Les hydrocarbures aromatiques polycycliques (HAP) constituent un groupe de polluants organiques persistants (POP). Ils sont produits par la combustion incomplète de la matière organique et peuvent être transférés sur de longues distances. Leur nature organique et hydrophobe leur confère une affinité vis-à-vis des surfaces lipidiques et des compartiments contenant de la matière organiques. Ils peuvent donc s’adsorber sur les matrices végétales et sur la matière organique des sols. La canopée, du fait de sa rugosité aérodynamique importante, constitue un puits important de HAP. Dans cette étude, nous avons suivi l’évolution de la concentration en HAP, sur une période de 20 ans (de 1993 à 2011), dans la végétation (feuilles/aiguilles), les couches organiques (OL, OF et OH) et les couches organo-minérales (0 – 10 cm, 10 – 20 cm et 20 - 40 cm) des sites forestiers français suivis par le réseau RENECOFOR (Réseau National de suivi à long terme des ECOsystèmes FORestiers).14 sites répartis sur tout le territoire français ont été choisis selon des caractéristiques différentes (type de l’essence forestière, climat, altitude, latitude, longitude, type d’humus forestier, contenu en carbone organique, etc.). Ainsi, 14 HAP considérés comme prioritaires par l’agence de protection de l’environnement des Etats-Unis (US-EPA) ont été quantifiés.Cette étude a mis en évidence l’efficacité de la végétation dans l’enregistrement de l’historique de la qualité de l’air des sites forestiers. Une baisse générale de la concentration en HAP dans la végétation et dans l’atmosphère pendant les 20 dernières années a été constatée. La végétation joue le rôle d’une pompe qui accumule les HAP et les transferts vers les sols forestiers. Le contenu en carbone organique semble être le paramètre contrôlant l’accumulation des HAP dans les sols forestiers. Ainsi, la dynamique des HAP entre les différents compartiments forestiers semble dépendre de leurs propriétés physico-chimiques et de leur capacité à subir des mécanismes de dissipation (lixiviation, dégradation, etc). Enfin, cette étude montre que les couches organo-minérales des sols forestiers constituent un puits plus important de HAP (légers, intermédiaires et lourds) que la végétation et les couches organiques dans l’écosystème forestier. / Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic pollutants (POPs). They are produced by incomplete combustion of organic matter and can be transported over long distances in the atmosphere. Because of their organic and hydrophobic nature they have a strong affinity for lipid surfaces and compartments containing organic matter. They can therefore be adsorbed on the plant matrices and on the organic matter of the soils. The canopy, due to its important aerodynamic roughness, constitutes an important sink for PAHs.In this study, we have monitored the evolution of PAHs concentrations over a period of 20 years (from 1993 to 2011) in 3 compartments of the forest ecosystem: vegetation (leaves / needles), organic layers (OL, OF and OH) and organo-mineral layers (0-10 cm, 10-20 cm and 20-40 cm) of French forest sites monitored by the RENECOFOR network (National Network for Long-term FOrest ECOsystem Monitoring).4 sites distributed throughout France were selected according to different characteristics (type of forest species, climate, altitude, latitude, longitude, type of forest humus, organic carbon content, etc.). Thus, 14 PAHs, considered as priority pollutants by the United States Environmental Protection Agency (U.S. EPA), were quantified.This study highlights the efficiency of vegetation in registering the history of air quality at forest sites. A general decrease in the concentration of PAHs in vegetation and in the atmosphere over the past 20 years has been observed.Vegetation plays the role of a pump that accumulates PAHs and transfers them to forest soils. Organic carbon content appears to be the most important parameter controlling PAHs accumulation in forest soils. Thus, PAHs transfer between the different forest compartments appears to depend on their physico-chemical properties and their ability to undergo dissipation mechanisms (leaching, degradation, etc.).Finally, this study shows that the organo-mineral layers of forest soils constitute a more important sink of (light, intermediate and heavy) PAHs than vegetation and organic layers in the forest ecosystem.

