Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.

Os hidrocarbonetos policíclicos aromáticos (HPAs) são uma classe de substâncias químicas que podem ser geradas na combustão de matéria orgânica, e 17 destas substâncias são consideradas como poluentes prioritários pela Agência de Proteção Ambiental dos EUA (US EPA), em função de seu potencial tóxico e carcinogênico. Para identificar e quantificar os HPAs no ambiente de trabalho por cromatografia gasosa / espectrometria de massas, foram realizadas coletas de amostras ambientais em um incinerador de resíduos de serviços de saúde localizado na região da grande São Paulo e em três fundições de metais nas cidades de Loanda e Santa Isabel do Ivaí, no Estado do Paraná. A concentração de HPAs totais nas amostras ambientais coletadas no Incinerador foi de 0,36 - 1,72 µg/m3; na Fundição 1 de 0,72 - 4,56 µg/m3; na Fundição 2 de 2,32 - 6,52 µg/m3; na Fundição 3 de 0,19 - 3,72 µg/m3. Foi realizada também a coleta de amostras biológicas de trabalhadores expostos e não expostos, para a identificação e quantificação do indicador biológico de exposição o 1-hidroxipireno por cromatografia líquida com detector de fluorescência. A concentração de 1-hidroxipireno nas amostrados biológicas dos trabalhadores no Incinerador apresentou diferença estatisticamente siginificante entre o grupo exposto e o grupo controle; na Fundição 1 houve diferença estatisticamente pouco significante entre os grupos estudados; nas Fundições 2 e 3 não houve diferença estatisticamente significante entre os grupos estudados. / The Polycyclic Aromatics Hydrocarbons (PAH) are a class of chemicals that can to be generated by organic matter burning, and 17 of that chemicals are considered priority pollutant by Environmental Protection Agency (US EPA), because their toxicity and carcinogenicity potentials. To identify and to quantitify the PAH at workplace atmosphere by gas chromatography/mass spectrometry (GC/MS), it was conducted a environmental sampling in a waste incinerator, settled in the metropolitan São Paulo Region, and at three metals foundry settled in the Loanda City and Santa Isabel do Ivaí City, at Paraná State. The concentration of total PAH in the environmental samples collected in a waste incinerator was 0,36 - 1,72 µg/m3; at metals foundry 1 was 0,72 - 4,56 µg/m3; at metals foundry 2 was 2,32 - 6,52 µg/m3; at metals foundry 3 was 0,19 - 3,72 µg/m3. The biological samples of the workers was collected to identify and to quantitify the biological indicator urinary 1-hydroxypyrene (1-OH-Pyr) by high performance liquid chromatography/fluorescence detector (HPLC-Flu). The 1-OH-Pyr concentration in the biological samples of the exposed and control group in a waste incinerator presented a statistical significant difference; in the metals foundry 1 was observed a slight statistical significant difference; and in the metals foundry 2 and metals foundry 3 was not observed statistical significant difference.

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.

