The rapid urbanization and industrialization has led to an increase of disposal petroleum hydrocarbons (PHC) and trace elements (TE) into the environment. These pollutants are considered as the most toxic contaminants in the world due to their persistence in the environment, and the long range of toxicological effects for living beings. Recent concerns regarding the environmental contamination have initiated the development of several remediation technologies, including physical, chemical, and biological approaches. In this thesis, gentle soil remediation options (GRO) were investigated at different scales for the reclamation of PHC and TE co-contaminated soil. In the first part of this thesis, laboratory experiments were performed to characterize PHC and TE contaminated soil as well as the indigenous microorganisms (bacteria and fungi) present inside these contaminated soil. It was found that the studied aged contaminated soil had a negative effect on earthworm’s development and L. sativum biomass. Moreover, a high respiration of microorganisms attributed to the transformation/ mineralization of organic matter or/and organic pollutants was observed. This presence of viable microorganisms suggested an adaptation of microorganisms to the contaminant. Further results showed that the long-term exposure of soil microorganisms to high PHC concentration and the type of isolation culture media did not influence the ability of isolates to effectively degrade PHC. However, phylogenic affiliation had a strong on PHC biodegradation. In the second part of this thesis, preliminary studies in greenhouse were assessed to investigate the ability of M. sativa assisted by compost in the greenhouse aided-phytoremediation of PHC and TE. It was found that compost incorporation into the soil promoted PHC degradation, M. sativa growth and survival, and phytoextraction of TE. Residual risk assessment after the phytoremediation trial also showed a positive effect of compost amendment on plant growth and earthworm development. Pilot scale ecopile experiment carried out in the third part of this thesis allow a reduction of up to 80% of PHC and 20% of metals after 17 months. This research demonstrated that M. sativa and H. annus were suitable for phytodegradation of PHC and phytoextraction of TE.  Results from this thesis are helpful for further full-scale phytoremediation studies.

A citometria de fluxo é um método preciso, rápido e eficaz na avaliação de múltiplos parâmetros celulares, tanto estruturais como funcionais, propiciando a separação e o estudo de diferentes populações e sub-populações de células. No presente estudo, foram empregados métodos citométricos para caracterização dos diferentes tipos celulares sangüíneos de Centropomus parallelus, assim como para verificação da viabilidade celular, avaliação da atividade fagocítica e ativação do burst respiratório. Foram identificadas três sub-populações representativas de leucócitos: linfócitos, monócitos e trombócitos. Estas células foram estimuladas, in vitro, com Staphylococcus aureus, marcado com iodeto de propídeo (SAPI), lipopolissacarídeo de Escherichia coli (LPS) e zymosan, partículas de Saccharomyces cerevisiae. As respostas frente aos estímulos foram distintas de acordo com o tipo celular e o estímulo apresentado. Os monócitos apresentaram maiores percentuais de fagocitose frente ao estímulo provocado pela SAPI e pelo Zymosan; já a população que continha trombócitos entre outros tipos celulares (por exemplo: linfócitos), apresentou \"fagocitose\" significativa apenas para SAPI. O burst oxidativo detectado pela fluorescência emitida pelo diacetato 2´7´diclorofluoresceína (DCFH) foi significativo apenas quando estimulado com PMA (miristato-acetato de forbol), não apresentando resposta estatisticamente significante para os estímulos SAPI e LPS. Após a exposição ao naftaleno nas concentrações 10-3, 10-6, 10-9 M durante quatro horas, in vitro, houve um aumento na fagocitose realizado pelos monócitos e trombócitos, porém uma diminuição no burst oxidativo apresentado nas concentrações 10-6 e 10-9 M de naftaleno. Este resultado reflete, in vitro, uma resposta ao contaminante com significado imunológico desfavorável para o peixe, já que as células estão aumentando a atividade fagocítica sem conseguirem, teoricamente, destruir o agente invasor. A partir desses resultados preliminares, podemos avaliar melhor, algumas características da resposta imune inata desta espécie de peixe, presente na costa litorânea brasileira. Estes parâmetros imunofisiológicos podem servir como base para futuros estudos ecotoxicológicos tanto em laboratório mas também a campo. Estudos que utilizam a imunidade inata como indicador biológico de alterações ambientais causadas por poluentes diversos, podem evidenciar o grau de impacto toxicológico sobre esta espécie e agregar valor a sua importância econômica e ecológica. / Flow cytometry is a precise, fast, and effective method for the evaluation of several cellular parameters, both structural and functional, allowing the sorting and analysis of particular populations and sub-populations of cells. In this study, we employed cytometric methods on the caractherization of different blood cell types from Centropomus parallelus, and also verified cell viability, phagocytic activity and oxidative burst in these cells. Three sub-populations were identified: lymphocytes, monocytes, and trombocytes. These cells were stimulated in vitro with propidium iodide-conjugated Staphylococcus aureus (SAPI), Escherichia coli lipopolysaccharide (LPS), or zymosan (Saccharomyces cerevisae particles). Responses