The large conductance Ca++-activated K+ channel (BKCa) regulates neuronal excitability through the efflux of K+, in response to membrane depolarization and increases in intracellular Ca++. The activity of the BKCa channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie, in part, the influence of the drug on peptide hormone release from neurohypophysial nerve terminals (Dopico et al., 1996, 1998). Moreover, chronic EtOH exposure attenuates acute drug action on hormone release, and reduces the sensitivity of BKCa channels to acute EtOH exposure (Knott et al., 2002). The factors regulating EtOH action on BKCa channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels, including BKCa (Chang et al., 1995a, b; Moczydlowski et al., 1985; Park et al., 2003; Turnheim et al., 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distribution of membrane cholesterol (CHS) (Chin et al., 1978; Chin et al., 1979; Wood et al., 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKCa channel activity and EtOH sensitivity. To address this, we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKCa channels retain EtOH sensitivity in this reductionist preparation (Chu et al., 1998), and we extend the study here to examine cloned human brain (hslo) BKCachannels. We show here that hslo channels maintain their characteristic large conductance, voltage and Ca++-dependent gating, and sensitivity to 50 mM EtOH in bilayers cast from a 3:1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels, in a concentration-dependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings, we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKCa channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structure itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore, reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel, we demonstrate that decreases in negative surface charge, increases in the proportion of unsaturated acyl chains, and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslochannels, relative to control POPE/POPS (3:1) bilayers. Overall, these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S.N., O'Connell, R.J., and Crowley, J.J. (2002). Artificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research, D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J.J., Treistman, S.N., and Dopico, A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKCA channels in binary phospholipid bilayers. Mol. Pharma. 64(2):364-372.

Les Technosols issus de l’arrêt des activités industriels présentent des propriétés physico-chimiques très contrastées de celles de sols naturels et peuvent renfermer de fortes teneurs en polluants organiques (e.g. composés aromatiques polycycliques (CAP)). Leur abandon pendant des décennies a entrainé l’apparition d’une pédogenèse, particulièrement sous l’influence de facteurs climatiques et supposé entrainer un phénomène d’atténuation naturelle des polluants organiques bien que ces effets ne soient rarement identifiés. Nous proposons ainsi de modéliser l’évolution de la disponibilité des CAP au cours du temps et sous l’influence du facteur climatique. La première étape a été de transcrire des données climatiques atmosphériques en conditions pédoclimatiques et ce, selon les effets de la localisation à l’échelle mondiale, de scénarios de changements climatiques (GIEC), des propriétés des sols et de la profondeur. L’effet de modalités pédoclimatiques sur la disponibilité des CAP a ensuite été testée en conditions contrôlées et sur 11 terres industrielles contrastées (cokeries, usines à gaz). Les conséquences de ces variations de disponibilité des CAP sur la toxicité des sols ont ensuite été validées par un test de croissance de Zea mays L sur les terres vieillies. Les résultats ont été incrémentés dans les modélisations des pédoclimats permettant ainsi de prédire l’évolution de la disponibilité des CAP. Si les conditions climatiques actuelles participent à une diminution de la disponibilité des CAP, le phénomène inverse se produit suite à un réchauffement climatique, que nous définirons comme amplification naturelle, soulignant à nouveau les risques potentiels liés aux changements climatiques sur les sols / Technosols linked with the end of industrial activities depict contrasted properties from those of natural soils and may contain high concentration of organic pollutants (e.g. polycyclic aromatic compounds (PAC)). Their neglect led to the apparition of pedogenetic processes particularly under the influence of climate which potentially contribute to the