Abstract available in the Clinical Pharmacology in Drug Development.

A produção de espécies reativas de oxigênio (ROS) nos sistemas biológicos é contrabalanceada pelos sistemas antioxidantes enzimáticos e não-enzimáticos. Quando há um desequilíbrio entre a geração de ROS e esses sistemas, ocorre um aumento dessas espécies reativas causando estresse oxidativo, que pode levar a danos a macromoléculas, como lipídios, proteínas e o DNA. Os fármacos doxorrubicina (antineoplásico) e benzonidazol (antiparasitário) são conhecidos por induzir efeitos colaterais que podem estar relacionados ao aumento de ROS. Além disso, ensaios de mutagenicidade demonstram que esses fármacos apresentam atividade mutagênica por danos oxidativos. O Grupo NEQUIMED desenvolve substâncias com potencial atividade antineoplásica e antiparasitária, as quais ainda não foram avaliadas em relação às propriedades tóxicas e genotóxicas. Dessa forma, o objetivo deste trabalho foi analisar as propriedades mutagênicas e de estresse oxidativo dessas novas substâncias, comparando aos fármacos benzonidazol e doxorrubicina. Para detecção de ROS foi realizado o ensaio fluorimétrico utilizando o marcador 2,7-diacetato de diclorofluoresceína (DCFH-DA) em linhagens celulares de hepatocarcinoma humano (HepG2) e fibroblasto de camundongo (Balb/C 3T3 clone A31). O estado redox destas células foi avaliado através da quantificação da expressão gênica e do conteúdo proteico das enzimas antioxidantes através das técnicas de qRT-PCR e Western blot, respectivamente. A atividade mutagênica foi analisada com o ensaio Ames miniaturizado Salmonella/microssoma utilizando a linhagem TA102 de Salmonella typhimurium que detecta agentes mutagênicos que causam danos por oxidação. Os resultados mostraram que as substâncias estudadas pelo Grupo não induzem aumento na produção de ROS ou induzem em menores níveis do que doxorrubicina e benzonidazol, além de levar a alterações menos proeminentes que os fármacos para a expressão das proteínas antioxidantes. No ensaio mutagênico, o benzonidazol apresentou o pior perfil, doxorrubicina e Neq0438 somente foram mutagênicos com ativação enzimática, enquanto Neq0551 foi inativo. Assim, as novas substâncias (Neq0438 e Neq0551) apresentaram um perfil melhor do que os fármacos de referência, tornando-os candidatos promissores para estudos in vitro e in vivo subsequentes. / The production of reactive oxygen species (ROS) in biological systems is compensated by the enzymatic and non-enzymatic antioxidant systems. The excessive ROS production causes oxidative stress, which can damage important cellular macromolecules such as lipids, proteins and DNA. The drugs doxorubicin (antineoplastic) and benzonidazole (antiparasitic) are both known for their side effects, which can be related to the increase of ROS. Besides, mutagenicity assays show that these drugs have a mutagenic activity via oxidative damages. The research group NEQUIMED studies new substances with potential antineoplastic and antiparasitic activities, but their toxic and genotoxic properties have not been fully evaluated yet. Thus, the aim of this work is to assess the mutagenic potential and the oxidative stress generated by these substances, comparing them to benzonidazole and doxorubicin. The fluorimetric assay using the probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for ROS detection in human hepatocarcinoma (HepG2) and mouse fibroblast (Balb/C 3T3 clone A31) cell lines. The redox state of these cells was evaluate by qRT-PCR and Western blot methods to quantifying gene expression and protein content of the antioxidant enzymes. The mutagenic potential was assessed by the miniaturized Ames text with the Salmonella/microssome mutagenicity assay, using the TA102 strain of Salmonella typhimurium, which detects oxidation damages to the DNA. The new substances did not induce an increase on ROS production, or did in lower levels when compared to doxorubicin and benzonidazole. Moreover, reference drugs also induced greater changes on the expression of the antioxidant enzymes. Benznidazole had a higher mutagenic activity, while Neq0438 and doxorubicin were mutagenic only when incubated with enzymatic activation. Neq0551 was inactive for Ames assay. Therefore, these new substances (Neq0438 and Neq0551) had a better overall profile than the reference drugs, turning out to be promising candidates for further in vitro and in vivo studies.