to each individual stimulus differed according to cell type. Monocytes displayed the higher percentages of phagocytosis in presence of SAPI or zymosan; on the other hand, the population that included trombocytes, among other cell types (such as lymphocytes) only performed phagocytosis in a relevant level in the presence of SAPI. The oxidative burst, detected by fluorescence emitted by 2´7´dichlorofluorescein diacetate (DCFH) was significant only after stimulation with PMA (phorbol myristate-acetate), but not when the stimulus was SAPI or LPS. Based on these preliminary results, the innate immune response in these animals (ubiquitous coastal waters of the Brazilian shore) can be further evaluated. Immunophysiological parameters in these species can build a solid ground for future ecotoxicological studies. Approaching innate immunity as a biological indicator of environmental changes induced by pollutants may support the degree of toxicological impact over these animals, and aggregate value to its current ecological and economical importance.

Investigar, em motores veiculares pesados, alimentados com combustível diesel, a emissão de dioxinas e furanos clorados (PCDD/Fs) e hidrocarbonetos policíclicos aromáticos (PAHs), através da análise de material particulado e gases do sistema de exaustão. Investigar a mutagenicidade do material particulado e gases coletados. Material e Métodos: Os testes de emissão foram feitos em banco de provas dinamométrico de bancada de acordo com Ciclo 13 Pontos de testes, utilizando-se motores Diesel nacionais de aplicação veícular. Um mini-túnel de diluição foi usado para obtenção de amostras de material particulado. As análises de PCDD/Fs e PAHs foram realizadas em laboratório nacional. Para a análise de PCDD/Fs utilizou-se o método USEPA 8290 (1994) e para a análise de PAHs foi utilizado o método USEPA TO 13A (1999). A análise de mutagenicidade foi realizada de acordo com o Teste de Ames (TA98). Resultados: As medições de PCDD/Fs indicaram em geral valores abaixo do limite de detecção do método analítico. Em uma das amostras foi verificado que o OCDD ficou acima do limite de detecção, enquanto em outra isto ocorreu para o OCDF e para o 1,2,3,7,8-PeCDF. Em relação aos PAHs analisados verificou-se a presença dos seguintes compostos: Naftaleno, Acenaftileno, Acenafteno, Fluoreno, Fenantreno, Antraceno, Fluoranteno, Pireno, Benzo[a]antraceno, Criseno, Benzo[b]fluoranteno, Benzo[k]fluoranteno. A média encontrada para 5 amostras analisadas foi de 3.580,5 µg/g de material particulado. Os PAHs encontrados neste trabalho indicam alguma similaridade com a literatura disponível, para valores em % e µg/g de material particulado total coletado. O teste de Ames indicou que o material particulado coletado de motores diesel tem alta atividade mutagênica. Conclusões: A emissão de PCDD/Fs, para o nível de tecnologia dos motores Diesel testados mostrou-se pouco significativa, considerando-se combustível conforme especificado e método e limite de detecção utillizados. Em relação aos PAHs, os resultados indicaram que motores diesel têm uma contribuição importante na formação destes compostos. A alta atividade mutagênica observada mostra que as emissões de veículos Diesel são potencialmente tóxicas. Recomenda-se a continuidade de estudos para definição da regulamentação desses poluentes. / To investigate emission of chlorinated dioxins and furans (PCDD/Fs) and polycyclic aromatic hydrocarbons (PAHs) in heavy duty diesel engines, through the analysis of particulate material and gases in the exhaust.To verify mutagenicity of collected particulate material and gasesous sample extracts. Material and Methods: The engine emissions tests were done in dynamometer test bench according to 13-Mode Cycle, using brazilian engines, vehicle application. A mini dilution tunnel was used to obtain particulate matter and gaseous sample. PCDD/Fs and PAHs analyses were carried out in a certified national laboratory. Dioxins and furans were analyzed according with Method 8290 (USEPA, 1994) and PAHs by Method TO13A (USEPA, 1999). The mutagenicity analyses were done in accordance with Ames test (TA98) Results: The measurements of dioxins and furans in diesel engines operating in controlled conditions in dynamomter bench test, had indicated emissions values in general below the detention limit of the analytical method. In one of the samples it was verified that the OCDD was above the detention limit, while in another one this occurred for OCDF and 1,2,3,7,8-PeCDF. In relation to 16 PAHs it was verified the presence of the following compounds: Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Antracene, Fluorantene, Pyrene, Benzo[a]antracene, Chrysene, Benzo[b]fluoranthene and Benzo[k]fluorantene. The average value of the sum of PAHs found in five samples was 3,580.5 µg.g-1. The PAHs found in this study indicates some similarity with available literature for values in % and µg/g of particulate matter.The Ames test showed that the collected particulate material from diesel engines presents high mutagenic activity. Conclusion: The emission of PCDD/Fs, for the level of technology of the tested Diesel engines revealed little significant, considering the specified fuel, method of analysis and limit of detention used. In relation to the 16 PAHs, the tests results had indicated that diesel engines has an important contribution in the formation of these compounds. The high mutagenic activity observed shows that the emissions of Diesel vehicles are potentially toxic. The continuity of studies for definition of the regulation of these pollutants is recommended.

