar alternativas que superem este impasse; no entanto, as formulações obtidas até o momento não são completamente satisfatórias. Assim, nossa proposta neste estudo, foi omitir ou substituir os componentes xenobióticos tradicionalmente utilizados no meio para queratinócitos, por similares humanos. Como resultado, padronizamos um meio de cultura onde omitimos o emprego de toxina colérica, substituímos o soro fetal bovino por lisado de plaquetas humanas na concentração de 2,5% e a insulina de origem bovina foi substituída por insulina recombinante humana na mesma concentração do método original (5 μg/mL). Com os resultados obtidos foi possível concluir ser viável o cultivo de queratinócitos humanos, mantidos em meio de cultura livre de componentes xenobióticos. / Confluent epithelia of autologous keratinocytes, cultivated according to standardized methodology by Rheinwald and Green in 1975, have been used as grafts in different clinical situations. However, the presence of xenobiotics components applied in this method implies the possibility of transmission of zoonoses, príons, and viruses to patients, besides involving ethical issues related to the use of animals for components obtainment. Such concerns have driven researchers to seek alternatives that overcome this deadlock, as the formulations obtained so far are not completely satisfactory. Thus, our proposal in this study was to omit or replace the xenobiotics components traditionally used in the medium to keratinocytes culture, by human similar. As a result, we have standardized a culture medium whereby we omitted the use of cholera toxin, replaced fetal bovine serum by human platelet lysate at a 2.5% concentration and bovine insulin was replaced by recombinant human insulin at the same concentration as the original method (5 μg/mL). With the results obtained we could conclude that the method to be viable to cultivate human keratinocytes, kept in culture medium free of xenobiotics components.

Epitélios confluentes de queratinócitos autólogos, cultivados segundo metodologia padronizada por Rheinwald e Green em 1975, têm sido usados como enxertos em diferentes situações clínicas. Contudo, a presença de componentes xenobióticos empregados nesta metodologia implica na possibilidade de transmissão de zoonoses, príons e viroses aos pacientes, além de envolver questões éticas relacionadas à utilização de animais. Tais preocupações têm direcionado pesquisadores a buscar alternativas que superem este impasse; no entanto, as formulações obtidas até o momento não são completamente satisfatórias. Assim, nossa proposta neste estudo, foi omitir ou substituir os componentes xenobióticos tradicionalmente utilizados no meio para queratinócitos, por similares humanos. Como resultado, padronizamos um meio de cultura onde omitimos o emprego de toxina colérica, substituímos o soro fetal bovino por lisado de plaquetas humanas na concentração de 2,5% e a insulina de origem bovina foi substituída por insulina recombinante humana na mesma concentração do método original (5 μg/mL). Com os resultados obtidos foi possível concluir ser viável o cultivo de queratinócitos humanos, mantidos em meio de cultura livre de componentes xenobióticos. / Confluent epithelia of autologous keratinocytes, cultivated according to standardized methodology by Rheinwald and Green in 1975, have been used as grafts in different clinical situations. However, the presence of xenobiotics components applied in this method implies the possibility of transmission of zoonoses, príons, and viruses to patients, besides involving ethical issues related to the use of animals for components obtainment. Such concerns have driven researchers to seek alternatives that overcome this deadlock, as the formulations obtained so far are not completely satisfactory. Thus, our proposal in this study was to omit or replace the xenobiotics components traditionally used in the medium to keratinocytes culture, by human similar. As a result, we have standardized a culture medium whereby we omitted the use of cholera toxin, replaced fetal bovine serum by human platelet lysate at a 2.5% concentration and bovine insulin was replaced by recombinant human insulin at the same concentration as the original method (5 μg/mL). With the results obtained we could conclude that the method to be viable to cultivate human keratinocytes, kept in culture medium free of xenobiotics components.

É grande a preocupação na identificação de compostos envolvidos no desenvolvimento de processos deletérios, como o câncer. O tetraidrofurano (THF) é um solvente amplamente utilizado em indústria e pesquisa e estudos recentes indicaram uma atividade carcinogênica em animais experimentais, incentivando a investigação da sua possível interação com biomoléculas. Adutos de DNA servem como marcadores para a identificação da atividade genotóxica de substâncias e para o monitoramento da exposição humana aos agentes genotóxicos. Estudos realizados in vitro, em células em cultura, bem como em animais experimentais, permitem que mecanismos sejam desvendados e metodologias sejam desenvolvidas para uma posterior avaliação da exposição humana. O contínuo desenvolvimento de técnicas de análise de adutos em DNA para monitoramento biológico constitui importante preocupação para pesquisadores envolvidos com a saúde ocupacional e a medicina preventiva. No presente trabalho foi investigada a reação de produtos de oxidação do THF com os nucleosídeos 2\'-desoxiadenosina (dAdo) e 2\'-desoxicitidina (dCyd). As estruturas dos adutos formados foram elucidadas por análises de espectros de massas (ESI/MS-MS) e ressonância magnética nuclear (RMN) após purificação de quantidade suficiente dos produtos por cromatografia líquida de alta eficiência (HPLC). Com isso, foi aberta a possibilidade de validação de novos marcadores de exposição a THF. Tais lesões foram detectadas em DNA incubado com THF oxidado in vitro, após o desenvolvimento de uma metodologia sensível e seletiva baseada em HPLC/ESI/MS-MS. Além disso, camundongos foram expostos a vapor de THF e foi feita análise de dano oxidativo (8-oxo-2-desoxiguanosina) no DNA de fígado e rim. Um aumento de aproximadamente oito vezes no nível de 8-oxo-2-desoxiguanosina foi observado no DNA dos órgãos dos camundongos expostos ao THF. Nossos estudos in vitro apontam para a possibilidade de ação genotóxica do THF oxidado (formação de adutos com o DNA). A ocorrência dessa via in vivo será investigada em estudos posteriores. Adicionalmente, há aumento de dano oxidativo em órgãos alvos de carcinogênese induzida por esse solvente. / There is a huge concern to identify compounds involved in the development of harmful processes, like cancer. Tetrahydrofuran (THF) is a solvent widely used in industries and recent researches and studies indicated a carcinogenic activity in experimental animals, instigating the investigation of its possible interaction with biomolecules. DNA adducts are used as markers for the identification of genotoxic activity of substances and monitoring the human exposition to genotoxic agents. Studies carried out in vitro, in culture cells and in experimental animals allow to disclose mechanisms and develop methodologies for a posterior evaluation of the human exposition. The continuous development of techniques for the analysis of DNA adducts for biological monitoring constitutes a remarkable concern for the researchers involved with occupational health and preventive medicine. In the present paper, the reaction of THF oxidation products with the nucleosides 2\'-deoxyadenosine (dAdo) and 2\'-deoxycitidine (dCyd) were investigated. The structures of the formed adducts were elucidated by mass spectra analysis (ESI/MS-MS) and nuclear magnetic resonance (RMN) after purification of sufficient quantities of the products by high-performance liquid chromatography (HPLC). Thus, the validation of new markers of exposition to THF was made possible. Such damages were detected in DNA incubated with oxidized THF in vitro, after the development of a sensible and selective methodology based on HPLC/ESI/MS-MS. Moreover, mice were exposed to THF vapors and an oxidative damage analysis (8-oxo-2\'-deoxyguanosine) was carried out on DNA from liver and kidney. An approximate 8-fold increase in the level of 8-oxo-2\'-deoxyguanosine was observed on DNA from the mices organs exposed to THF. Our in vitro studies indicate the possibility of oxidized THF genotoxic action (formation of adducts with DNA). The occurrence of this path in vivo will be investigated in future studies. In addition, the oxidative damage in target organs of carcinogenesis induced by this solvent was increased.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix PER/ARNT/SIM (bHLH-PAS) transcription factor that binds ligands typified by 2,3,7,8-tetracholordibenzo-p-dioxin, translocates to the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT) and associates with specific cis xenobiotic response elements to activate transcription of genes involved with xenobiotic metabolism. AHR-mediated signal transduction has been evaluated thoroughly in the C57BL/6J mouse model system. This model system, however, may not be the most accurate model for human comparisons as the AHRb-1 allele carried by C57BL/6J contains a point mutation that prematurely truncates the receptor at 805 amino acids, while the AHRb-2, rat, and human AHR all contain an additional 42-45 amino acids at their carboxy-terminus that have 70% identity. This carboxy-terminal region could be functionally significant and the analysis of AHR-mediated signal transduction in the rat, human, or other mouse strains may better represent the physiology of the AHR pathway. ARNT is another member of the bHLH-PAS family of proteins that is essential in several distinct signal transduction pathways mediated by its dimerization with a variety of bHLH-PAS proteins. Several isoforms of ARNT have been identified in mammalian and aquatic species. While ARNT and ARNT2 exhibit >90% amino acid identity in the bHLH and PAS domains, gene knock-out of either ARNT or ARNT2 results in embryonic/perinatal lethality characterized by distinct phenotypes. This suggests that neither protein can compensate fully for the loss of the other. Since overlapping tissue specific expression of ARNT and ARNT2 does exist, but neither ARNT can compensate fully for loss of the other, this suggests that the two proteins have distinct functions in the presence of various dimerization partners. Thus, the focus of these studies is to examine the discrepancies between the rat, human, or AHRb-2 possessing the extended carboxy-terminal region and that of the AHRb-1 and also to examine the role of both ARNT and ARNT2 during AHR-mediated signal transduction.