natural attenuation phenomenon. Hence, we suggest a new method to predict the evolution of PACs’ availability over time and under the influence of climate. The first step was to build a transcription model to predict the pedoclimate evolution derived from weather datasets and under the influence of climate location, climate change scenarios (IPCC), soil properties and depth. The effect of pedoclimatic events on the evolution of PAC availability was then tested in controlled conditions and on 11 contrasted industrial soils (coking plant and gas plant). This allowed predicting the frequency and occurrence of major pedoclimatic events (e.g. freeze-thaw, wetting-drying cycles and periods of high temperature) that drive pedogenesis. The consequence of these availability variation on toxicity were then tested using a growth test of Zea mays L. on aged soils, confirming that PAC availability and soil phytotoxicity are linked. The aging results were then incremented into the pedoclimate model to predict the evolution of PAC availability over time. As the current climatic conditions lead to a decrease in PAC availability (natural attenuation), the opposite effect is observed under the warmer climate of 2100. We described this phenomenon as natural amplification which underlines the upcoming threat of climate change on soils

Orientador: Mary Rosa Rodrigues de Marchi Santiago da Silva / Banca: Lilian Rotschild de Carvalho / Banca: Fernando Mauro Lanças / Banca: Maria Lucia Ribeiro / Banca: Alberto José Cavalheiro / Resumo: município de Campo Grande, MS, com uma população de aproximadamente 600 mil habitantes concentrados na área urbana, todos os anos, entre os meses de julho a novembro, período muito seco, fica encoberto pela fumaça proveniente da queima de biomassa, a qual tem sido apontada como responsável pelo aumento da incidência de casos de doenças respiratórias na população. Neste trabalho, é reportada a composição química de aerossóis, partículas inaláveis (1 μm ≤ dp≤ 15 μm), de emissões do forno utilizado para a produção de carvão vegetal e de 14 amostras de ar da cidade de Campo Grande / MS. O particulado atmosférico foi amostrado de junho a novembro de 1998 no campus da UFMS e as amostras de emissão direta foram coletadas a 1,5 m de um forno construído com tijolos e saibro, similar aos utilizados nas carvoarias da região. Nos dois tipos de amostra, o material particulado foi coletado sobre filtro de Fluorepore em PTFE com 37 mm de diâmetro e as substâncias semivoláteis em tubos do adsorvente XAD-2, utilizando-se amostrador de baixo volume (low-vol). As substâncias foram extraídas com diclorometano/metanol (4:1) em banho ultra-sônico. As análises foram realizadas por CG/EM nos modos SCAN e SIM. A concentração de HPAs na atmosfera próxima ao forno, proveniente da queima de madeira para produção de carvão, foi estimada em 23,6 μg.m-3 de ar para a soma de 15 HPAs, e em 310,1 ng.m-3 para o BaP. A concentração média dos 15 HPAs nas amostras ambientais foi de 21,05 ng.m-3 e a concentração média do BaP de 0,25 ng.m-3...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Campo Grande City (Mato Grosso do Sul State, Brazil) has around 600,000 inhabitants, concentrated in the urban area. Every year, between July and November, the dry season, the city is covered with biomass burning smoke. The biomass burning is result of the vegetal carbon production, agricultural handling, or de-florestation operation into Brazilian Savannah ("cerrado"). The smoke has been indicated as main cause of respiratory disease increasing of the population. In this work it was reported the aerosol composition (1μm ≤ dp≤ 10 μm), from emission of vegetal carbon production and 14 air samples of Campo Grande city. The particulate material was sampled from June until November, 1998 at campus of Federal University of Mato Grosso do Sul (UFMS) while the emission samples were collected at 1.5 m far from an oven made with brick and a mixture of clay and sand, similar to ones used by vegetal carbon producers of region. For both kind of samples, the particulate material was collected with Fuorepore/PTFE filters (37 mm diameter) while volatile fraction was sampled into adsorbent tubes (XAD-2), using low vol sampler. The extracts were obtained by ultrasonic bath using dichloromethane:methanol (4:1) and were analyzed by GC/MS, SCAn and SIM modes. The total HPAs emission of vegetal carbon oven was estimated in 23.6 μg.m-3 and 310 ng.m-3 for benzo(a)pyrene. On the other hand, the city air samples shown concentration (average) of 21,05 ng.m-3 for total HPAs, and 0.25 ng.m-3 for benzo(a) pyrene. HPAs, OXI-HPAs, phenols e Metoxy-phenols were identified...(Complete abstract, click electronic access below) / Doutor