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2016. / Polycyclic aromatic hydrocarbons (PAHs) are an alarming group of organic substances for humans and environmental organisms due to their ubiquitous presence, toxicity, and carcinogenicity. They are semi-volatile substances which result from the fusion of carbon and hydrogen atoms and constitute a large group of compounds containing two to several aromatic rings in their molecule. Natural processes and several anthropogenic activities involving complete or incomplete combustion of organic substances such as coal, fossil fuel, tobacco and other thermal processes, generally result in the release of the PAHs into the environment. However, the fate of the PAHs is of great environmental concern due to their tendency to accumulate and their persistence in different environmental matrices and their toxicity. Animal studies have revealed that an excessive exposure to PAHs can be harmful. Evidence of their carcinogenic, mutagenic, and immune-suppressive effects has been reported in the literature. In the soil environment, they have the tendency to be absorbed by plants grown on soil being contaminated by the PAHs. It is, therefore, important to evaluate their occurrence levels in different environmental matrices such as soil concentrations.

A citometria de fluxo é um método preciso, rápido e eficaz na avaliação de múltiplos parâmetros celulares, tanto estruturais como funcionais, propiciando a separação e o estudo de diferentes populações e sub-populações de células. No presente estudo, foram empregados métodos citométricos para caracterização dos diferentes tipos celulares sangüíneos de Centropomus parallelus, assim como para verificação da viabilidade celular, avaliação da atividade fagocítica e ativação do burst respiratório. Foram identificadas três sub-populações representativas de leucócitos: linfócitos, monócitos e trombócitos. Estas células foram estimuladas, in vitro, com Staphylococcus aureus, marcado com iodeto de propídeo (SAPI), lipopolissacarídeo de Escherichia coli (LPS) e zymosan, partículas de Saccharomyces cerevisiae. As respostas frente aos estímulos foram distintas de acordo com o tipo celular e o estímulo apresentado. Os monócitos apresentaram maiores percentuais de fagocitose frente ao estímulo provocado pela SAPI e pelo Zymosan; já a população que continha trombócitos entre outros tipos celulares (por exemplo: linfócitos), apresentou \"fagocitose\" significativa apenas para SAPI. O burst oxidativo detectado pela fluorescência emitida pelo diacetato 2´7´diclorofluoresceína (DCFH) foi significativo apenas quando estimulado com PMA (miristato-acetato de forbol), não apresentando resposta estatisticamente significante para os estímulos SAPI e LPS. Após a exposição ao naftaleno nas concentrações 10-3, 10-6, 10-9 M durante quatro horas, in vitro, houve um aumento na fagocitose realizado pelos monócitos e trombócitos, porém uma diminuição no burst oxidativo apresentado nas concentrações 10-6 e 10-9 M de naftaleno. Este resultado reflete, in vitro, uma resposta ao contaminante com significado imunológico desfavorável para o peixe, já que as células estão aumentando a atividade fagocítica sem conseguirem, teoricamente, destruir o agente invasor. A partir desses resultados preliminares, podemos avaliar melhor, algumas características da resposta imune inata desta espécie de peixe, presente na costa litorânea brasileira. Estes parâmetros imunofisiológicos podem servir como base para futuros estudos ecotoxicológicos tanto em laboratório mas também a campo. Estudos que utilizam a imunidade inata como indicador biológico de alterações ambientais causadas por poluentes diversos, podem evidenciar o grau de impacto toxicológico sobre esta espécie e agregar valor a sua importância econômica e ecológica. / Flow cytometry is a precise, fast, and effective method for the evaluation of several cellular parameters, both structural and functional, allowing the sorting and analysis of particular populations and sub-populations of cells. In this study, we employed cytometric methods on the caractherization of different blood cell types from Centropomus parallelus, and also verified cell viability, phagocytic activity and oxidative burst in these cells. Three sub-populations were identified: lymphocytes, monocytes, and trombocytes. These cells were stimulated in vitro