O material particulado atmosferico é reconhecido como mutagênico. Embora vários compostos já tenham sido identificados nessa matriz, os mesmos não explicam o efeito observado na maioria dos bioensaios realizados. O ensaio de mutagenicidade Salmonella/microssoma tem sido amplamente utilizado para a avaliação de amostras atmosféricas o qual é muito sensível na detecção de vários tipos de mutágenos, pois é realizado com diferentes linhagens de Salmonella typhimurium na presença e ausência de ativação metabólica exógena. O principal objetivo deste trabalho foi verificar a aplicabilidade da utilização de combinações seletivas de linhagens S. typhimurium na caracterização da mutagenicidade de amostras de material particulado total de ar coletado em Limeira, uma cidade do interior do estado de São Paulo sob influência de intenso tráfego, emissões industriais e da agricultura, incluindo o plantio de cana de açúcar. Foram avaliados seis extratos orgânicos obtidos com diclorometano por ultrasonicação, utilizando o método Salmonella/microssoma em microssuspensão na presença e ausência de ativação metabólica (mistura S9) em experimentos de dose-resposta, com quantidade máxima de 50 µg de material orgânico extraído (MOE) por placa. Os pares de linhagens utilizados foram: YG1041/TA98, para verificar a presença de nitrocompostos, como 1- nitropireno e/ou aminas aromáticas; YG5161/TA1538, altamente sensivel aos hidrocarbonetos policíclicos aromáticos (HPA) não substituídos como o benzo[a]pireno (BaP); e YG7108/TA1535, para a indicação de agentes alquilantes. Os resultados indicaram que os compostos que podem melhor explicar os efeitos observados são os nitro e/ou aminas aromáticas e não os HPA não substituídos como o BaP. O par YG7108/TA1535 forneceu respostas negativas para todas as amostras. As combinações seletivas de linhagens S. typhimurium (YG1041/TA98 e YG5161/TA1538) mostraram-se eficientes na caracterização da mutagenicidade de amostras de ar coletadas na cidade de Limeira, podendo ser utilizadas como uma importante ferramenta de auxílio do ensaio Salmonella/microssoma em microssuspensão em estudos de análises quimicas direcionadas pelo efeito biológico (ADEB). As potências mutagênicas observadas com a linhagem TA98 para a cidade de Limeira foram similares aquelas verificadas na Região Metropolitana de São Paulo. A comparação das respostas de mutagenicidade observadas para as amostras analisadas com o perfil de compostos puros, encontrados na literatura, não forneceu candidatos que podem representar a mistura. Mais estudos de mutagenicidade com compostos puros utilizando os pares de linhagens propostos (TA98/YG1041 e TA1538/YG5161) e análises quimicas de extratos fracionados são necessários para verificar se esta abordagem pode ser útil em estudos de ADEB, e consequentemente na descoberta de novos poluentes atmosféricos. / The atmospheric particulate matter is recognized as mutagenic. Although several compounds have been identified in this matrix, they do not explain the observed effect in most bioassays that have been performed. The Salmonella/microsome assay has been widely used for the evaluation of atmospheric samples, The assay is very sensitive in the detection of various types of mutagens, because it is performed with different strains of Salmonella typhimurium in the presence and absence of exogenous metabolic activation. The main objective of this work was to verify the applicability of selective combination of strains in the characterization of mutagenicity of total airborne particulate matter collected in Limeira, a São Paulo State city in a site influenced by heavy traffic, industrial and agricultural emissions, including sugar cane plantations. We tested six extracts obtained with methylene chloride by ultrasonication using the Salmonella/microsome microsuspension protocol in the presence and absence of rat liver S9 in dose response experiments, using 50 µg of extractable organic matter (EOM) as a maximum dose per plate. The following pairs of strains were used: YG1041/TA98, to verify the presence of nitro-compounds, as 1-nitropyrene and/or aromatic amines; YG5161/TA1538, highly sensitive to non-substituted polycyclic aromatic hydrocarbons (PAHs) as benzo[a]pyrene (BaP); and YG7108/TA1535, for the indication of alkylating agents. The results indicated that the compounds that can better explain the observed effect are the nitro and/or aromatic amines and not nonsubstituted PAHs such as BaP. The pair YG7108/TA1535 provided negative responses for all the samples. Selective combinations of S. typhimurium strains (YG1041/TA98 and YG5161/TA1538) proved efficient in the characterization of mutagenicity of airborne samples collected in the city of Limeira and can be used as an important tool for the Salmonella/microsome microsuspension assay in effect directed chemical analysis (EDA) studies. The mutagenic potencies observed with TA98 strain for the city of Limeira were similar to those verified in the Metropolitan Region of São Paulo. The comparison of the observed mutagenicity responses for the samples analyzed with the profiles of pure compounds found in the literature did not provide any candidates that could alone represent the mixture. More mutagenicity studies with pure compounds using the proposed pair of strains (TA98/YG1041 and TA1538/YG5161) along with chemical analysis of fractioned extracts are needed to verify if this approach can be useful in EDA studies, and consequently in the discovery of new air priority pollutants.