A produção de espécies reativas de oxigênio (ROS) nos sistemas biológicos é contrabalanceada pelos sistemas antioxidantes enzimáticos e não-enzimáticos. Quando há um desequilíbrio entre a geração de ROS e esses sistemas, ocorre um aumento dessas espécies reativas causando estresse oxidativo, que pode levar a danos a macromoléculas, como lipídios, proteínas e o DNA. Os fármacos doxorrubicina (antineoplásico) e benzonidazol (antiparasitário) são conhecidos por induzir efeitos colaterais que podem estar relacionados ao aumento de ROS. Além disso, ensaios de mutagenicidade demonstram que esses fármacos apresentam atividade mutagênica por danos oxidativos. O Grupo NEQUIMED desenvolve substâncias com potencial atividade antineoplásica e antiparasitária, as quais ainda não foram avaliadas em relação às propriedades tóxicas e genotóxicas. Dessa forma, o objetivo deste trabalho foi analisar as propriedades mutagênicas e de estresse oxidativo dessas novas substâncias, comparando aos fármacos benzonidazol e doxorrubicina. Para detecção de ROS foi realizado o ensaio fluorimétrico utilizando o marcador 2,7-diacetato de diclorofluoresceína (DCFH-DA) em linhagens celulares de hepatocarcinoma humano (HepG2) e fibroblasto de camundongo (Balb/C 3T3 clone A31). O estado redox destas células foi avaliado através da quantificação da expressão gênica e do conteúdo proteico das enzimas antioxidantes através das técnicas de qRT-PCR e Western blot, respectivamente. A atividade mutagênica foi analisada com o ensaio Ames miniaturizado Salmonella/microssoma utilizando a linhagem TA102 de Salmonella typhimurium que detecta agentes mutagênicos que causam danos por oxidação. Os resultados mostraram que as substâncias estudadas pelo Grupo não induzem aumento na produção de ROS ou induzem em menores níveis do que doxorrubicina e benzonidazol, além de levar a alterações menos proeminentes que os fármacos para a expressão das proteínas antioxidantes. No ensaio mutagênico, o benzonidazol apresentou o pior perfil, doxorrubicina e Neq0438 somente foram mutagênicos com ativação enzimática, enquanto Neq0551 foi inativo. Assim, as novas substâncias (Neq0438 e Neq0551) apresentaram um perfil melhor do que os fármacos de referência, tornando-os candidatos promissores para estudos in vitro e in vivo subsequentes. / The production of reactive oxygen species (ROS) in biological systems is compensated by the enzymatic and non-enzymatic antioxidant systems. The excessive ROS production causes oxidative stress, which can damage important cellular macromolecules such as lipids, proteins and DNA. The drugs doxorubicin (antineoplastic) and benzonidazole (antiparasitic) are both known for their side effects, which can be related to the increase of ROS. Besides, mutagenicity assays show that these drugs have a mutagenic activity via oxidative damages. The research group NEQUIMED studies new substances with potential antineoplastic and antiparasitic activities, but their toxic and genotoxic properties have not been fully evaluated yet. Thus, the aim of this work is to assess the mutagenic potential and the oxidative stress generated by these substances, comparing them to benzonidazole and doxorubicin. The fluorimetric assay using the probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for ROS detection in human hepatocarcinoma (HepG2) and mouse fibroblast (Balb/C 3T3 clone A31) cell lines. The redox state of these cells was evaluate by qRT-PCR and Western blot methods to quantifying gene expression and protein content of the antioxidant enzymes. The mutagenic potential was assessed by the miniaturized Ames text with the Salmonella/microssome mutagenicity assay, using the TA102 strain of Salmonella typhimurium, which detects oxidation damages to the DNA. The new substances did not induce an increase on ROS production, or did in lower levels when compared to doxorubicin and benzonidazole. Moreover, reference drugs also induced greater changes on the expression of the antioxidant enzymes. Benznidazole had a higher mutagenic activity, while Neq0438 and doxorubicin were mutagenic only when incubated with enzymatic activation. Neq0551 was inactive for Ames assay. Therefore, these new substances (Neq0438 and Neq0551) had a better overall profile than the reference drugs, turning out to be promising candidates for further in vitro and in vivo studies.

Arrabidaea chica Verlot (Bignoniaceae), conhecida como crajiru, fornece pigmentos vermelhos utilizados pelos índios no Brasil como corante e agente cicatrizante. Este estudo visou otimizar a extração de compostos fenólicos de A. chica e avaliar sua atividade farmacológica. Extratos de A. chica foram obtidos através de tratamento com xilanases de Bacillus pumilus previamente à extração. Os ensaios foram monitorados por CLAE e ESI-MS. O tratamento enzimático forneceu extratos enriquecidos em antocianidinas. Extratos sem tratamento enzimático apresentaram maior teor de antocianosídeos. O estudo farmacológico demonstrou que as atividades anticâncer e antioxidante in vitro estão diretamente relacionadas ao maior teor de agliconas. O ensaio in vitro de indução de crescimento de fibroblastos indicou que o maior teor da aglicona carajurina é inversamente proporcional à ação cicatrizante. Portanto, foi desenvolvida uma metodologia inovadora, através de processos biotecnológicos, para extração de antocianidinas que apresentam propriedades corantes e terapêuticas. / Arrabidaea chica Verlot (Bignoniaceae), known as crajiru, produces red pigments used by Brazilian indians as dye and as healing agent. This study has aimed the optimization of phenolic compounds extraction from A. chica and to evaluate its pharmacological activities. Extracts from A. chica were obtained through treatment with xylanases from Bacillus pumilus before the extraction. The assays were monitored by HPLC and ESI/MS-MS. The enzymatic treatment has produced more concentrated in anthocyanidins extracts. Those obtained without enzymatic treatment have presented higher glycosilated anthocyanins content. The pharmacologic study has demonstrated that the antitumoral and the antioxydant in vitro properties for A. chica are directly related to the higher contents of aglycones. In vitro assay for fibroblasts growth induction has demonstrated that a higher content of carajurin is inversely proportional to the healing action. In conclusion, a novel approach has been developed, through biotechnological process, aiming the extraction of anthocyanidins presenting dye and therapeutic properties.