with propidium iodide-conjugated Staphylococcus aureus (SAPI), Escherichia coli lipopolysaccharide (LPS), or zymosan (Saccharomyces cerevisae particles). Responses to each individual stimulus differed according to cell type. Monocytes displayed the higher percentages of phagocytosis in presence of SAPI or zymosan; on the other hand, the population that included trombocytes, among other cell types (such as lymphocytes) only performed phagocytosis in a relevant level in the presence of SAPI. The oxidative burst, detected by fluorescence emitted by 2´7´dichlorofluorescein diacetate (DCFH) was significant only after stimulation with PMA (phorbol myristate-acetate), but not when the stimulus was SAPI or LPS. Based on these preliminary results, the innate immune response in these animals (ubiquitous coastal waters of the Brazilian shore) can be further evaluated. Immunophysiological parameters in these species can build a solid ground for future ecotoxicological studies. Approaching innate immunity as a biological indicator of environmental changes induced by pollutants may support the degree of toxicological impact over these animals, and aggregate value to its current ecological and economical importance.

Mit Hilfe der Rastertunnelmikroskopie (STM, Scanning Tunneling Microscopy) und Tunnelspektroskopie (STS, Scanning Tunneling Spectroscopy) wurden in situ das Ordnungsverhalten und die elektronischen Eigenschaften verschiedener alkylsubstituierter, polyzyklischer Aromaten (PAHs, Polycyclic Aromatic Hydrocarbons) an der Grenzfläche zwischen organischer Lösung und der Basalfläche von Graphit untersucht. Die Anzahl der pro Molekül vorhandenen pi-konjugierten Kohlenstoffatome reicht von 42 (Hexabenzocoronen, HBC) bis 114. Zusätzliche Informationen zur Struktur der geordneten Systeme lieferten kraftfeldbasierte Molekülsimulationen. Die molekularen Systeme bilden durch Adsorption und Selbstaggregation hochgeordnete Monolagen auf der Graphitoberfläche aus, wobei die polyzyklischen Kohlenstoffstrukturen der aromatischen Bereiche wie das darunter liegende Graphitgitter orientiert und die Molekülkristalle kommensurabel mit dem Substrat sind. Die peripheren Alkylketten sind nicht notwendigerweise an der Adsorption auf dem Graphitgitter beteiligt. Dies führt in bestimmten Fällen zu hexagonalen Anordnungen der HBC-Moleküle. Periodische Helligkeitsmodulationen im Tunnelbild der Aromaten werden über unterschiedliche Adsorptionspositionen auf dem Substrat erklärt, die in regelmäßiger Weise durch die molekularen Spezies eingenommen werden. In vielen Fällen ist die Adsorption nicht auf Monolagen begrenzt. Attraktive pi--pi-Wechselwirkungen zwischen den Aromaten werden als Ursache für die Stapelung der Moleküle angeführt. Es besteht die Vermutung, dass auch ein signifikanter Überlapp von Orbitalen vorhanden ist. Folglich wird der an der Grenzfläche zum Graphit existierende molekulare Ordnungszustand auf die zusätzlich adsorbierenden Schichten übertragen. Die Beobachtung nichtperiodischer Kontrastmodulationen wird auf kleinste Unterschiede in den Stapelweisen der Aromaten zurückgeführt, die zu Unterschieden in den elektronischen Eigenschaften führen. Hochaufgelöste STM-Bilder zeigen verschiedene Punktmuster und Streifen innerhalb der aromatischen Bereiche einzelner Moleküle. Diese Muster können nicht direkt der elektronischen Struktur der Aromaten zugeordnet werden. Sie werden vielmehr als Ergebnis einer Faltung der elektronischen Eigenschaften von Substrat und Adsorbat betrachtet. Zwei der Punktmuster, die für ein HBC-Derivat beobachtet wurden und abhängig von der Anzahl der pro Aromatenstapel involvierten Moleküle sind, konnten als sqrt(3) x sqrt(3) R 30°- bzw. als 2 x 2 -Überstruktur des Graphits beschrieben werden. Die STS-Experimente waren auf die Aufnahme lokaler I(U)-Kennlinien gerichtet. Die Aromaten weisen eine asymmetrische I(U)-Charakteristik auf, die Ähnlichkeiten mit dem Kennlinienverlauf einer Diode zeigt. Die Asymmetrie wird durch resonantes Tunneln über das HOMO bei negativen Probenspannungen erklärt. Die Verstärkung der Asymmetrie mit zunehmender Größe des Aromaten wird durch den kleiner werdenden energetischen Abstand zwischen HOMO und Fermi-Niveau des Graphits verursacht. / Self-assembled structures and electronic properties of alkylsubstituted, polycyclic aromatic hydrocarbons (PAHs) at the solid liquid interface