Ce manuscrit décrit la synthèse et les caractérisations physico-chimiques de nouveaux systèmes hétérocycliques π-conjugués contenant un atome de phosphore. Le chapitre d'introduction présente les différentes voies de synthèse du cycle phospholes pouvant être incorporé dans des systèmes π-conjugués. Le chapitre I décrit la synthèse et les propriétés physico-chimiques de dérivés du pyrène contenant soit un phosphole, soit un silole. La synthèse de ces nouvelles structures a été réalisée à l'aide de réactions métallo-catalysées. Deux types de régioisomères ont ainsi été synthétisés afin de déterminer l'impact de la nature de l'hétéroatome (P ou Si) sur leurs propriétés optiques et électrochimiques et sur l'organisation des molécules à l'état solide. Le chapitre II décrit la synthèse et les propriétés physico-chimiques de nouveaux complexes d'Ir(III) ortho-métallés contenant un ligand phosphole. La présence de deux centres stéréogènes induit la formation de diastéréoisomères, dont la formation peut être contrôlée par les conditions réactionnelles. De plus, les propriétés électroniques du ligand phosphole permettent de moduler à façon les propriétés redox et optiques du complexe. Le chapitre III se focalise sur l'utilisation de la phosphépine comme sous-unité pour la construction de nouveaux systèmes π-conjugués. Deux systèmes π-conjugués ont été synthétisés : un squelette de PAH contenant la phosphépine et les dithieno-phosphépines. Les propriétés optiques et redox de ces dérivés ont été étudiées et discutées dans le manuscrit. / This manuscript describes the synthesis and characterisation of new heterocyclic π-conjugated systems built around the phosphorus atom. Firstly, an introduction to the phosphole chemistry (synthesis methods and integration into π-conjugated systems) is presented. The chapter I describes the synthesis and physico-chemical properties of pyrene functionalised either by a phosphole or silole ring. The synthesis of these new structures was achieved by using a transition-metal catalysis. Two types of regioisomers were synthesised in order to study the effect of the heteroatom (P or Si) on the optical and electrochemical properties together with the impact on the solid-state organisation. The chapter II describes the synthesis and the physical properties of the first phosphole-based Ir(III) ortho-metallated complexes. The presence of two stereogenic centres in the complex induced diastereoselectivity issues that could be solved by carefully choosing the reaction conditions. Furthermore, it was demonstrated that the nature of the ligands can tune the optical and the redox properties. The chapter III describes the use of P-containing seven-membered rings (phosphepines) for the development of new π-conjugated systems. Two original π-systems have been synthesised and studied: (i) phosphepine based PAH and (ii) dithieno-phosphepines. The optical and redox properties have been investigated and discussed in the manuscript.

According to the Waste Framework Directie 2008/98/ES and the Czech law on waste 185/2001 Sb., waste preienton is the main priority in waste management. Using biodegradable plastcs seems to represent a suitable approach in fulflling the strategy. The subject of this diploma thesis is biodegradable lightweight polyurethanes (PURs), which might represent suitable materials for repellent fences in agricultural landscapes. Biodegradaton processes are assumed to begin in the agricultural soil afer their lifetme. The biodegradability of new lightweight PURs (BIO 8-10), presented as material mineralizaton, was tested in agricultural soil under laboratory conditons according to the ASTM Internatonal standard method D5988-03. Afer the nine-month experiment the material with the highest biodegradability (BIO 10) containing starch (28 wt. %) was mineralized to 42.0 ± 4.2 %. Cellulose (the positie control) was completely mineralized; thus, the ialidity criterion (positie control mineralizaton > 70 %) was accomplished. Afer the biodegradability test, degradaton products (residual PURs) were separated from the soil by a two-step method using fuidisaton and fotaton, according to a modifed procedure described in Nuelle et al. (2014). The procedure enabled a subsequent analysis of PUR residues with Fourier transform...

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.