Arrabidaea chica Verlot (Bignoniaceae), conhecida como crajiru, fornece pigmentos vermelhos utilizados pelos índios no Brasil como corante e agente cicatrizante. Este estudo visou otimizar a extração de compostos fenólicos de A. chica e avaliar sua atividade farmacológica. Extratos de A. chica foram obtidos através de tratamento com xilanases de Bacillus pumilus previamente à extração. Os ensaios foram monitorados por CLAE e ESI-MS. O tratamento enzimático forneceu extratos enriquecidos em antocianidinas. Extratos sem tratamento enzimático apresentaram maior teor de antocianosídeos. O estudo farmacológico demonstrou que as atividades anticâncer e antioxidante in vitro estão diretamente relacionadas ao maior teor de agliconas. O ensaio in vitro de indução de crescimento de fibroblastos indicou que o maior teor da aglicona carajurina é inversamente proporcional à ação cicatrizante. Portanto, foi desenvolvida uma metodologia inovadora, através de processos biotecnológicos, para extração de antocianidinas que apresentam propriedades corantes e terapêuticas. / Arrabidaea chica Verlot (Bignoniaceae), known as crajiru, produces red pigments used by Brazilian indians as dye and as healing agent. This study has aimed the optimization of phenolic compounds extraction from A. chica and to evaluate its pharmacological activities. Extracts from A. chica were obtained through treatment with xylanases from Bacillus pumilus before the extraction. The assays were monitored by HPLC and ESI/MS-MS. The enzymatic treatment has produced more concentrated in anthocyanidins extracts. Those obtained without enzymatic treatment have presented higher glycosilated anthocyanins content. The pharmacologic study has demonstrated that the antitumoral and the antioxydant in vitro properties for A. chica are directly related to the higher contents of aglycones. In vitro assay for fibroblasts growth induction has demonstrated that a higher content of carajurin is inversely proportional to the healing action. In conclusion, a novel approach has been developed, through biotechnological process, aiming the extraction of anthocyanidins presenting dye and therapeutic properties.

Les substances pharmaceutiques font partie du groupe des contaminants émergents du fait de leur intérêt récent dans les études environnementales comparativement à des polluants étudiés depuis plus longtemps tels que les pesticides. Elles correspondent aux principes actifs des médicaments et, à ce titre, sont responsables des propriétés pharmacologiques des médicaments. Ce sont donc des molécules biologiquement actives qui peuvent agir sur les organismes vivants présents dans les écosystèmes impactés. L’origine des substances pharmaceutiques dans l’environnement est variable mais les principales sources sont liées à leur utilisation en médecine humaine ou vétérinaire. Une fois consommées, les substances pharmaceutiques sont excrétées dans les urines ou les fèces et se retrouvent dans les eaux usées (consommation humaine) ou dans les déchets d’élevage (consommation vétérinaire). Dans le premier cas, elles peuvent être rejetées directement dans le milieu, ou indirectement, avec les eaux usées traitées ou les boues résiduaires, après traitement dans les stations d’épuration (STEP). Dans le deuxième cas, elles atteignent directement le milieu lorsque les animaux sont élevés en prairie ou indirectement lorsque les déchets d’élevage sont épandus sur les sols agricoles pour les fertiliser. Ces travaux de thèse se sont attachés à étudier l’origine et le devenir de ces substances dans ces 2 cas de figure. Ainsi en se basant sur des critères de consommation, de présence dans l’environnement par rapport à des études antérieures, de toxicité et d’écotoxicité, d’originalité et de disponibilité des composés standards de référence, 32 puis 78 molécules appartenant aux classes thérapeutiques des antibiotiques, des anticancéreux, des béta-bloquants, des anti-VIH et des inhibiteurs de phosphodiestérase de type 5 (PDE 5) ont été étudiées dans 2 continuums : i) effluents hospitaliers - eaux usées brutes et traitées – eaux de surface, et ii) eaux usées brutes et traitées - eaux de surface - eaux de captage souterraines. En s’appuyant sur les mêmes critères de sélection, le devenir de 7 antibiotiques a été étudié dans des lisiers porcins dans des filières simples de traitement du lisier (fosse de stockage), dans des filières complexes de traitement du lisier (système de traitement ressemblant à des mini STEP) et dans des mésocosmes en conditions contrôlées. Pour pouvoir réaliser l’ensemble de ces études, des protocoles analytiques mettant en œuvre une étape d’extraction par SPE (Solide Phase Extraction) ou d’extraction ASE (Extraction Accélérée par Solvant) puis de purification par SPE et d’analyse par LC/MS/MS (Chromatographie