between a solution and the basal plane of graphite have been studied in situ by scanning tunneling microscopy (STM) and spectroscopy (STS). The number of pi-conjugated carbon atoms per investigated molecular species ranges from 42 (hexabenzocoronene, HBC) to 114. Supplementary structural information was obtained by forcefield based simulations. Upon adsorption and self-assembly, the molecules form ordered monolayers on the graphite surface. Predominantely, the polycyclic carbon structures of the aromatic moieties exhibit the same orientation as the underlying graphite lattice and the molecular lattices are commensurate with the substrate. The peripheral alkyl chains do not necessarily adsorb on the substrate which leads to hexagonal packings of the HBCs in some cases. Periodic contrast modulations of the aromatic moieties in the STM images are attributed to different adsorption sites on the graphite lattice. These different positions are occupied by the molecules in a regular way. In many cases, the adsorption is not limited to monolayers. It is proposed that the stacking is governed by pi--pi-interactions between the aromatic regions including a significant overlap of orbitals. As a consequence, the molecular order from the solid liquid interface is transferred to the additionally adsorbing layers. Nonperiodic contrast modulations indicate slight differences in the stacking giving rise to modified electronic properties. Highly resolved STM images show different dot patterns and stripes within the aromatic region of single molecules. These patterns do not directly stem from the electronic structure of the aromatic species. Instead, they are rather explained by a convolution of the electronic properties of both, substrate and adsorbate. Two of the dot patterns observed for an HBC derivative could be described as sqrt(3) x sqrt(3) R 30°- and as 2 x 2 -superstructure of the graphite lattice, respectively, depending on the number of stacked molecules. STS involved the measurement of I(U)-curves at specific locations. The curves of the aromatic moieties exhibit an asymmetric, diode like behavior explained by resonant tunneling via the HOMO at negative substrate voltages. The asymmetry is enhanced with increasing size of the aromatic moiety. This is due to the reduced distance between the HOMO and the graphite Fermi level for the larger PAHs.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that can be modified to oxygenated PAH (oxyPAHs) derivatives. It is well known that oxyPAHs tend to be much more reactive than their parent compounds. Toxicity can be attributed to direct interaction with target molecules or generation of reactive oxygen species (ROS). Metals are another class of contaminant found ubiquitously throughout the environment. Some metals are toxic at levels below the 1:1 ratio predicted by the biotic ligand model and are thought to manifest toxicity through ROS generation. Often metals and PAHs occur as co-contaminants in industrialized environments, yet little is known about their potential co-toxicity or mechanisms of action in mammalian reproductive function. Previously, we described that a PAH, 9, 10-phenanthrenequinone (PHEQ), inhibited LH-stimulated progesterone secretion in dispersed rat corpus luteal (CL) cells (Nykamp et al., 2001). Viability was decreased in CL cells exposed to PHEQ and 1,2-dihydroxy-anthraquinone (1,2-dhATQ), but not their parent compounds phenanthrene (PHE) or anthracene (ANT). Similarly, LH-stimulated progesterone production in CL cells was inhibited by PHEQ and 1,2-dhATQ, but not PHE. Further investigation revealed that PHEQ, but not PHE, ANT nor 1,2-dhATQ generated ROS in CL cells. Viability experiments were repeated using the choriocarcinoma cell line JEG-3 with similar results. Various metals were assessed for their toxicity to both CL and JEG-3 cells. The endpoints used to measure viability were metabolic activity and membrane integrity. In general, metabolic activity was a more sensitive indicator of toxicity than membrane integrity. The order of toxicity for metals in CL cells was Hg2+ > Cd2+ > Zn2+ > Ni2+ > Cu2+ for metabolic activity and Hg2+ ≈ Zn2+ > Cd2+ > Cu2+ > Ni2+ for membrane integrity. Only Hg2+ and Cu2+ were tested in JEG-3 cells. While Cu2+ was non-toxic, EC50s for Hg2+ metabolic activity and membrane integrity were 20 mM and 23 mM, respectively. Experiments