A exaustão de um veículo de motor a diesel é uma importante fonte de poluentes atmosféricos, pois forma uma matriz complexa composta de poluentes regulados e não regulados pelos órgãos governamentais. Dentre os poluentes regulados podemos citar óxidos de nitrogênio (NOx) e material particulado. Os poluentes não regulados são pouco estudados até hoje e dentre estes encontra-se a classe dos hidrocarbonetos policíclicos aromáticos e seus derivados nitrados (nitro-HPA). Estes são encontrados na exaustão do diesel na forma gasosa ou agregados ao material particulado. Hoje, o interesse em estudos destes compostos vem aumentando, devido às suas atividades carcinogênicas e mutagênicas às quais estão sujeitas as populações dos centros urbanos. O impacto causado pelos nitro-HPA emitidos por motores a ciclo diesel ao ambiente não está ainda completamente estabelecido. Este estudo consiste na modelagem e simulação do processo de combustão de hidrocarbonetos na faixa de C1 a C4 com o objetivo de descrever a formação de compostos aromáticos, principalmente HPA, e óxidos de nitrogênio a partir de modelos cinéticos de combustão propostos na literatura como referência e fazendo uso do software de simulação Kintecus. Este projeto tem como objetivo em longo prazo propor um modelo cinético para combustão do óleo Diesel. Foi iniciada a construção de um modelo cinético de combustão a partir de modelos de hidrocarbonetos simples de C1 a C4, com formação de aromáticos, HPA e óxidos de nitrogênio. Os modelos originais foram avaliados e modificados a fim de estudar como parâmetros do modelo afetam a concentração das espécies de interesse. Foi observado a tendência de formação de benzeno e fulveno em baixas temperaturas e a tendência de formação de antraceno, pireno, fenantreno a temperaturas mais altas. Foi avaliado que a conversão NO-NO2 ocorre em maiores proporções em reações iniciadas a baixas temperaturas, 600 K. Os resultados indicam que propano é o maior responsável por esta conversão. O modelo final obtido resultou da união dos modelos de combustão Hori e Marinov mais inclusão do GRI-Mech 3.0 e reações adicionais de NOx retiradas da base de dados NIST / The diesel engine exhaust is an important source of air pollutants, which comprises a complex matrix of regulated and unregulated pollutants. The nitrogen oxides (NOx) and the particulate material are examples of regulated pollutants found in the engine exhaust. The unregulated pollutants are poorly studied until today, being the polycyclic aromatic hydrocarbons and its nitrated derivatives (nitro-PAH) a class of unregulated pollutant observed in diesel exhaust. The nitro-PAHs are observed in gas phase or aggregated to the particulate material. At the present, the interest in those compounds raised due to their carcinogenic and mutagenic properties, which the population of urban centers are subject. The environmental impact caused by nitro-PAH is not fully established. This study consists in the modeling and simulation of the hydrocarbons combustion process comprising the C1 to C4 hydrocarbons using kinetic models proposed in the literature as reference models and the Kintecus software. The purpose of the present study is to describe the formation of aromatic compounds, mainly PAH, and nitrogen oxides. The long term goal is to build a combustion model to Diesel oil. The kinetic model was constructed based on known combustion models of C1 to C4 hydrocarbons, which includes the formation of aromatics, PAH and nitrogen oxides. The original models were evaluated and modified to analyze how the model parameters affects the species concentration. There is a trend to the formation of benzene and fulvene at low temperatures, and a tendency to formation of antracene, pyrene and antracene at high temperatures. The NO-NO2 conversion was evaluated and the high rates of conversion was obtained in simulations started at temperature. The results indicates that propene is the major hydrocarbon that promotes the NO-NO2 conversion. The final model proposed is based in the combination of Marinov and Hori models with the inclusion of GRI-Mech 3.0 plus additional reactions extracted from NIST database

Bile acid metabolism is integrally involved in cholesterol homeostasis in mammals because it is the major means by which cholesterol is eliminated from the body. We have undertaken an effort to study the molecular mechanisms underlying the regulation of bile acid metabolism by isolating and characterizing the cDNA and gene for an enzyme that hydroxylates lithocholic acid (LCA) at position 6β, lithocholic acid 6β-hydroxylase; the first bile acid-induced gene reported. LCA is a very hydrophobic, toxic bile acid formed from chenodeoxycholic acid in the gut lumen upon reduction of the 7α-hydroxy group by microbial enzymes. The proper elimination of LCA is essential for maintenance of the bile acid pool and for prevention of cholestasis which results from LCA precipitating in the cannaculi of the liver when its concentration is high. The LCA 6β-hydroxylase cDNA was isolated by differential hybridization of hamster liver libraries prepared from animals fed either a cholic acid enriched diet or a cholestipol-rich chow and was named CYP3A10 based on its homology with other cytochrome P450s (P450) in family 3A. We found that CYP3A10 was essentially expressed only in males. A statistical analysis of RNA from young males fed with cholic acid and normal chow showed that the cholic acid induction was about 50% at the RNA level. We determined the biological nature of the protein encoded by CYP3A10 by expression of the cDNA in COS cells. Microsomes prepared from transfected cells were assayed with LCA as a substrate and found to hydroxylate LCA predominantly at position 6β. We examined whether CYP3A10 could hydroxylate other steroid compounds by assays with testosterone, progesterone and androstenedione and found that, although 6β-hydroxylase (as well as others) activity was observed with all three, LCA was the preferred substrate based on kinetic analysis. A developmental time course of CYP3A10 expression in males showed little expression before puberty, a striking induction of expression at puberty and a fourfold induction thereafter through adulthood. We then examined the male-specific expression of CYP3A10 in hamster liver. We disrupted the pattern of GH secretion in male hamsters by hypophysectomy, neonatal glutamate treatment and by continuous infusion of GH via osmotic minipumps (to mimic the female pattern of GH secretion) and found no significant effect on CYP3A10 expression. Conversely, in females, hypophysectomy and neonatal glutamate treatment significantly induced CYP3A10 expression 5- to 10-fold. Additionally, when females treated neonatally with glutamate were injected twice daily with GH as adults (to mimic the male pattern of GH secretion), the levels of CYP3A10 expression were not significantly different from those of normal males. These results led us to conclude that the pattern of GH secretion in males does not control the male-specific expression of CYP3A10 but that in females expression can be induced by altering the tonic secretion of GH. No significant effect on CYP3A10 expression was observed by castration of adult males, indicating that circulating androgens were not required for expression. We found that gonadal hormones (e.g. estrogen and progesterone) do not have a suppressive effect on CYP3A10 expression in females since ovariectomy did not induce expression. Many genes are "imprinted" neonatally by exposure to a given effector for developmental-, tissue- or sexually regulated expression. We investigated whether neonatal androgen exposure was required for male-specific expression of CYP3A10 by castrating hamsters neonatally and determining the level of CYP3A10 expression in adulthood. Our results indicate that androgens are required neonatally for CYP3A10 expression since no expression was observed in neonatally castrated hamsters. We were unable to induce expression in neonatally castrated hamsters by either GH or testosterone injections. These results suggest several notable points 1) that CYP3A10 expression is programmed neonatally by androgen exposure; 2) that androgens exert their effect directly on the liver and not via the hypothalamus; 3) that neither testosterone nor GH can restore CYP3A10 expression when males have not been exposed to androgens neonatally; and 4) that in experimental conditions, females can be induced to express CYP3A10, which indicates that there are two modes for regulating expression: by "imprinting" in males and by GH and testosterone in females. We are now studying the molecular mechanisms involved in the bile acid-mediated induction and the male-specific expression of CYP3A10. We have cloned approximately 8 kb of 5' flanking DNA from a hamster genomic library and sequenced about 1 kb of proximal DNA. Primer extension and S1 digestion analyses indicate that the mRNA for CYP3A10 has multiple transcription initiation sites clustered about 90 bp from the initiator methionine codon. We have also prepared CYP3A10 promoter/lacZ chimeric constructs to begin delineating the cis-acting elements controlling CYP3A10 expression and regulation. We used H2.35 cells as recipients because they are a mouse hepatocyte cell line that has been transformed with a temperature sensitive SV40. These cells can be grown at the permissive temperature and can be induced to behave like liver cells, the differentiated condition, by switching to a nonpermissive temperature. We have found that the construct with 1 kb of proximal CYP3A10 5' flanking DNA was able to express the reporter gene at higher levels under differentiated conditions, which were consistent with higher expression of an albumin promoter/lacZconstruct, upon switching the cells to the more liver phenotype. The system characterized and described here is ideally suited for dissecting the molecular details governing bile acid-mediated regulation and sexually dimorphic expression of liver genes. Very little is known about both these very important biological phenomena. Much could be learned about transcriptional regulation of liver genes by investigating the cis-elements and trans-acting factors mediating regulation of CYP3A10 expression.

Les Hydrocarbures Aromatiques Polycycliques (HAP) sont responsables de cancers du poumon, de la vessie et de la peau. Parmi eux, seul le benzo(a)pyrène (BaP) est classé cancérogène certain pour l'homme. La surveillance biologique de leur exposition passe par le dosage du 1-hydroxypyrène urinaire (1-OHP), métabolite du pyrène non cancérogène. L'objectif de cette thèse était de développer un nouveau biomarqueur plus proche du risque cancérogène. Le 3-hydroxybenzo(a)pyrène (3-OHBaP) est apparu comme le meilleur candidat car c'est le métabolite majoritaire du BaP, car ses concentrations sont liées aux taux d'adduits à l'ADN mesurés dans le poumon de rat et car son dosage est réalisable dans les urines de sujets exposés. Nous avons développé une méthode analytique de routine associant l'extraction en phase solide à la chromatographie liquide avec une détection en fluorescence, qui nous a permis d'atteindre une limite de détection 0,02 ng/L. La médiane des concentrations de 3-OHBaP dans les urines de sujets fumeurs atteignait 0,02 nmole/mole de créatinine et était deux fois plus élevée que chez les non-fumeurs. Dans les secteurs de production du silicium, des cathodes et des anodes, de 30 à 70% des niveaux étaient supérieurs à la valeur recommandée en France, ce qui confirme le risque sanitaire lié à ces activités professionnelles. Lors d'une exposition répétée aux HAP pendant une semaine de travail, le prélèvement des urines doit être réalisé en fin de semaine fin de poste, du fait de l'atteinte d'un équilibre entre les doses de BaP absorbées et celles de 3-OHBaP éliminées. La corrélation entre les niveaux urinaires de 3-OHBaP et de 1-OHP est variable en fonction du secteur et des activités professionnelles. Ceci démontre la difficulté d'établir une seule valeur limite professionnelle en lien avec le risque cancérogène des HAP pour le 1-OHP et souligne tout l'intérêt de doser le 3-OHBaP. / Polycyclic Aromatic Hydrocarbons (PAH) are responsible for lung, skin and bladder cancers. Among them, only benzo(a)pyrene (BaP) is classified as carcinogenic to humans. Their biomonitoring is performed by the determination of urinary 1-hydroxypyrene (1-OHP), a metabolite of the non-carcinogenic pyrene. The aim of this thesis was to develop a new biomarker closest carcinogenic risk. The 3-hydroxybenzo(a)pyrene (3-OHBaP) seemed to be the best candidate because it is the major metabolite of BaP, these urinary levels are related to the rate of DNA adducts measured in the lung of rats and its analysis is achievable in the urine of exposed subjects. We have developed a simple analytical method combining solid phase extraction and liquid chromatography with fluorescence detection, which achieved a detection limit of 0.02 ng/L. The median concentrations of 3-OHBaP in the urine of smokers reached 0.02 nmol/mol of creatinine and was two times higher than among non-smokers. In the industries of silicon, cathodes and anodes, from 30 to 70 % of levels were above the recommended value in France, which confirms the health risk associated with these occupational activities. In case of repeated exposure to PAH during a workweek, urine sampling should be performed at the end of the shift the last workday, due to a balance between the amounts of BaP absorbed and those of 3-OHBaP eliminated. The correlation between urinary levels of 3-OHBaP and 1-OHP is variable depending on the sector and occupational activities. This demonstrates the difficulty of establishing a single limit value in connection with the carcinogenic risk of PAH for 1-OHP and highlights the interest of the 3-OHBaP.