<p>In this work, a simple, less time consuming electrochemical method in the form of an electrochemical sensor has been developed for the detection of PAHs. The sensor was fabricated by the deposition of silver-gold (1:3) alloy nanoparticles (Ag-AuNPs) on ultrathin overoxidized polypyrrole (PPyox) film which formed a PPyox/Ag-AuNPs composite on glassy carbon electrode (PPyox/Ag-AuNPs/GCE). The silver-gold alloy nanoparticles deposited to form the composite were chemically prepared by simultaneous reduction of silver nitrate (AgNO3) and chloroauric acid (HAuCl4) using sodium citrate and characterized by UV-visible spectroscopy technique which confirmed the homogeneous formation of the alloy nanoparticles.</p>

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system. The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence. Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA &gt; dG &gt; dT &gt; dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

Introduction: Prenatal exposure to polycyclic aromatic hydrocarbons (PAH) and to acrylamide has been suggested to reduce fetal growth. The role of diet, the main source of exposure to these compounds in the general population, remains uncertain. This thesis aimed to characterize women’s exposure during pregnancy to dietary acrylamide and the genotoxic PAH benzo(a)pyrene [B(a)P], and to assess the effects of prenatal exposure to these compounds on fetal growth indicators. Methods: This thesis was done within two large European population-based cohort studies conducted in Spain and Norway: the INfancia y Medio Ambiente (INMA; n=657) and The Norwegian Mother and Child Cohort (MoBa; n=50651). Dietary B(a)P and acrylamide intakes were estimated based on information from food frequency questionnaires and the concentrations of these compounds in foods. Results: (i) Smokers had higher dietary intakes of B(a)P and acrylamide compared to non-smokers; (ii) the main predictors of B(a)P intake were shellfish and processed/cured meats while the main predictors of acrylamide intake were snacks, fried potatoes, and crisp bread; (iii) higher prenatal exposure to dietary B(a)P and acrylamide may reduce birth weight and increase the risk of small for gestational age, independently of smoking-related exposure and (iv) stronger effects of dietary B(a)P on fetal growth were observed among women with low vitamin C intake. Conclusions: Dietary B(a)P and acrylamide intakes during pregnancy may impair fetal growth. Therefore, reducing the intake of these compounds from the diet should be recommended in dietary guidelines for pregnant women. Likewise, increasing intakes of fruits and vegetables rich in vitamin C should be recommended given its potential to prevent adverse effects from exposure to such contaminants. / Introducción: La exposición prenatal a los hidrocarburos aromáticos policíclicos (HAP) y a la acrilamida ha sido asociada con la reducción del crecimiento fetal. El papel de la dieta, la principal fuente de exposición a estos compuestos en la población general, sigue siendo incierto. Los objetivos de esta tesis son caracterizar la exposición a través de la dieta a la acrilamida y a los HAP, específicamente el compuesto genotóxico benzo(a)pireno [B(a)P], durante el embarazo, y evaluar los efectos de la exposición prenatal a estos compuestos sobre indicadores del crecimiento fetal. Métodos: Esta tesis se realizó dentro del marco de dos grandes estudios europeos de cohortes de base poblacional realizados en España y Noruega: INfancia y Medio Ambiente (INMA; n=657) y The Norwegian Mother and Child Cohort (MoBa; n=50651). La ingesta de B(a)P y acrilamida fue estimada a partir de información de cuestionarios de frecuencia alimentaria y las concentraciones de estos compuestos en los alimentos. Resultados: (i) Las fumadoras tuvieron mayor ingesta de B(a)P y acrilamida a través de la dieta en comparación con las no fumadoras; (ii) los principales predictores de la ingesta de B(a)P fueron los mariscos y los embutidos, mientras que los principales predictores de la ingesta de acrilamida fueron los aperitivos, las patatas fritas y el pan crujiente; (iii) la exposición prenatal al B(a)P y la acrilamida pueden reducir el peso al nacer y aumentar el riesgo de pequeño para la edad gestacional, independientemente de la exposición relacionada con el tabaco y (iv) los efectos de la ingesta de B(a)P a través de la dieta sobre el crecimiento fetal fueron más fuertes entre las mujeres con baja ingesta de vitamina C. Conclusiones: La ingesta de B(a)P y acrilamida a través de la dieta durante el embarazo, puede perjudicar el crecimiento fetal. Consecuentemente, recomendaciones para la reducción de la ingesta de estos compuestos a través de la dieta deberían ser incluidas en las guías dietéticas para mujeres embarazadas. Asimismo, debería recomendarse un aumento en la ingesta de frutas y verduras con alto contenido en vitamina C por su potencial para prevenir efectos relacionados con la exposición a estos contaminantes.

A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program.

<p>In this work, a simple, less time consuming electrochemical method in the form of an electrochemical sensor has been developed for the detection of PAHs. The sensor was fabricated by the deposition of silver-gold (1:3) alloy nanoparticles (Ag-AuNPs) on ultrathin overoxidized polypyrrole (PPyox) film which formed a PPyox/Ag-AuNPs composite on glassy carbon electrode (PPyox/Ag-AuNPs/GCE). The silver-gold alloy nanoparticles deposited to form the composite were chemically prepared by simultaneous reduction of silver nitrate (AgNO3) and chloroauric acid (HAuCl4) using sodium citrate and characterized by UV-visible spectroscopy technique which confirmed the homogeneous formation of the alloy nanoparticles.</p>

Le benzo[a]pyrène (BaP) est un contaminant environnemental de la famille des hydrocarbures aromatiques polycycliques ayant été classé cancérogène chez l’humain. Cependant, la relation entre l’exposition et les effets est toujours mal documentée. L’objectif de cette thèse était de mieux documenter la relation quantitative entre l’exposition au BaP, l’évolution temporelle des biomarqueurs d’exposition et l’apparition d’altérations biologiques précoces, à partir d’études expérimentales chez le rat. Dans un premier temps, nous avons déterminé l’effet de 4 doses de BaP (0.4, 4, 10 et 40 µmol/kg) sur plusieurs biomarqueurs d’exposition (3- et 7-OHBaP, 4,5- et 7,8-diolBaP, BaPtétrol et 1,6-, 3,6- et 7,8-diones-BaP), les adduits à l’ADN et l’expression de gènes impliqués dans le métabolisme du BaP, la réparation de l’ADN et le stress oxydatif. Le BaP et ses métabolites ont été mesurés dans le sang, les tissus et les excrétas, 8 h et 24 h après l’administration intraveineuse de BaP par chromatographie liquide à ultra haute performance (UHPLC) couplée à la fluorescence. Les adduits à l’ADN ont été quantifiés dans les poumons par immuno-essai en chémoluminescence. L’expression des gènes dans les poumons a été réalisée par PCR quantitative en temps réel (qRT-PCR). Les résultats ont révélé une bonne relation dose-excrétion pour le 3-OHBaP, le 4,5-diolBaP et le 7,8-diolBaP et ils ont également renforcé l’utilité du 4,5-diolBaP comme potentiel biomarqueur du BaP en plus du 3-OHBaP. De plus, l’augmentation dose-dépendante de la formation des adduits et de l’expression de certains gènes impliqués dans le métabolisme et le stress oxydatif a suggéré l’intérêt de ces derniers comme biomarqueurs d’effet précoce. Dans un second temps, nous avons évaluer le profil cinétique des biomarqueurs en lien avec la modulation temporelle de la formation des adduits à l’ADN et de l’expression génique, en utilisant la dose de 40 µmol/kg de BaP telle qu’établie dans l’étude précédente, avec une série de mesures sur une durée de 72 h après l’injection intraveineuse de BaP. Il est apparu que le 3- et le 7-OHBaP ainsi que le 4,5- et le 7,8-diolBaP semblaient être de bons biomarqueurs d'exposition; les hydroxyBaP et diolBaP présentaient des cinétiques différentes, mais tous ces métabolites étaient corrélés de façon significative aux adduits BaPDE dans les poumons. L’expression de certains gènes et l’étude de leur profil cinétique a également permis de faire des liens avec la formation des adduits et de mieux comprendre le métabolisme du BaP. Après ces résultats, il semblait alors logique de s’intéresser à l’effet de la voie d’exposition dans un context d’exposition multiple de la population. Les données mesurées dans le sang et les excréta, après administration de 40 µmol/kg de BaP par voie intraveineuse, intratrachéale, orale, et cutanée, ont encore une fois montré l'intérêt de mesurer plusieurs métabolites pour l’évaluation de l’exposition en raison des différences en fonction de la voie d’administration du composé et des différences dans la cinétique de plusieurs biomarqueurs, notamment entre les hydroxy (3- et 7-OHBaP) et les diols-BaP (4,5- et 7,8-diolBaP). Les résultats suggèrent aussi que la mesure de ratios de concentrations de différents métabolites pourrait aider à indiquer le moment et la principale voie d’exposition. Ces données ont permis une meilleure compréhension du continuum entre l’exposition et les effets. / Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that has been classified as carcinogenic to humans. However, the relationship between exposure and effect is still poorly documented. The aim of this thesis was to document the quantitative relationship between exposure to BaP, the temporal evolution of biomarkers of exposure and the appearance of early biological alterations, by conducting experimental studies in rats. First, we determined the effect of four doses of BaP (0.4, 4, 10 and 40 µmol / kg) on several biomarkers of exposure (3- and 7-OHBaP, 4,5- and 7,8 -diolBaP, BaPtetrol and 1,6 -, 3,6 - and 7,8-dione-BaP), on DNA adducts and the expression of genes involved in the metabolism of BaP, DNA repair and oxidative stress. BaP and its metabolites were measured in blood, tissues and excreta, 8 h and 24 h after intravenous administration of BaP by ultra high performance liquid chromatography (UHPLC) coupled to fluorescence. The DNA adducts were quantified in lungs by chemiluminescence immunoassay. Gene expression in lungs was achieved by quantitative real time PCR (qRT-PCR). The results showed a good dose-excretion relationship for 3- OHBaP , 4,5- and 7,8- diolBaP and they also showed the usefulness of 4,5- diolBaP as a potential biomarker of BaP in addition to 3- OHBaP. Furthermore, the dose-dependent increase in the formation of adducts and the expression of certain genes involved in metabolism and oxidative stress highlighted the latter as potentially interesting biomarkers of effect. The following study was designed to evaluate the toxicokinetic profile of biomarkers of exposure related to the temporal modulation of the formation of DNA adducts and gene expression, using a dose of 40 µmol/kg BaP as set out in the previous study, but with regular measurements during the 72-h period following intravenous injection of BaP. It appeared that the 3 - and 7- OHBaP and 4,5 - and 7,8- diolBaP seemed to be good biomarkers of exposure; hydroxyBaPs and diolBaPs exhibited different kinetics but all the metabolites were significantly correlated with BaPDE adducts in the lungs. The expression of genes and the study of their kinetic profile also allowed to assess the relationship with the formation of adducts and better understand the metabolism of BaP. Based on these results, it seemed logical to focus on the effect of the route of exposure. The data measured in the blood and excreta after intravenous, intratracheal, oral, and cutaneous administration of 40 mmol/kg BaP have once again demonstrated the importance of measuring several metabolites due to kinetic differences depending on the route of administration of the compound and among biomarkers, in particular between OHBaPs (3 - and 7 - OHBaP) and diolBaPs (4,5 - and 7,8- diolBaP). Results also suggest that measurements of concentration ratios of different metabolites could help indicate the time and the main route of exposure. Overall, these data allowed a better understanding of the continuum between BaP exposure and early effects.