were designed to study the mixtures of metals and PAHs on viability, ROS production, and LH-stimulated progesterone production in CL cells. Mixtures of each metal with either PHEQ or 1,2-dhATQ were incubated with CL cells and their effect on metabolic activity and membrane integrity assessed. Generally, most metal/oxyPAH mixtures displayed only additive toxicity. However, mixtures of Cu2+ and PHEQ showed synergistic toxicity to both metabolic activity and membrane integrity. Mixture studies in JEG-3 cells used only combinations of Cu2+ or Hg2+ with PHEQ or 1,2-dhATQ. Similar results to metabolic activity and membrane integrity in CL cells were observed. Mixtures of Cu2+ and PHEQ or 1,2-dhATQ were tested in CL cells for their effect on LH-stimulated progesterone secretion and ROS production. Additive effects were observed in both LH-stimulated progesterone secretion and ROS production for Cu2+/1,2-dhATQ mixtures while synergistic effects for both parameters were seen with Cu2+/PHEQ. Efforts to determine the site of action for mixtures of Cu2+/PHEQ involved adding the cholesterol analogue, 22-OH cholesterol (22-OHC) to CL cells in the absence of LH. Cytochrome P450 side-chain cleavage (CYP450scc) enzyme operates constitutively and the addition of 22-OHC to CL cells resulting in a 5-fold increase in progesterone production without added LH. Kinetic assays with 22-OHC show that while progesterone secretion was inhibited with PHEQ addition alone, a further significant reduction with both Cu2+ and PHEQ was not observed. The use of forskolin, an activator of adenylate cyclase, did not show any significant enhancement of progesterone secretion with the addition of Cu2+/PHEQ compared to PHEQ alone. The potential targets of Cu2+/PHEQ mixture include any step in the steroidogenic cascade from activation of protein kinase A onward with the proteins of the mitochondria, cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein, being the most likely. Differential display polymerase chain reaction (ddPCR) was a molecular approach taken to determine the effect of PHEQ on JEG-3 gene expression. The genes whose expression appeared to be up-regulated with PHEQ exposure were serine protease inhibitor, Alu repeat sequence, heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 3 (eIF3), nucleoporin-like protein, eukaryotic translation elongation factor 1a1 (eEF1 a 1), autophagy-linked FYVE domain (Alfy), spectrin, and proteasome. Apparent down-regulated genes in JEG-3 cells after PHEQ exposure included poly(ADP-ribose) polymerase 10 (PARP10), polyglutamine binding protein-1 (PQBP-1), heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 5A (eIF5A), and keratin. In both cell types, oxyPAHs were more toxic than their parent compounds. Metals showed greater toxicity to metabolic activity than to membrane integrity. Of the combinations tested, only PHEQ and Cu2+ exhibited synergistic toxicity. ROS generation was the likely mechanism behind PHEQ/Cu2+ toxicity. Both cell types used represent critical roles in human reproductive health. The proper production of progesterone, a critical hormone for the maintenance of pregnancy in mammals, represents a unique endpoint for the assessment of toxicity. These results illustrate the need to study modified oxyPAHs, metals and metal/oxyPAH mixtures for their potential impact on human reproductive health.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that can be modified to oxygenated PAH (oxyPAHs) derivatives. It is well known that oxyPAHs tend to be much more reactive than their parent compounds. Toxicity can be attributed to direct interaction with target molecules or generation of reactive oxygen species (ROS). Metals are another class of contaminant found ubiquitously throughout the environment. Some metals are toxic at levels below the 1:1 ratio predicted by the biotic ligand model and are thought to manifest toxicity through ROS generation. Often metals and PAHs occur as co-contaminants in industrialized environments, yet little is known about their potential co-toxicity or mechanisms of action in mammalian reproductive function. Previously, we described that a PAH, 9, 10-phenanthrenequinone (PHEQ), inhibited LH-stimulated progesterone secretion in dispersed rat corpus luteal (CL) cells (Nykamp et al., 2001). Viability was decreased in CL cells exposed to PHEQ and 1,2-dihydroxy-anthraquinone (1,2-dhATQ), but not their parent compounds phenanthrene (PHE) or anthracene (ANT). Similarly, LH-stimulated progesterone production in CL cells was inhibited by PHEQ and 1,2-dhATQ, but not PHE. Further investigation revealed that PHEQ, but not PHE, ANT nor 1,2-dhATQ generated ROS in CL cells. Viability experiments were repeated using the choriocarcinoma cell line JEG-3 with similar results. Various metals were assessed for their toxicity to both CL and JEG-3 cells. The endpoints used to measure viability were metabolic activity and membrane integrity. In general, metabolic activity was a more sensitive indicator of toxicity than membrane integrity. The order of toxicity for metals in CL cells was Hg2+ > Cd2+ > Zn2+ > Ni2+ > Cu2+ for metabolic activity and Hg2+ ≈ Zn2+ > Cd2+ > Cu2+ > Ni2+ for membrane integrity. Only Hg2+ and Cu2+ were tested in JEG-3 cells. While Cu2+ was non-toxic, EC50s for Hg2+ metabolic activity and membrane integrity were 20 mM and 23 mM, respectively. Experiments were designed to study the mixtures of metals and PAHs on viability, ROS production, and LH-stimulated progesterone production in CL cells. Mixtures of each metal with either PHEQ or 1,2-dhATQ were incubated with CL cells and their effect on metabolic activity and membrane integrity assessed. Generally, most metal/oxyPAH mixtures displayed only additive toxicity. However, mixtures of Cu2+ and PHEQ showed synergistic toxicity to both metabolic activity and membrane integrity. Mixture studies in JEG-3 cells used only combinations of Cu2+ or Hg2+ with PHEQ or 1,2-dhATQ. Similar results to metabolic activity and membrane integrity in CL cells were observed. Mixtures of Cu2+ and PHEQ or 1,2-dhATQ were tested in CL cells for their effect on LH-stimulated progesterone secretion and ROS production. Additive effects were observed in both LH-stimulated progesterone secretion and ROS production for Cu2+/1,2-dhATQ mixtures while synergistic effects for both parameters were seen with Cu2+/PHEQ. Efforts to determine the site of action for mixtures of Cu2+/PHEQ involved adding the cholesterol analogue, 22-OH cholesterol (22-OHC) to CL cells in the absence of LH. Cytochrome P450 side-chain cleavage (CYP450scc) enzyme operates constitutively and the addition of 22-OHC to CL cells resulting in a 5-fold increase in progesterone production without added LH. Kinetic assays with 22-OHC show that while progesterone secretion was inhibited with PHEQ addition alone, a further significant reduction with both Cu2+ and PHEQ was not observed. The use of forskolin, an activator of adenylate cyclase, did not show any significant enhancement of progesterone secretion with the addition of Cu2+/PHEQ compared to PHEQ alone. The potential targets of Cu2+/PHEQ mixture include any step in the steroidogenic cascade from activation of protein kinase A onward with the proteins of the mitochondria, cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein, being the most likely. Differential display polymerase chain reaction (ddPCR) was a molecular approach taken to determine the effect of PHEQ on JEG-3 gene expression. The genes whose expression appeared to be up-regulated with PHEQ exposure were serine protease inhibitor, Alu repeat sequence, heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 3 (eIF3), nucleoporin-like protein, eukaryotic translation elongation factor 1a1 (eEF1 a 1), autophagy-linked FYVE domain (Alfy), spectrin, and proteasome. Apparent down-regulated genes in JEG-3 cells after PHEQ exposure included poly(ADP-ribose) polymerase 10 (PARP10), polyglutamine binding protein-1 (PQBP-1), heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 5A (eIF5A), and keratin. In both cell types, oxyPAHs were more toxic than their parent compounds. Metals showed greater toxicity to metabolic activity than to membrane integrity. Of the combinations tested, only PHEQ and Cu2+ exhibited synergistic toxicity. ROS generation was the likely mechanism behind PHEQ/Cu2+ toxicity. Both cell types used represent critical roles in human reproductive health. The proper production of progesterone, a critical hormone for the maintenance of pregnancy in mammals, represents a unique endpoint for the assessment of toxicity. These results illustrate the need to study modified oxyPAHs, metals and metal/oxyPAH mixtures for their potential impact on human reproductive health.