en phase liquide couplée à la spectrométrie de masse en tandem) ont été développés. Ces protocoles, en remplissant des critères de qualité tels que des limites de détection et de quantification compatibles avec des analyses environnementales (de l’ordre du ng/l à la dizaine de ng/l), une bonne linéarité, précision, justesse et performance, ont permis d’analyser la phase dissoute des échantillons d’eaux et la phase dissoute et solide des échantillons de lisiers. Il ressort des analyses des échantillons aqueux que : i) les béta-bloquants, les anti-VIH et les antibiotiques appartenant aux familles des macrolides, des fluoroquinolones et des sulfonamides, sont les molécules les plus représentatifs de la contamination du milieu naturel parmi les classes étudiées ; ii) les rejets de STEP sont une source majeure de la contamination des systèmes aquatiques ; iii) les eaux usées sont davantage contaminées en hiver qu’en été ; et iv) les eaux de surface sont davantage contaminées en été qu’en hiver. / Pharmaceutical substances belong to the group of emerging contaminants due to their recent interest in environmental studies in comparison with pollutants who have been studied for a longer time like pesticides. They correspond to the active ingredient of drugs and by this mean are responsible for their pharmacological properties. Consequently they are biologically active molecules that can act on living organisms present in impacted ecosystems. The origin of pharmaceuticals in the environment is variable but the main sources are related to their use in human and veterinary medicine. Once consumed, pharmaceutical substances are excreted in urine or feces and are found in wastewater (human consumption) or animal manure (veterinary consumption). In the first case, they can be discharged directly in the environment, or indirectly, with treated wastewater or sludge from sewage treatment plants (SWTP). In the second case, they directly reach the environment when animals are bred on grassland or indirectly when livestock wastes are spread on agricultural soils as fertilizer. This PhD work has been focused on the study of the origin and fate of pharmaceutical substances in these 2 cases. Thus according to consumption data, occurrence in the environment reported in previous studies, toxicity and ecotoxicity data, originality and availability of reference standard compounds, 32 then 78 molecules belonging to 5 different therapeutic classes (antibiotics, antineoplastics, beta-blockers, anti-HIV, phosphodiesterase type 5 inhibitors (PDE 5 inhibitors)) were studied in 2 continuums : i) hospital wastewater effluents – raw and treated wastewater – surface water, and ii) raw and treated wastewater – surface water – ground water. Based on the same selection criteria, the fate of 7 antibiotics was studied in pig manure in simple manure storage facilities (storage tank), in aerobic manure treatment facilities (treatment system like in small SWTP) and in mesocosms under controlled conditions. In order to achieve all these studies, analytical protocols implementing an extraction step by SPE (Solid Phase Extraction) or an ASE extraction (Accelerated Solvent Extraction) followed by a SPE purification and an analytical step by LC / MS / MS (liquid chromatography tandem mass spectrometry) have been developed. These protocols, by filling out quality criteria such as limits of detection and quantification compatible with environmental analysis (ng/l to dozen of ng/l), good linearity, precision, accuracy and performance, were used to analyze the dissolved phase of water samples and dissolved and solid phases of pig manure samples. The water samples analysis shows : i) beta-blockers, anti-HIV and antibiotic belonging to the families of macrolides, fluoroquinolones and sulfonamides are the most representative molecules of the environmental contamination from the classes studied; ii) SWTP releases are a major source of aquatic systems’ contamination; iii) wastewaters are more contaminated in winter than in summer; and iv) surface water are more contaminated in summer than in winter. The pig manure samples analysis shows : i) the levels of contamination of manure by antibiotics are high, from a few µg/l to mg/l; ii) the manure level of contamination is not related to the physiological stage of pigs; iii) the interest to store manure before spreading in order to reduce the antibiotics contamination is not highlighted; iv) oxytetracycline, tetracycline, tylosin and marbofloxacin are mainly present in the solid phase whereas sulfadiazine, lincomycin and monensin are mainly present in the liquid phase of manure; v) the separation of solid and liquid phases reduce manure contamination in aerobic treatment facilities; and vi) antibiotics degradation is mainly aerobic.Key words: ,