Alkylphenanthrenes such as retene (7-isopropyl-1-methylphenanthrene) are aquatic contaminants commonly found in anthropogenically-, industrially-, and petroleum-contaminated environments, and have been implicated in crude oil toxicity. In the early life stages (ELS) of fish, exposures to alkylphenanthrenes produce signs of toxicity typical of those observed in exposures to halogenated aromatic hydrocarbons, particularly to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD, the most toxic congener, serves as the basis of the current mechanism-based risk assessment model. The model assumes that congeners that produce TCDD-like toxicity share a common mode of action and act additively. The mechanism of TCDD-like toxicity is assumed to be mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved in the xenobiotic response (e.g., induction of cytochrome P450 1A enzymes; CYP1A) and in normal development. CYP1A enzymes are not involved in the mechanism of TCDD toxicity. Alkylphenanthrenes toxic to the ELS of fish are AhR ligands, but in contrast to TCDD, are readily metabolized by CYP1A enzymes. The byproducts of CYP1A metabolism have been implicated in retene toxicity. However, the target tissue of retene and the direct roles of AhR and CYP1A in retene toxicity are unknown, but are expected to be similar to those of TCDD. The results presented in this thesis suggest that in the ELS of fish: (1) the primary target of retene is the cardiovascular system (Chapters 2 & 5); (2) retene toxicity is stage-specific (Chapter 2); (3) the mechanism of retene toxicity is mediated by AhR2, and is independent of CYP1A enzymes (Chapter 5); (4) multiple CYP1A-independent toxicities can result from exposures to different mixtures of CYP1A inducing (retene) and CYP1A inhibiting (alpha-naphthoflavone or 2-aminoanthracene) PAHs (Chapters 3 & 4); and (5) multiple concentration-dependent mechanisms of toxicity (i.e., synergism and response addition) can occur in co-exposures of a CYP1A inducer (retene) with a range of CYP1A inhibitor (alpha-naphthoflavone) concentrations (Chapter 3). Thus, retene toxicity is mechanistically similar to that of TCDD toxicity, suggesting alkylphenanthrenes can be included in the current risk assessment model. However, the observed variable mixture toxicities and species differences in retene toxicity raise questions about the effectiveness of this model. / Thesis (Ph.D, Biology) -- Queen's University, 2009-01-13 12:10:31.373

Les activités humaines sont à l’origine de nombreuses pollutions par des hydrocarbures au niveau des écosystèmes, et plus particulièrement au niveau des sols. Afin de préserver la santé humaine et environnementale, il est nécessaire d’éliminer les polluants présents. Dans ce but, les techniques de bioremédiation apparaissent aujourd’hui comme de réelles alternatives aux techniques classiques, invasives et onéreuses. Cependant, l’utilisation optimale de tels procédés nécessite une meilleure connaissance des capacités métaboliques des communautés microbiennes impliquées dans la biodégradation de ces polluants. Dans ce cadre, l’utilisation des biopuces ADN fonctionnelles pour analyser ces écosystèmes semble très appropriée.Cependant, une de ses limitations actuelles est la détermination des sondes, qui ne ciblent que les gènes dont les séquences ont été caractérisées. Pour cela, un outil informatique (Metabolic Design) a été mis au point, afin de déterminer des sondes exploratoires pour biopuces fonctionnelles. L’étude, avec notre biopuce fonctionnelle, des capacités métaboliques de dégradation des HAP de la souche Sphingomonas paucimobilis sp. EPA505 a permis de mettre en évidence la sensibilité et la spécificité des sondes développées, ainsi que leur aspect exploratoire. Puis, nous nous sommes attachés à caractériser les capacités métaboliques des communautés bactériennes d’un sol pollué principalement par des HAP, sans à priori sur les séquences ou les organismes présents, montrant l’efficacité de notre approche. / Soil ecosystems are sensitive to damage from pollutions, and there is an increasing need to develop better methods for removing pollutants from soils. The removal of pollutants, such as polycyclic aromatic hydrocarbons, by bioremediation, is a less invasive and expensive process than classical decontamination. However, use and optimization of bioremediation treatments require knowledge on metabolic capacites of microbial communities involved in the biodegradation of such pollutants. To assess their huge metabolic potentialities, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences and so, fail to cover the full microbial gene diversity present in complex environments. We have thus developed a program, named Metabolic Design, to design efficient explorative probes for functional microarrays. Then, we successfully validated our new functional microarray studying metabolic capacities of Sphnigomonas paucimobilis sp. EPA505 able to degrade polycyclic aromatic hydrocarbons. Finally, we assessed metabolic capacities of microbial communities in soil, contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently.