Teses / dissertações sobre o tema "CYP2E1"

Orientador: Janete Dias Almeida
Coorientador: Celina Faig Lima Carta
Banca: Emília Ângela Lo Schiavo Arisawa
Banca: Ana Lia Anbinder
Banca: Alberto José de Araújo
Banca: José Benedito Oliveira Amorim
Resumo: Os carcinógenos do tabaco estão relacionados a diversos tipos de câncer incluindo o carcinoma de células escamosas (CCE) bucal. Aliado ao álcool, o tabaco contribui para o desfecho desfavorável destes casos. A susceptibilidade individual ao câncer pode estar relacionada a expressão das enzimas que metabolizam tais carcinógenos. O objetivo deste trabalho é avaliar a expressão dos genes CYP1A1, CYP1B1, CYP2A6 e CYP2E1 no CCE bucal por meio de qPCR. Foram coletadas amostras de 32 indivíduos com CCE e de 15 controles submetidos a cirurgias bucais por lesões benignas. Foram constituídos quatro grupos: Grupo CCE fumante (n=26), Grupo CCE não fumante (n=6), Grupo controle fumante (n=9) e Grupo controle não fumante (n=6). O Teste de Fagerström para Dependência a Cigarros (TFDC) foi usado para avaliar a dependência nicotínica (DN) e AUDIT para avaliação do consumo de etílicos. Houve diminuição da expressão do gene CYP1B1 nos casos de CCE comparados aos controles. Foram encontradas diferenças estaticamente significativas de expressão gênica de CYP1B1 entre os Grupos CCE fumante e controle fumante (p=0,0018), Grupo CCE não fumante e controle não fumante (p=0,0079) e CCE fumante com CCE não fumante (p=0,0385) e entre os quatro grupos (p<0,0001). Houve diminuição da expressão do CYP2A6 no Grupo CCE fumante em relação ao Grupo controle, mas apenas um paciente do Grupo controle expressou este gene. Houve aumento da expressão de CYP2E1 entre os Grupos CCE fumante e controle fumante (p=0,0424... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Tobacco carcinogens are related to various types of cancer, including oral squamous cell carcinoma (OSCC). Allied to alcohol, tobacco contributes to the unfavorable outcome of the cases. Individual cancer susceptibility may be related to an expression of the enzymes that metabolize such carcinogens. The aim of this work is to evaluate the expression of the genes CYP1A1, CYP1B1, CYP2A6 and CYP2E1 on OSCC by qPCR. Samples were collected from 32 individuals with OSCC and 15 controls submitted to oral surgeries due to benign lesions. There were four groups: Smoker SCC group (n = 26), nonsmoker SCC group (n = 6), Smoker control group (n = 9) and nonsmoker control group (n = 6). The Fagerström Test for Cigarette Dependence (TFCD) was used to evaluate nicotinic dependence (ND) and AUDIT for the evaluation of alcohol consumption. There was a decrease in CYP1B1 gene expression in cases of SCC compared to controls. (P = 0.0018); smoker CCE and non-smoker control (p = 0.0079); smoker SCC with nonsmoker SCC (p = 0.0385) and between the four groups (p <0.0001). There was a decreased expression in CYP2A6 in the smoker SCC Group compared to the control group, but only one control group patient expressed this gene. There was an increased expression of CYP2E1 between the smoking and nonsmoking SCC groups (p = 0.0424). In conclusion, large interindividual variability was found in the study of the expression of the genes studied. There was greater expression of CYP1A1 and CYP2E1 in samples from... (Complete abstract click electronic access below)
Doutor

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Os carcinógenos do tabaco estão relacionados a diversos tipos de câncer incluindo o carcinoma de células escamosas (CCE) bucal. Aliado ao álcool, o tabaco contribui para o desfecho desfavorável destes casos. A susceptibilidade individual ao câncer pode estar relacionada a expressão das enzimas que metabolizam tais carcinógenos. O objetivo deste trabalho é avaliar a expressão dos genes CYP1A1, CYP1B1, CYP2A6 e CYP2E1 no CCE bucal por meio de qPCR. Foram coletadas amostras de 32 indivíduos com CCE e de 15 controles submetidos a cirurgias bucais por lesões benignas. Foram constituídos quatro grupos: Grupo CCE fumante (n=26), Grupo CCE não fumante (n=6), Grupo controle fumante (n=9) e Grupo controle não fumante (n=6). O Teste de Fagerström para Dependência a Cigarros (TFDC) foi usado para avaliar a dependência nicotínica (DN) e AUDIT para avaliação do consumo de etílicos. Houve diminuição da expressão do gene CYP1B1 nos casos de CCE comparados aos controles. Foram encontradas diferenças estaticamente significativas de expressão gênica de CYP1B1 entre os Grupos CCE fumante e controle fumante (p=0,0018), Grupo CCE não fumante e controle não fumante (p=0,0079) e CCE fumante com CCE não fumante (p=0,0385) e entre os quatro grupos (p<0,0001). Houve diminuição da expressão do CYP2A6 no Grupo CCE fumante em relação ao Grupo controle, mas apenas um paciente do Grupo controle expressou este gene. Houve aumento da expressão de CYP2E1 entre os Grupos CCE fumante e controle fumante (p=0,0424). Concluindo, foi encontrada grande variabilidade interindividual no estudo da expressão dos genes estudados. Houve maior expressão de CYP1A1 e CYP2E1 em amostras de indivíduos fumantes com CCE. Os genes CYP1B1 e CYP2A6 estavam menos expressos no Grupo CCE fumante em relação ao Grupo controle. Para os genes CYP1B1 e CYP2E1 foram encontrados valores significativos na correlação entre a expressão gênica e parâmetros demográficos e de perfil tabágico no Grupo controle fumante, e do AUDIT no Grupo CCE não fumante. O gene CYP2E1, além de estar relacionado ao metabolismo do álcool, também deve ser considerado importante marcador do metabolismo dos carcinógenos derivados do tabaco.
Tobacco carcinogens are related to various types of cancer, including oral squamous cell carcinoma (OSCC). Allied to alcohol, tobacco contributes to the unfavorable outcome of the cases. Individual cancer susceptibility may be related to an expression of the enzymes that metabolize such carcinogens. The aim of this work is to evaluate the expression of the genes CYP1A1, CYP1B1, CYP2A6 and CYP2E1 on OSCC by qPCR. Samples were collected from 32 individuals with OSCC and 15 controls submitted to oral surgeries due to benign lesions. There were four groups: Smoker SCC group (n = 26), nonsmoker SCC group (n = 6), Smoker control group (n = 9) and nonsmoker control group (n = 6). The Fagerström Test for Cigarette Dependence (TFCD) was used to evaluate nicotinic dependence (ND) and AUDIT for the evaluation of alcohol consumption. There was a decrease in CYP1B1 gene expression in cases of SCC compared to controls. (P = 0.0018); smoker CCE and non-smoker control (p = 0.0079); smoker SCC with nonsmoker SCC (p = 0.0385) and between the four groups (p <0.0001). There was a decreased expression in CYP2A6 in the smoker SCC Group compared to the control group, but only one control group patient expressed this gene. There was an increased expression of CYP2E1 between the smoking and nonsmoking SCC groups (p = 0.0424). In conclusion, large interindividual variability was found in the study of the expression of the genes studied. There was greater expression of CYP1A1 and CYP2E1 in samples from smokers with SCC. The CYP1B1 and CYP2A6 genes were less expressed in the smoker SCC Group. Significant values were found for the CYP1B1 and CYP2E1 genes in the correlation between a gene expression and a parameter and a non-smoker control group, non-smoker control group and AUDIT. The CYP2E1 gene, besides being related to alcohol metabolism, should also be considered an important marker of the metabolism of the carcinogens derived from tobacco.
2016/08633-0

Les objectifs de ce travail ont ete l'etude de l'ontogenese du cyp2e1 hepatique humain et la mise en evidence des mecanismes moleculaire impliques dans le controle de l'expression du cyp2e1 au cours du developpement. Le cyp2e1 presente une expression uniquement postnatale : la proteine et les activites enzymatiques correspondantes apparaissent au cours des heures qui suivent la naissance, et augmentent graduellement jusqu'a l'age adulte. Le taux d'arnm cyp2e1 reste faible au cours de la periode perinatale, bien que la concentration de la proteine cyp2e1 augmente immediatement apres la naissance. Ceci suggere l'intervention d'un mecanisme post-transcriptionnel de regulation, probablement par stabilisation de la proteine par les corps cetoniques. A partir de l'age d'un mois, l'augmentation de l'arnm cyp2e1 est parallele a celle du contenu en cyp2e1 microsomal et permet de supposer que le controle de l'expression du cyp2e1 a lieu au niveau transcriptionnel. Les profils de methylation des sites hpall/mspl dans la partie 5', l'exon 1 et l'intron 1 du gene cyp2e1 ont ete compares. Des modifications de l'etat de methylation ont ete observees au niveau d'un site situe a 34 pb en aval de la boite tata et sites localises a l'interieur de l'intron 1. Ces sites sont methyles dans le foie de foetus et de nouveau-nes exprimant des taux faibles d'arnm cyp2e1. Leur demethylation est correlee a l'expression de l'arnm cyp2e1 chez les nouveau-nes et les adultes. Dans le poumon et le rein de foetus, de nouveau-ne et d'adulte, qui expriment des taux faibles d'arnm cyp2e1, les sites hpall/mspl sont egalement methyles, tandis qu'une demethylation partielle existe dans le placenta, exprimant des taux variables de transcrit. En conclusion, deux mecanismes interviennent dans le controle de l'expression du cyp2e1 : la stabilisation de la proteine au cours des jours qui suivent la naissance et l'activation transcriptionnelle du cyp2e1 associee a la demethylation des residus cpg.

Polychlorinated biphenyls (PCBs) continue to pose a health concern because of their predominance in the diet and air as well as in environmental samples and humans. PCB congeners with 3 or 4 chlorine substituents in ortho position have been associated with neurodevelopmental disorders. Hydroxylated metabolites (OH-PCBs) of these PCBs are also potentially toxic to the developing brain. Metabolism studies have mainly focused on animal models. However, preliminary data from this dissertation work have revealed PCB metabolism differences between laboratory animal models and humans in terms of metabolite profiles, chiral signatures. More concerning, biotransformation of chiral PCBs is poorly investigated in humans. The objective of this dissertation research was to study the biotransformation of chiral and prochiral PCBs to chiral hydroxylated metabolites in humans and rats and to identify individual human P450 enzymes involved in the metabolism of these PCBs. I chose chiral PCB congeners 2,2',3,4',6-pentachlorobiphenyl (PCB 91); 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) and 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) for this investigation because they are environmentally relevant and their metabolism has been studied in rodents and other laboratory animal species (Kania-Korwel et al., 2016a). Prochiral PCB congeners 2,2′,4,6′-tetrachlorobiphenyl (PCB 51) and 2,2′,4,5,6′-pentachlorobiphenyl (PCB 102) were selected because their considerable presence in technical PCB mixtures. To test the hypothesis that P450 enzyme and species differences mediate the congener-specific enantioselective metabolism of chiral PCBs to hydroxylated metabolites, I sought to establish structure-metabolism relationships by studying the enantioselective metabolism of structurally diverse chiral PCBs by human liver microsomes (HLMs). Racemic PCB 91, PCB 95 and PCB 132 were incubated in vitro with pooled or individual donor HLMs at 37 °C, and levels and chiral signatures of the parent PCB and its hydroxylated metabolites were determined by high-resolution gas chromatography equipped with time-of-flight mass spectrometry (GC/TOF-MS) or electron capture detection (GC-ECD). Hydroxylated metabolites formed were identified and metabolic schemes for these PCBs proposed. I found inter-individual differences in the formation of OH-PCBs by individual donor HLMs. Comparison of the metabolite profiles of PCB 91, PCB 95, PCB 132 and PCB 136 (PCB 136 metabolism by HLMs was investigated by other researchers) revealed congener-specific differences in the oxidation of PCBs by human cytochrome P450 enzymes. PCB 91 and PCB 132 were mainly hydroxylated in meta position, with the 1,2-shift metabolites being the major metabolites formed from both PCB congeners by HLMs. In contrast, PCB 95 and PCB 136 were primarily hydroxylated in the para position. Moreover, we determined human P450 isoforms involved in the metabolism of neurotoxic PCBs using in silico and in vitro approaches. In silico predictions suggested that chiral PCBs are metabolized by CYP1A2, CYP2A6, CYP2B6, CYP2E1, and CYP3A4. Experimentally we found that CYP2A6, CYP2B6 and to a minor extent CYP2E1 were the enzymes involved in the metabolism of these chiral PCBS. We also investigated nonchiral sources of chiral OH-PCBs by studying the P450- and species-dependent biotransformation of prochiral PCB 51 and PCB 102 to chiral OH-PCB metabolites. Prochiral PCB 51 and PCB 102 were incubated with liver microsomes prepared from male Sprague-Dawley rats pretreated with various inducers of P450 enzymes including phenobarbital (PB), dexamethasone (DEX), isoniazid (INH), β-naphthoflavone (BNF), clofibric acid (CFA) or corn oil (CO); and untreated male cynomolgus monkeys, Hartley albino guinea pigs, New Zealand rabbits, golden Syrian hamsters; and untreated female Beagle dogs. PCB 51 and PCB 102 were metabolized to 2,2',4,6'-tetrachlorobiphenyl-3'-ol (OH-PCB 51) and 2,2',4,5,6'-pentachlorobiphenyl-3'-ol (OH-PCB 102), respectively. The formation of both metabolites was P450 isoforms- and species-dependent. Moreover, OH-PCB 51 and OH-PCB 102 were chiral and were formed enantioselectively in all microsomes investigated. Taken together, my findings demonstrate (1) considerable inter-individual variability in the congener-specific metabolism of PCBs to OH-PCBs; (2) the enantioselective formation of OH-PCBs by human CYP2A6, CYP2B6, and CYP2E1; and (3) that chiral PCB metabolites are formed enantioselectively from prochiral PCB congeners. Interestingly, the metabolism of PCBs by CYP2A6 appears to involve arene oxide intermediates, as suggested by the formation of 1,2-shift products as major metabolites of PCB 91 and PCB 132. In contrast, 1,2-shift products are minor PCB metabolites formed in rodents. Therefore extrapolation of hepatic metabolism across species may not be consistent and these differences should be considered in future toxicity and risk assessment studies.

Class of 2007 Abstract
Objectives: To characterize the difference in hepatic gene expression between Cyp2e1 +/+ and Cyp2e1 -/- mice after exposure to hydrazine in order to elucidate the functional pathway(s) for hydrazine-induced steatosis. Methods: The project was designed by Dr. Charlene McQueen and consisted of the following aims: (1) to characterize the hepatic pathology induced by hydrazine in CYP2E1 +/+ and -/- mice, (2) to evaluate hepatic gene expression profiles following exposure to hydrazine, and (3) to determine the expression of CYP2E1 and CYP4A14. The animal exposure and data collection have been completed and aim #2 is awaiting data analysis. Aim #2 consisted of treating CYP2E1 +/+ and CYP2E1 -/- mice to saline and hydrazine at doses of 100 mg/kg. Livers were collected at six and 24 hours and the mRNA was isolated with an Absolutely RNA RT-PCR Miniprep Kit. The transcriptome was determined using the Affymetrix GeneChip Expression Analysis System using total mouse genome GeneChips. The GeneChips were scanned using an Agilent GeneArray Scanner and the image was quantitated and archived awaiting data analysis. The data was collected by the SWEHSC Microarray Facility on June 20, 2005 was analyzed. The data analysis was completed by both Kristjon Lindgren and Dana Seng with the help and training from Dr. George Watts. The six sets of data from aim #2 was analyzed using Agilent's GeneSpring 7.3.1 software to characterize the two-fold differences in mice (n = 2 per group) hepatic gene expression. Genes of interest were identified as containing the keywords cyp, fatty, glutathione, hepat, lipid, liver, oxid, perox, steroid, and phosphatidylinositol in the Gene Ontology Biological Process, Cellular Component, or Molecular Function descriptions. Lastly, pathway mining of/for genes of interest was performed using Bioresource for array of genes (BioRag) available at www.biorag.org and maintained by the AzCC/SWEHSC Bioinformatics Facility. Results: The amount of information extracted from this research project is too immense to be described or summarized on this form. For more information, please obtain a copy of this research project from the University of Arizona College of Pharmacy or from the project co-authors Kristjon Lindgren (kristjon.lindgren@gmail.com) or Dana Seng (dana.seng@gmail.com). Conclusions: The effects of Cyp2e1 on hepatic gene expression in 129/Sv-Cyp2e1tm1Gonz/J and 129S1/SvImJ mice exposed to hydrazine was analyzed. Data showing that Cyp2e1 was protective against HD-induced hepatotoxicity was consistent with the proposed hypothesis. Hepatic gene expression results show that Cyp2e1 -/- mice have decreased expression of microsomal ω-oxidation genes (Cyp4a10 and Cyp4a14) compared to Cyp2e1 +/+ at 6h (both increased at 24h) and peroxisomal β–oxidation genes (Ehhadh) at 6h like Cyp2e1 +/+ (but increased at 24h only in Cyp2e1 -/-). Conversely, an increased expression of mitochondrial β-oxidation genes (Cpt1a) in both genotypes at 6 and 24h and cholesterol synthesis genes (Fdft1, Hmgcr, Hmgcs1, Idi, Lss, Mvk, Nsdhl, Sc4mol, and Sqle) in Cyp2e1 -/- at 24h was observed. These results support mechanisms by which ω-oxidation or PPARγ is protective or peroxisomal β- oxidation is damaging. Additional studies are needed to further eludidate the mechanisms of HD-induced steatosis.

Cytochrome P4502E1 (CYP2E1), the ethanol-inducible isoform of cytochrome P450 superfamily, catalyzes many low molecular weight endogenous and exogenous compounds, including ethanol, acetone, drugs like acetaminophen and chlorzoxazone, and industrial solvents like benzene and styrene, most of which are carcinogenic. Besides, it has a high capacity to produce reactive oxygen species. CYP2E1 is induced by ethanol and isoniazid, as well by some pathophysiological conditions like diabetes and starvation. CYP2E1 gene shows genetic polymorphisms which are thought to play a major role in interindividual variability in drug response and in susceptibility to chemical-induced diseases, like several types of cancers. It is well established that CYP2E1 polymorphisms vary markedly in frequency among different ethnic and racial groups. Therefore, in this study, the frequency of two important CYP2E1 polymorphisms
the single nucleotide polymorphisms C-1019T / G-1259C in 5&rsquo
-flanking region and T7678A poymorphism in intron 6, in Turkish population was investigated. For this purpose, whole blood samples were collected from 132 healthy volunteers representing Turkish population and genomic DNA for each subject was isolated in intact form. The genotypes were determined by PCR amplification of corresponding regions followed by restriction endonuclease RsaI, PstI (for C-1019T / G-1259C SNPs) and DraI (for T7678A SNP) digestions. The genotype frequencies, for C-1019T / G-1259C SNPs, which are in complete linkage disequilibrium, were investigated on 116 DNA samples, and determined as 97.4% for homozygous wild type (c1/c1), 2.6% for heterozygotes (c1/c2) and 0.0% for homozygous mutants (c2c2). The allele frequency of wild type allele (c1) was calculated as 98.7% and that of mutated allele (c2) as 1.3%. The genotype frequencies for T7678A SNP, investigated in 108 DNA samples were determined as 80.6% for homozygous wild type (DD), 19.4% for heterozygotes (CD) and 0.0% for homozygous mutants (CC). The corresponding allele frequencies were 90.3% for wild type allele (D), and 9.7% for mutated allele (C). Genotype frequencies of both polymorphisms fit Hardy-Weinberg equation and showed no significant difference with respect to gender. The genotype distributions of both polymorphisms showed similarity when compared to other Caucasian populations like French, Swedish, German, and Italian populations, while both polymorphisms studied differed significantly from Chilean, Japanese, Taiwanese and Chinese populations, as compared with Chi-Square test.

The relevant literature concerning nonalcoholic steatohepatitis (NASH), the morphologically similar condition of alcoholic liver disease (ALD), and the hepatic cytochromes P450 (P450 or CYP) was reviewed with particular emphasis on CYP2E1. CYP2E1 is induced in ALD. It plays an important role in the pathogenesis of this condition by generating reactive oxygen species (ROS). In turn, ROS produce lipid peroxidation, which contributes to the cellular injury in ALD. CYP2E1 is constitutively expressed in acinar zone 3. The early lesions observed in both ALD and NASH are most pronounced in the same acinar region of the liver. Since NASH and ALD have similar histological appearances, the possibility that CYP2E1 may play a significant role in the pathogenesis of NASH was considered. One of the limitations in evaluating the pathogenesis of NASH has been the absence of an appropriate animal model.

L'exposition humaine aux contaminants environnementaux est inévitable du fait de leur présence dans l'eau, l'air et l'alimentation. La plupart d'entre eux sont reconnus comme étant mutagènes et/ou carcinogènes chez l'animal mais ils sont souvent seulement suspectés de l'être chez l'Homme. Le manque de connaissance vis-à-vis des substances chimiques a conduit l'UE à lancer le programme REACH avec l'objectif d'évaluer la toxicité d'environ 30 000 molécules. Cette évaluation nécessiterait l'utilisation de plus de 4 millions d'animaux et la pertinence controversée de ces modèles pourrait aboutir à des conclusions discutables. Les méthodes in vitro sont considérées comme une alternative potentielle à l'expérimentation animale. Néanmoins, le choix du modèle cellulaire et des conditions expérimentales restent à préciser. Les hépatocytes humains en culture primaire représentent le modèle le plus pertinent en toxicologie malgré de nombreuses contraintes (variabilité inter-individuelle, changements phénotypique précoces, obtention aléatoire). La lignée HepaRG constitue une alternative intéressante puisque ces cellules peuvent proliférer de manière illimitée et expriment les EMXs à des niveaux proches des hépatocytes humains. L'expression de ces enzymes restant stable pendant plusieurs semaines, ce modèle permet l'évaluation du risque lié à une exposition chronique aux contaminents environnementaux, essentielle en génotoxicité. Il reste cependant nécessaire de caractériser plus amplement cette lignée vis-à-vis des EMXs et de l'adapter aux tests de toxicologie actuels. Dans ces travaux, nous avons développé un test haut débit utilisant la quantification in situ des histones phosphorylées γH2AX avec l'objectif de pouvoir évaluer le risque génotoxique d'une exposition unique ou répétée aux contaminants environnementaux. Ce test a été validé avec succès par l'évaluation de la génotoxicité associée à une exposition de 1, 7 et 14 jours pour 10 polluants. Nous avons ensuite généré des lignées recombinantes biosenseurs, dérivées du modèle HepaRG et permettant d'identifier les xénobiotiques altérant l'expression transcriptionnelle des EMXs. Par transfection transitoire, nous avons dans un premier temps validé à l'aide d'inducteurs prototypiques et de nos 10 contaminants nos constructions contenant le gène rapporteur de la luciférase sous le contrôle des promoteurs de plusieurs EMXs. Ensuite, nous avons généré des lignées stables exprimant la GFP comme gène rapporteur et permettant une détection rapide des xénobiotiques capables d'induire l'expression des EMXs. Parmi les EMXs, le CYP2E1 joue un rôle important en santé humaine. En effet, cette enzyme induite dans certaines conditions physiopathologiques comme le diabète et l'obésité est responsable de l'activation de nombreux procarcinogènes et est à l'origine d'une production d'EROs. Les cellules HepaRG pourraient constituer un modèle pertinent pour l'étude du CYP2E1. Cependant, l'expression et l'activité de cette enzyme au sein de ce modèle nécessitent d'être mieux caractérisées en regard des données discordantes de la littérature. A l'aide de la chlorzoxazone, un marqueur spécifique de l'activité du CYP2E1, nous avons démontré l'influence du métabolisme de phase II sur l'activité apparente du CYP2E1. Nous proposons ici quelques recommandations afin de mieux quantifier l'activité du CYP2E1 sur les hépatocytes humains et sur le modèle HepaRG à l'aide la chlorzoxazone
Human exposure to toxic chemicals is virtually unavoidable due to contamination of air, water and food. A number of environmental contaminants are recognized as mutagenic and/or carcinogenic in animal but they are often only suspected to have similar effects in Humans. The lack of knowledge on the effects of most industrial-made chemicals has led the EU to launch the REACH program with the aim of evaluating the toxicity of more than 30.000 molecules. Such evaluation would require the use of at least 4 millions of animals for an estimated cost of 2.8 billions €. While the relevance of these in vivo models remains controversial

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The Cytochrome P4502E1 (CYP2E1) gene encodes an enzyme which is the main constituent of microsomal ethanol oxidizing system. Genetic variation present in the CYP2E1 gene result in significant differences in enzyme activity, however the functional significance of these variations in the metabolism of ethanol remains controversial, since there is not yet a clear correlation with differences in catalytic activity of the enzyme. In this study, the variability of the promoter region of the CYP2E1 gene was analyzed in samples of alcoholics of a Psychosocial Attention Center (CAPS) in the state of Goiás, in the city of Goiânia and the results were compared with the data obtained by the 1000 Genomes project. We conducted a study of association between polymorphisms of the promoter region of the CYP2E1 and genotoxic damage in alcoholics, for this sequencing reaction and the comet assay were performed. In our analysis there was no correlation of data polymorphisms found with genotoxic damage. However, genotoxicity analysis of the groups showed greater genotoxic damage in the case group (alcoholic) compared to the control group (p<0,05). Considering all populations evaluated, 17 points of variation were found. These points of variation are arranged in 22 different haplotypes. A high linkage disequilibrium was observed between the variants at positions -1295 and -1055, in the case and control groups and the American, European and Asian populations. Considering the complex interaction between genetic and environmental factors in response of interindividual alcoholic beverages, it is desirable to conduct studies about this interaction in populations with different genetic characteristics.
O gene citocromo P4502E1 (CYP2E1) codifica uma enzima que é a principal constituinte do Sistema microssomal de oxidação do etanol. Variações genéticas presentes no gene CYP2E1 resultam em diferenças significativas na atividade enzimática, entretanto o significado funcional dessas variações no metabolismo do etanol permanece controverso, uma vez que ainda não existe uma correlação evidente com diferenças na atividade catalítica da enzima. No presente estudo, a variabilidade da região promotora do gene CYP2E1 foi analisada em amostras de alcoolistas de um Centro de Atenção Psicossocial (CAPS) do Estado de Goiás, da cidade de Goiânia e os resultados foram comparados com os dados obtidos pelo projeto 1000 Genomas. Foi realizado um estudo de associação entre polimorfismos da região promotora do CYP2E1 e o dano genotóxico em alcoolistas, para isso foram realizados a reação de sequenciamento e o ensaio cometa. Nas nossas análises não houve relação dos dados de polimorfismos encontrados com o dano genotóxico. Entretanto, a análise de genotoxidade dos grupos analisados mostrou maior dano genotóxico no grupo caso (alcoolistas) em relação ao grupo controle (p<0,05). Considerando todas as populações avaliadas, 17 pontos de variação foram encontrados. Estes pontos de variação estão arranjados em 22 haplótipos diferentes. Um elevado desequilíbrio de ligação foi observado entre as variantes nas posições -1295 e - 1055, nos grupos caso e controle e nas populações americana, europeia e asiática. Considerando a complexa interação entre fatores genéticos e ambientais na resposta interindividual ao uso de bebidas alcoólicas, é desejável a realização de estudos acerca dessa interação em populações com características genéticas distintas.

Background: Childhood acute lymphoblastic leukemia (ALL) is a complex disease whose etiology remains largely unknown. Both genetic and environmental factors are believed to be involved in leukemogenesis. It has long been suspected that organic solvents are carcinogens. They are common in the workplace and are potentially important sources of exposure in mothers during various time periods: preconception, pregnancy and postnatal. These time windows are vital for the developing fetus and exposures to carcinogens through the placenta or breast milk could lead to DNA damage. In addition, variants in xenobiotic metabolizing genes that biotransform various chemicals entering the body, in particular CYP (cytochrome P450) and GST (glutathione S-transferase) genes, have equally been linked to the development of ALL. As such, it is quite possible that variants in CYP and GST genes affect the biotransformation of chemicals such as organic solvents in the fetus or infant, leading to increased DNA damage and potentially cancer. Methods: I analyzed the effects of maternal occupational exposure to organic solvents during pregnancy and breastfeeding on the risk of developing ALL in the offspring. The effects of organic solvents from household activities were also investigated in breastfeeding mothers during the postnatal period. In addition, I analyzed the joint effects of case genetic variants in certain likely functional xenobiotic metabolizing genes (CYP1A1, CYP2E1 and GSTM1) with organic solvent exposures. The data was taken from a large population based case-control with 790 cases and 790 controls recruited from Quebec, Canada. The data included state-of-art determination of occupational exposures, household exposures to various environmental exposures, and genotyped DNA samples from the study participants and their parents. The data was analyzed using logistic regression and Poisson log-linear models based on case-control, case-only and case parent-trio designs. Results: Significant main effects were found between case GSTM1 null and CYP1A1 *4 variants and ALL. Additionally, individuals with one copy of the CYP1A1 *2A variant and GSTM1 null had a significant odds ratio of developing ALL at 1.68 (95% CI: 1.03-2.75) as compared to an individual with neither. Offspring with the GSTM1 null variant whose mothers were occupationally exposed to aliphatic alcohols and aliphatic ketones, specific chemical families of organic solvents, during pregnancy had a lower risk of developing ALL than carriers of the wild type carriers. The case-parent trio analysis did detect a harmful interaction effect between offspring with the CYP1A1 *2B variant and maternal occupational exposure to any type of organic solvent during pregnancy. Similarly, the case-only analysis found important harmful interaction effects between the CYP1A1 *2A and *4 variants and maternal occupational exposures to any type of organic solvent during pregnancy and protective interaction effects between GSTM1 null variants and this same exposure. Among mothers who breastfed, exposure to organic solvents from household activities from one year before pregnancy to date of diagnosis did not generally increase the risk of ALL; however, there was evidence to suggest that the GSTM1 null and CYP1A1 *2A variants modified the effect of solvent exposure from furniture stripping, and likewise for the CYP2E1 *5 variant with certain activities involving exposure to electronics. The CYP1A1 *2A variant also appeared to significantly modify the effect of latex and/or acrylic paint exposures in a breastfeeding mother on the risk of ALL. Discussion: Although the study had limited power to uncover statistically significant interactions, the results suggest a role on the incidence of childhood ALL for gene variants involved in the metabolism of carcinogens in the presence of environmental prenatal or breastfeeding exposure to organic solvents.
Introduction: La leucémie lymphoblastique aiguë (LLA) chez l'enfant est une maladie complexe dont l'étiologie reste peu connue. Des chercheurs soupçonnent que les solvants organiques sont des carcinogènes importants et qu'ils sont présents dans plusieurs lieux de travail. Ces produits chimiques ont le potentiel d'être une source d'exposition chez la mère durant les différentes périodes du développement du foetus, notamment, la préconception, la gestation et le postnatal. De plus, quelques variantes dans les gènes xénobiotiques du métabolisme, qui transforment une multitude de produits chimiques absorbés par le corps, en particularité des variantes dans les gènes CYP (cytochrome P450) et GST (glutathion S-transférase), ont été associées avec la LLA chez l'enfant. Tout cela suggère que les variantes dans les gènes CYP et GST perturbent la biotransformation des produits chimiques, tel que les solvants organiques, chez le fœtus et le nouveau-né, menant à des dommages à l'ADN et possiblement des néoplasies. Méthodes : J'ai analysé les effets de l'exposition maternelle aux solvants organiques pendant la gestation et l'allaitement sur le risque de développer la LLA. Les effets de l'exposition aux solvants organiques retrouvés au domicile ont aussi été investigués chez les mères qui allaitent. De plus, j'ai analysé l'effet cumulatif des variantes dans les gènes CYP et GST et les expositions aux solvants organiques au travail de la mère pendant la gestion et celles au domicile pour les mères qui ont allaité. Les données ont été sélectionnées d'une étude cas-témoin réalisée au Québec, recrutant 790 cas et 790 témoins. Des échantillons d'ADN ont été recueillis des participants et de leurs parents. Les données ont été analysées avec des modèles de régression logistiques et log-linéaire (régression de Poisson) basés sur les concepts de cas-témoins, cas-seul et trio cas-parent. Résultats : Des effets statistiquement significatifs ont été trouvés entre les variantes GSTM1 nulle et CYP1A1 *4 chez l'enfant et la LLA. Les individus qui possèdent une copie de la variante CYP1A1 *2A et de la variante GSTM1 nulle, ont un risque significatif de développer la LLA comparé à des individus avec aucune de ces variantes. Les progénitures d'une mère qui a été exposée à des solvants organiques au travail, particulièrement les alcools aliphatiques et les cétones aliphatiques, qui possèdent la variante GSTM1 nulle, ont un risque inférieur de développer la LLA comparées aux progénitures qui ont le gène de type sauvage. Le trio cas-parent suggérait un effet d'interaction nocive entre la variante CYP1A1 *2B chez la progéniture et l'exposition de la mère à n'importe quel solvant organique au travail pendant la gestation. De même, le type d'étude cas-seul a détecté un effet d'interaction nocive entre les variantes CYP1A1 *2A et *4 et l'exposition de la mère à n'importe quel solvant organique au travail et une interaction protectrice entre la variante GSMT1 nulle et cette même exposition. Le risque de développer la LLA pour les progénitures des mères qui ont allaité et qui ont été exposées à des solvants organiques lors d'activités au domicile pendant une année avant la gestation jusqu'à la date de diagnostic n'était pas élevé. Par contre, les variantes GSMT1 nulle et CYP1A1 *2A ont modifié l'effet des solvants relié à des activités de décapage de meubles et d'électroniques. La variante CYP1A1 *2A a aussi modifié l'effet de l'exposition chez la mère qui allaite aux peintures de type latex et/ou acrylique sur le risque de développer la LLA chez l'enfant. Discussion: Ces résultats suggèrent la possibilité que les variantes des gènes étudiés interagissent avec l'exposition aux solvants organiques pendant la gestation ou l'allaitement chez la mère, pour influencer le risque de développer la LLA chez l'enfant, malgré le pouvoir limité de l'étude pour détecter des interactions statistiquement significatives.

Um grande número de pacientes em tratamento para tuberculose desenvolve algum tipo de reação adversa. A mais grave destas, hepatotoxicidade, é, muitas vezes, atribuída aos metabólitos tóxicos gerados durante a metabolização da isoniazida (INH), principal fármaco do tratamento. A INH é metabolizada pelas enzimas N-acetiltransferase 2 (NAT2), citocromo P450 2E1 (CYP2E1) e glutationa S-transferase (GSTM1 e GSTT1). Polimorfismos nos genes que codificam estas enzimas parecem influenciar na sua atividade e toxicidade, além de estarem distribuídos de forma variável entre as populações. Este estudo foi elaborado com o objetivo de estimar a frequência dos polimorfismos nos genes CYP2E1, GSTM1 e GSTT1, e avaliar a relação destes genes e de fatores de risco com o desenvolvimento de hepatotoxicidade. Foram incluídos no estudo 245 pacientes que fizeram tratamento para tuberculose no ambulatório do Hospital Sanatório Partenon (Porto Alegre, RS) com rifampicina, isoniazida e pirazinamida (esquema RHZ). Esses pacientes forneceram termo de consentimento livre e esclarecido, entrevista para coleta de dados clínicos e epidemiológicos e amostra de sangue para exames de provas de função hepática e perfil genético. A identificação dos genótipos foi realizada através das técnicas de PCR para os genes GSTM1 e GSTT (presença ou ausência) e PCR-RFLP para CYP2E1 (SNPs nas posições -1053C>T,-1293G>C e 7632T>A). Através da montagem de um banco de dados com as informações epidemiológicas e genéticas foram testadas possíveis relações entre as características. As frequências dos alelos variantes no gene CYP2E1 foram 8% (-1053), 8,5% (-1293) e 12% (7632). Os genes GSTM1 e GSTT1 estavam ausentes em 42.9% e 12.4% da população, respectivamente. Quinze (6,1%) pacientes desenvolveram hepatotoxicidade. Pacientes com genótipo selvagem em CYP2E1, sendo acetiladores lentos para NAT2, estão sob maior risco de desenvolver hepatotoxicidade. O genótipo nulo para GSTM1 e GSTT1 não teve influência na resposta ao tratamento para essa população. Os fatores de risco associados à hepatite induzida por tuberculostáticos foram: HIV positivo, tuberculose extrapumonar e níveis elevados de transaminases basais.
Patients under treatment for tuberculosis (TB) frequently suffer adverse drug reactions. The most severe, hepatotoxicity is often related to toxic metabolites produced during the main drug metabolism, isoniazid (INH). INH is metabolized by N-acetyltransferase 2 (NAT2), cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase (GSTM1 and GSTT1) enzymes. Polymorphisms in coding genes can modulate enzyme activity and toxicity, and are distributed in a variable way among populations. This study was designed to determine the frequency of CYP2E1, GSTM1 e GSTT polymorphisms, and to evaluate whether clinical risk factors and polymorphism are related to drug-induced hepatotoxicity. A total of 245 TB outpatients from Hospital Sanatorio Partenon (Porto Alegre, RS) using INH, RMP and PZA were included in the study. Patients provided a written informed consent, clinical records through an interview and blood sample for liver enzymes tests and genotyping. Individuals were genotyped using polymerase chain reaction (GSTM1 and GSTT) and restriction fragment length polymorphism (CYP2E1) methods. Using a database containing genetic and clinical information, possible relations were tested. The frequencies for the CYP2E1 polymorphic alelles RsaI, PstI and DraI are 8%, 8,5% and 12%, respectively. GSTM1 and GSTT1 genes are deleted in 42.9% and 12.4% of the population. Fifteen patients (6.1%) developed hepatotoxicity. Patients without polymorphisms in CYP2E1, having NAT2 slow acetylator profile, are at higher risk for drug-induced hepatotoxicity. Null genotype for GSTM1 and GSTT1 showed no influence in drug response. HIV, extrapulmonary TB and high baseline levels of transaminases are independent risk factors for hepatotoxicity in this population.

Cytochromes(CYP) P450 are class of heme-containing enzymes involved in Phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 is involved in the metabolism of many xenobiotics and procarcinogens including ethanol, acetone, nitrosamine, pyridine, isoniazid and carbon tetrachloride (CCl4), which are also CYP2E1-inducing agents. CYP2E1 comprises approximately 7% of the liver CYP content and it is also expressed in kidney, lung, brain, gastrointestinal tract and breast tissue implying that this enzyme is implicated in other biological processes aside from its role in Phase I metabolism. Several studies converge to the conclusion that CYP2E1 is induced under many pathological conditions including cancer, obesity, and type 2 diabetes. Increased hepatic ketogenesis and insulin resistance are the possible mechanisms mediating CYP2E1 induction in these conditions. Elevated CYP2E1 expression has been reported in the presence of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). CYP2E1 generates the highest level of reactive oxygen species (ROS) among the CYP450 superfamily and transient overexpression of this CYP family member in COS cells increases the production of mitochondrial ROS, even in the absence of substrate. Oxidative stress induced by CYP2E1 disturbs the folding capacity of the endoplasmic reticulum (ER), with concomitant alterations in the mRNA and protein expression of the ER stress proteins GRP78 and GRP94. Initially the unfolded protein response (UPR) restores ER homeostasis, and cell viability, but at later stages the accumulation of unfolded proteins stimulates pro-apoptotic signals. Cell death induced by ER stress has been reported in several conditions including hypoxia, and diseases such as diabetes and heart disease. To gain better understanding of the factors regulating CYP2E1 gene expression in breast cancer cells we studied CYP2E1 mRNA and protein levels in MCF7 (p53wt) and MDA-MB-231 (p53 mutated) breast cancer cells and identified that CYP2E1 was under p53 and HIF-1α transcriptional control in both of these cell lines treated with etoposide or desferrioxamine respectively. In addition, CYP2E1 was differentially expressed in the low metastatic potential MCF7 cell line compared to highly metastatic MDA-MB-231 cells in accord with clinical studies indicating that CYP2E1 isoenzyme is expressed in lower levels in patients at clinical stages II, III, and IV which exhibit higher metastatic potential than in patients at stage I of breast cancer. To further investigate the functional significance of the difference in CYP2E1 levels the generation of ROS in breast cancer cells ectopically overexpressing CYP2E1 or in cells in which CYP2E1 expression had been silenced was assessed. Our results indicated that CYP2E1 overexpression resulted in higher ROS generation, which coincided with increased autophagy biomarker expression, increased endoplasmic reticulum stress, detected by XBP1 mRNA splicing in fluorescent reporter assays, and inhibition of metastatic potential. These studies show that CYP2E1 exerts an important role in breast cancer cells by inducing generation of oxidants the levels of which differentially influence the unfolding protein response and apoptosis in high and low metastatic potential breast cancer cells and suggest that targeting CYP2E1 might be a powerful approach to modulate breast cancer initiation, progression, and metastasis.

INTRODUCTION: The liver is the primary organ responsible for the removal of toxic substances from the body by means of a variety of metabolic pathways. One class of proteins responsible for much of the body’s xenobiotic drug and alcohol metabolism is the Cytochrome P450 family of proteins. One protein, Cytochrome P450 Class E Subclass 2 (Cyp2E1), has an integral role in alcohol metabolism by the liver. Cyp2E1 becomes fully activated after an organism has consumed excessive amounts of alcohol excessive alcohol and works with aldehyde dehydrogenase (ALDH) to metabolize ethanol to acetaldehyde. Another metabolic protein, C1q TNF Related Protein 3 (CTRP3), has been shown to effectively prevent alcoholic fatty liver disease (AFLD), specifically with long-term alcohol-induced lipid accumulation. METHODS: In this experiment, 12-week old male mice were fed a Lieber-Decarli alcohol diet (5% ETOH by volume) for 6-weeks. The food intake and body weight of the mice was recorded each day. The mice in the experiment included both wild type and transgenic CTRP3 overexpressing mice. At the end of the 6-week period the mice were euthanized, and the liver was carefully removed, flash-frozen, and prepared for immunoblot analysis of the proteins. RESULTS: Cyp2E1 levels increased significantly in response to ethanol consumption. Cyp2E1 levels were further elevated in ethanol-fed CTRP3 transgenic overexpressing mice. Cyp2E1 levels in CTRP3 transgenic mice were nearly twice that of wild type ethanol-fed mice. CONCLUSIONS: The results of the experiment show a significant increase in Cyp2E1 in mice which overexpress CTRP3. This upregulation of Cyp2E1 with CTRP3 overexpression could explain the mechanism for reduced hepatic lipid accumulation in ethanol-fed CTRP3 transgenic mice.

The aetiology of Parkinson's disease (PD) is still unclear. Research findings suggest that both environmental and genetic factors may contribute to its development. The interactions between genes and the environment might exist and play a key role. Cigarette smoking was found to be one of the few factors exhibiting a protective effect. If chemical compounds found in cigarette smoke influence PD risk, the difference in the ability of certain individuals in metabolising these substances might alter their susceptibility to the risk of developing PD. Many metabolic enzyme genes exhibit polymorphic traits with alteration of gene function. These might be associated with an altered susceptibility of individuals to PD. Few studies have examined the hypothesis that metabolic enzyme gene polymorphisms might modulate the effect of smoking on PD risk. However, it is crucial to consider these potential interactions when we try to elucidate the aetiology of PD. Even if each factor only contributes a slight variation and influences a small portion of the whole population, non-linear and unpredictable interactions may account for a high proportion of the aetiological fraction. Previous studies have not been strictly designed to examine the interactions between smoking and metabolic enzyme genetic polymorphisms. These studies have not been able to elucidate the extent of the interaction. Therefore, this PhD project attempted to examine whether genetic factors, operating in the phase one and phase two metabolic pathways, interact with smoking to influence the development of PD. This is the first genetic epidemiological study of PD specifically addressing this issue. The research aids in further understanding the aetiology of PD and may be useful for identifying people at higher risk. A case-only design was chosen for this project for two reasons: first, PD is a relatively rare disease and the case-only design is much more efficient at detecting gene-environment interactions; second, the PD cases for the project were recruited over the past few years and represent a prevalence series, for which an appropriate comparison group for the cases is difficult to identify and recruit. In a case-only study, only cases are used to investigate the multiplicative effects of the exposures and susceptible genotypes of interest, while non-case subjects (traditionally controls) are solely used to test the independence between the exposure and the susceptible genotype. Therefore, this approach avoids the challenges of control selection, a major limitation inherent in the case-control approach. This thesis comprised of three independent studies: the first study investigated the interactions between genetic polymorphisms of GSTM1, P1, T1 and Z1 and smoking in PD; the second study examined the interactions between genetic polymorphisms of CYP2E1 and smoking in PD; and the third study examined the interactions between genetic polymorphisms of CYP2D6 and smoking in PD. The first two studies recruited 400 white Caucasian PD cases from both hospital wards and private neurology clinics (230 men and 170 women). The third study further included 142 white Caucasian PD cases newly recruited from the same sources (542 in total, 321 men, and 221 women). The mean age of cases was 67 years with the average onset age at 60 years. GSTM1, GSTP1, GSTT1, GSTZ1 AND CYP2E1 genotyping processes were performed using protocols previously published with minor modification, whereas CYP2D6 genotyping methods were mainly developed by me with assistance from associate supervisor Dr. George Mellick. Reliability and validity of the PCR and RFLP methods were assessed through re-conducting the genotype assays using at least a 10% sample of our DNA samples. The results for all re-assessments were 100% concordant. Crude bivariate analyses were adjusted for potential confounding effects of the variables, including age at onset, gender, family history of PD and pesticide exposures. Among our unaffected, aged subjects (mean age: 63.9 years, sd: 11.4 years), the genotype frequencies at each locus were similar to those reported in other Caucasian populations. The first study showed that the proportion of carriers of the GSTP1-114Val allele (mutant) increased with increasing smoking dose from 0 to > 30 pack-years. Homozygotes of the 114Ala allele (wild-type) decreased with increasing smoking dose (trend test: p=0.02). This trend existed both in male and female cases. This dose-effect relationship was most significant in the group of cases with late-onset PD (i.e., age at onset > 55 years) with the ORicase-only values of 1.88 (95%CI: 0.65-5.48) and 2.63 (95%CI: 1.07-6.49) for > 0-10 and > 10 pack-years, respectively. No similar trend was found among our unaffected, aged subjects (p=0.42). Haplotype analyses revealed significant differences for GSTP1 haplotypes between smoking and non-smoking PD cases (ORicase-only for *C haplotype=2.00 (95%CI: 1.11-3.60), p=0.03). In this case, smoking-exposed PD cases were more likely to posses the *C haplotype defined by A to G base-pair transition at nucleotide +313 and C to T base-pair transition at nucleotide +341 (at amino acid level, valine at both positions 105 and 114). The second study found no difference in CYP2E1 genotype frequencies between PD cases who ever smoked compared to those who never smoked (odds ratio for interaction (ORi) = 1.00 (95% CI: 0.39-2.51, p=0.99)). No CYP2E1 gene-smoking interactions were detected in relation to age at onset of PD. The third study found that among cases without regular pesticide exposures, CYP2D6 PMs who smoked more than 5 pack-years had a later mean age at disease onset (68.6 years) than those with extensive metaboliser phenotypes (EMs) (61.1 years, p=0.02) and non-smokers (60.5 years, p=0.01). Analysis of aged subjects without PD confirmed that neither smoking status nor CYP2D6 PM status was associated with age itself. Our data suggest: 1. smoking exposure is independent of GSTM1, P1, T1, Z1 and CYP2E1 genotypes; 2. smoking may be, to some extent, associated with CYP2D6 genotypes; 3. there are no multiplicative interactive effects linking smoking and GSTM1, T1, Z1 or CYP2E1 genotypes with the risk for PD; 4. there is a multiplicative interactive effect between smoking and GSTP1 haplotype - particularly for genotypes carrying the 114Val allele; and 5. there is a multiplicative interactive effect between smoking and CYP2D6 PMs - particularly for people who ever smoked cigarettes more than 5 pack-years. In general, this thesis provides a model for exploring the gene-smoking interactions in PD. Further studies need to consider the recruitment of a large number of population-based and randomly-selected samples and to pay more attention to measurement of environmental exposures. Further studies also need to examine simultaneously the impact of smoking, pesticide exposures and other potential risk factors on PD. These studies will build evidence for interactions contributing to this common neurological movement disorder.

Le stress oxydant est induit par le déséquilibre de la balance anti/pro-oxydante. Il est incriminé dans différentes pathologies (maladies neurodégénératives, rhumatoi͏̈des, athérosclérose, vieillissement accéléré. . . ), en tant que facteur de genèse ou phénomène associé. Bien que plusieurs systèmes oxydatifs aient été proposés comme médiateurs possibles des dommages dans ces états pathologiques, l'origine de l'oxydation et le mécanisme oxydatif n'ont pas encore été complètement élucidé. Les enzymes cytochromes P450 (P450)s, qui utilisent l'oxygène pour oxyder leurs substrats, sont une source importante de formation des espèces réactives de l'oxygène (ERO). Le cytochrome P450 2E1 (CYP2E1) se caractérise par une production d'ERO plus importante que les autres isoformes de P450s et est facilement induit par les xénobiotiques. Même si beaucoup d'études ont montré le rôle des P450s dans le métabolisme des composés endogènes et des xénobiotiques, peu d'études évaluent la capacité des P450s à oxyder les protéines et à réguler le système antioxydant. C'est pourquoi nous avons étudié les modifications de la protéine CYP2E1, de son activité en réponse aux xénobiotiques, et les cibles cellulaires d'ERO produits par les P450s, apolipoprotéine E (apoE) et gamma-glutamyltranspeptidase (CGT). . .

The aetiology of Parkinson's disease (PD) is still unclear. Research findings suggest that both environmental and genetic factors may contribute to its development. The interactions between genes and the environment might exist and play a key role. Cigarette smoking was found to be one of the few factors exhibiting a protective effect. If chemical compounds found in cigarette smoke influence PD risk, the difference in the ability of certain individuals in metabolising these substances might alter their susceptibility to the risk of developing PD. Many metabolic enzyme genes exhibit polymorphic traits with alteration of gene function. These might be associated with an altered susceptibility of individuals to PD. Few studies have examined the hypothesis that metabolic enzyme gene polymorphisms might modulate the effect of smoking on PD risk. However, it is crucial to consider these potential interactions when we try to elucidate the aetiology of PD. Even if each factor only contributes a slight variation and influences a small portion of the whole population, non-linear and unpredictable interactions may account for a high proportion of the aetiological fraction. Previous studies have not been strictly designed to examine the interactions between smoking and metabolic enzyme genetic polymorphisms. These studies have not been able to elucidate the extent of the interaction. Therefore, this PhD project attempted to examine whether genetic factors, operating in the phase one and phase two metabolic pathways, interact with smoking to influence the development of PD. This is the first genetic epidemiological study of PD specifically addressing this issue. The research aids in further understanding the aetiology of PD and may be useful for identifying people at higher risk. A case-only design was chosen for this project for two reasons: first, PD is a relatively rare disease and the case-only design is much more efficient at detecting gene-environment interactions; second, the PD cases for the project were recruited over the past few years and represent a prevalence series, for which an appropriate comparison group for the cases is difficult to identify and recruit. In a case-only study, only cases are used to investigate the multiplicative effects of the exposures and susceptible genotypes of interest, while non-case subjects (traditionally controls) are solely used to test the independence between the exposure and the susceptible genotype. Therefore, this approach avoids the challenges of control selection, a major limitation inherent in the case-control approach. This thesis comprised of three independent studies: the first study investigated the interactions between genetic polymorphisms of GSTM1, P1, T1 and Z1 and smoking in PD; the second study examined the interactions between genetic polymorphisms of CYP2E1 and smoking in PD; and the third study examined the interactions between genetic polymorphisms of CYP2D6 and smoking in PD. The first two studies recruited 400 white Caucasian PD cases from both hospital wards and private neurology clinics (230 men and 170 women). The third study further included 142 white Caucasian PD cases newly recruited from the same sources (542 in total, 321 men, and 221 women). The mean age of cases was 67 years with the average onset age at 60 years. GSTM1, GSTP1, GSTT1, GSTZ1 AND CYP2E1 genotyping processes were performed using protocols previously published with minor modification, whereas CYP2D6 genotyping methods were mainly developed by me with assistance from associate supervisor Dr. George Mellick. Reliability and validity of the PCR and RFLP methods were assessed through re-conducting the genotype assays using at least a 10% sample of our DNA samples. The results for all re-assessments were 100% concordant. Crude bivariate analyses were adjusted for potential confounding effects of the variables, including age at onset, gender, family history of PD and pesticide exposures. Among our unaffected, aged subjects (mean age: 63.9 years, sd: 11.4 years), the genotype frequencies at each locus were similar to those reported in other Caucasian populations. The first study showed that the proportion of carriers of the GSTP1-114Val allele (mutant) increased with increasing smoking dose from 0 to > 30 pack-years. Homozygotes of the 114Ala allele (wild-type) decreased with increasing smoking dose (trend test: p=0.02). This trend existed both in male and female cases. This dose-effect relationship was most significant in the group of cases with late-onset PD (i.e., age at onset > 55 years) with the ORicase-only values of 1.88 (95%CI: 0.65-5.48) and 2.63 (95%CI: 1.07-6.49) for > 0-10 and > 10 pack-years, respectively. No similar trend was found among our unaffected, aged subjects (p=0.42). Haplotype analyses revealed significant differences for GSTP1 haplotypes between smoking and non-smoking PD cases (ORicase-only for *C haplotype=2.00 (95%CI: 1.11-3.60), p=0.03). In this case, smoking-exposed PD cases were more likely to posses the *C haplotype defined by A to G base-pair transition at nucleotide +313 and C to T base-pair transition at nucleotide +341 (at amino acid level, valine at both positions 105 and 114). The second study found no difference in CYP2E1 genotype frequencies between PD cases who ever smoked compared to those who never smoked (odds ratio for interaction (ORi) = 1.00 (95% CI: 0.39-2.51, p=0.99)). No CYP2E1 gene-smoking interactions were detected in relation to age at onset of PD. The third study found that among cases without regular pesticide exposures, CYP2D6 PMs who smoked more than 5 pack-years had a later mean age at disease onset (68.6 years) than those with extensive metaboliser phenotypes (EMs) (61.1 years, p=0.02) and non-smokers (60.5 years, p=0.01). Analysis of aged subjects without PD confirmed that neither smoking status nor CYP2D6 PM status was associated with age itself. Our data suggest: 1. smoking exposure is independent of GSTM1, P1, T1, Z1 and CYP2E1 genotypes; 2. smoking may be, to some extent, associated with CYP2D6 genotypes; 3. there are no multiplicative interactive effects linking smoking and GSTM1, T1, Z1 or CYP2E1 genotypes with the risk for PD; 4. there is a multiplicative interactive effect between smoking and GSTP1 haplotype - particularly for genotypes carrying the 114Val allele; and 5. there is a multiplicative interactive effect between smoking and CYP2D6 PMs - particularly for people who ever smoked cigarettes more than 5 pack-years. In general, this thesis provides a model for exploring the gene-smoking interactions in PD. Further studies need to consider the recruitment of a large number of population-based and randomly-selected samples and to pay more attention to measurement of environmental exposures. Further studies also need to examine simultaneously the impact of smoking, pesticide exposures and other potential risk factors on PD. These studies will build evidence for interactions contributing to this common neurological movement disorder.

The effects of diabetes on cytochrome P450 dependent drug metabolizing enzymes have not to be clarified yet. The most widely used animals in these studies have been rats, and information regarding the effects of diabetes on cytochrome P450 dependent procarcinogen/carcinogen metabolism in rabbits is limited. In the present study, we investigated, for the first time, the influence of benzene on liver, kidney and lung microsomal cytochrome P450 dependent drug metabolizing enzyme activities, protein and mRNA levels in diabetic and non-diabetic rabbits. Male New Zealand rabbits were made diabetic by a single dose of alloxan treatment in this study. AST, ALT and LDH enzyme activities in the blood serum and lipid peroxidation in liver microsomes were found to increase in diabetic, benzene treated and benzene treated diabetic rabbits. Besides these, CYP2E1 dependent NDMA N-demethylase and p-nitrophenol hydroxylase activities and CYP2E1 protein level were found to increase in liver and kidney of diabetic and benzene-treated rabbits. The combined effects of benzene and diabetes on these activities and protein level were found to be additive. Although diabetes caused induction of pulmonary CYP2E1 protein level and associated enzyme activities, benzene treatment of rabbits resulted in no change in enzyme activities and protein level in lung. The level of mRNA was investigated by Real-Time PCR. Accordingly, hepatic CYP2E1 mRNA level was increased 6.71-, 10.53- and 12.93-fold in diabetic, benzene treated and benzene treated diabetic rabbits with respect to the control animals. Similarly, renal CYP2E1 mRNA level was found in increase in these rabbits. In addition to CYP2E1, CYP3A6 associated enzyme activity, erythromycin N-demethylase, CYP3A6 protein and mRNA level were found to increase in diabetic rabbit liver and lung. Unlike diabetes, benzene treatment caused suppression of CYP3A6 protein and inhibition of associated enzyme activity in liver. There was no significant change in the erythromycin N-demethylase activity and CYP3A6 level of liver and lung as a result of benzene treatment of diabetic rabbits. Moreover, diabetes induced CYP1A2 protein and mRNA level and CYP1A associated enzyme activities in the rabbit liver. On the other hand, benzene caused statistically insignificant decreases in CYP1A dependent enzyme activities and CYP1A2 protein level in liver. CYP1A associated enzyme activities, CYP1A2 protein and mRNA levels were not changed in the liver of benzene treated diabetics. The results of the present work indicate that both diabetes and benzene stimulate metabolic activation toxic chemicals metabolized by CYP2E1 such as NDMA and benzene by inducing CYP2E1 which results in the formation of increased amounts of reactive metabolites. Application of benzene to diabetic rabbits further elevates expression and activities of the CYP2E1. As a result of additive induction of the CYP2E1 in benzene treated diabetics, further increase the risk of hepatotoxicity produced by toxins may be observed when compared to the separate treatments. This may in turn further potentiate the risk of organ toxicity and mutagenesis in liver and kidney of these subjects. As in the case of CYP2E1, the risk of carcinogenesis due to induction of CYP1A may be increased in diabetic subjects. Moreover, in diabetic and benzene exposed subjects, alteration of drug clearance and clinical drug toxicity may be observed due to induction or suppression of CYP3A.

Muitos autores associam as doenças alcoólicas hepáticas às deficiências nutricionais. Por outro lado, trabalhos experimentais estabelecem que a hepatotoxidade alcoólica relaciona-se especialmente à geração de espécies reativas através do sistema microsomal que oxida etanol via citocromo 450, principalmente o CYP2E1. O CYP2E1 hepático tem a capacidade de ativar algumas drogas comumente utilizadas, como o acetaminofeno, em seus metabólitos mais tóxicos e promover carcinogênese. Além. disso, o metabolismo pelo CYP2E1 resulta num aumento na produção de espécies reativas, com diminuição nos sistemas de defesa antioxidantes, estabelecendo o estresse oxidativo. Como a expressão do CYP2E1 é muito influenciada por fatores nutricionais e hormonais, este trabalho descreve os efeitos do tratamento com etanol nos níveis de CYP2E1 e sua relação com alguns parâmetros pró- e antioxidantes, considerando três modelos experimentais diferentes. Ratos machos Sprague Dawley com cerca de três meses de idade receberam ad lib. ração Purina (Purina Ind., Brasil) e, separadamente, uma solução 25 % etanol-20%sacarose durante 1, 2, 3 ou 4 semanas. Os grupos controles foram isocaloricamente pareados aos animais que consumiram etanol, ou receberam quantidades de sacarose equivalentes às calorias recebidas com o consumo de etanol. 18 h antes do sacrifício os animais foram mantidos em abstinência alcoólica, recebendo água e ração ou apenas água ad lib. Os resultados indicam que o consumo de etanol pode ser associado à estabilização do CYP2E1. No entanto, nas nossas condições experimentais, a presença da isoforma não está associada ao estresse oxidativo. Esses resultados indicam que as deficiências nutricionais, especialmente o baixo consumo de carboidratos, são fundamentais na potenciação do estresse oxidativo induzido pelo etanol.
Many authors have attributed alcoholic liver disease to dietary deficiencies. On the other hand, experimental studies have established that alcohol hepatotoxicity is especially related to the generation of oxidant species through its microsomal metabolism via cytochrome P-450, mainly CYP2E1. Liver CYP2E1 has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis. Moreover, metabolism by CYP2E1 results in a significant reactive oxygen species (ROS) release, accompanied by the defense systems decrease against oxidative stress. Since the expression of CYP2E1 is very much influenced by hormonal and nutritional factors, this study describes the effects of ethanol treatment on CYP2E 1 levels and their relationship with some pro and antioxidant parameters considering three experimental models. Male Sprague-Dawley rats were fed ad. lib. for 1, 2, 3 or 4 weeks a commercial diet (Purina Ind., Brazil) plus a 25% ethanol-20% sucrose solution. Control groups were isocalorically pair-feed to the leading ethanol-consuming animals, or received isocaloric amounts of sucrose for pairing only ethanol calories. Eighteen hours before sacrifice ethanol was withdrawal and animals had only free access to tap water or they were offered food and water ad. lib. Results have shown that ethanol administration was associated with CYP2E1 stabilization although under our experimental condition it was not associated with any oxidative stress. These findings indicate that dietary deficiencies, especially low carbohydrate intake are crucial in the potentiation of the ethanol-induced oxidative stress.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Ethanol is considered the most consumed drug in the world, is part of several cultures. However, chronic ethanol consumption has become a social and public health problem, since it has serious health consequences and is a risk factor for several diseases and mortality. The CYP2E1 gene encodes an enzyme that is the main constituent of the microsomal oxidation system of ethanol. The CYP1A1 gene encodes an enzyme that, like CYP2E1, acts on the oxidative biotransformation of phase I substrates of the metabolism of xenobiotics such as ethanol. In addition, genes related to DNA repair such as OGG1 are also of great importance as they are involved in protecting the genome by helping to maintain the cellular functions of organisms. Problems in the repair process can interfere with aging and promote disease development. Genetic variations in these genes result in significant differences in enzyme activity. However, there is still no clear correlation between the functional significance of these variations in both ethanol metabolism and ethylene repair. In the present study, the variability of specific regions of the CYP2E1, CYP1A1 and OGG1 genes were analyzed in samples of alcoholics from a Psychosocial Care Center (CAPS) of the State of Goiás, in the city of Goiânia, and the results were compared with the data obtained by the 1000Genomas project. A study of the association between the polymorphism of these genes and the damage to the DNA in ethanol was carried out. Sequencing reactions and the comet assay were performed. There was no relation between the data of the polymorphism found with DNA damage. However, in the analysis of the DNA damage between the case and control groups, greater damage was observed in the alcoholics in relation to the control group. The genotypic frequencies adhered to that expected by the HardyWeinberg Equilibrium for all the polymorphism in the Brazilian samples. Based on the polymorphism of the CYP2E1 gene, 15 haplotypes were inferred. Of these, only 4 haplotypes were common in all populations. While based on the two polymorphism of the CYP1A1 and OGG1 genes, 3 haplotypes were inferred for each gene, all present in the Brazilian study population.
O etanol é considerado a droga mais consumida no mundo, cujo o consumo crônico tornou-se um problema social e de saúde pública, pois traz graves consequências a saúde, sendo fator risco a diversas doenças e mortalidade. O gene CYP2E1 codifica uma enzima que é a principal constituinte do sistema microssomal de oxidação do etanol. O gene CYP1A1 codifica uma enzima que como a CYP2E1 atua na biotransformação oxidativa de substratos de fase I do metabolismo de xenobióticos como o etanol. Além disso, genes relacionados ao reparo de DNA como o OGG1 também são de grande importância, pois estão envolvidos na proteção do genoma auxiliando na manutenção das funções celulares dos organismos. Problemas no processo de reparo podem interferir negativamente no envelhecimento e promover o desenvolvimento de doenças como vários tipos de câncer. Variações genéticas presentes nesses genes resultam em diferenças significativas na atividade enzimática. Porém, ainda não existe uma correlação evidente entre o significado funcional dessas variações tanto no metabolismo do etanol quanto no reparo em etilistas. No presente estudo, a variabilidade de regiões específicas dos genes CYP2E1, CYP1A1 e OGG1 foram analisadas em amostras de etilistas de um Centro de Atenção Psicossocial (CAPS) do município de Goiânia, do Estado de Goiás e os resultados foram comparados com os dados obtidos pelo projeto 1000Genomas. Foi realizado um estudo de associação entre pontos de variação desses genes e o dano ao DNA em etilistas, para isso foram realizadas reações de sequenciamento e o ensaio cometa, respectivamente. Não houve relação entre os dados dos pontos de variação encontrados com o dano ao DNA. Entretanto, na análise do dano ao DNA entre os grupos caso e controle foi observado maior dano nos etilistas em relação ao grupo controle. As frequências genotípicas aderiram ao esperado pelo Equilíbrio de Hardy-Weinberg para todos os pontos de variação nas amostras brasileiras. Com base nos pontos de variação do gene CYP2E1, 15 haplótipos foram inferidos. Desses, apenas 4 haplótipos foram frequentes em todas as populações. Enquanto que com base nos dois pontos de variação dos genes CYP1A1 e OGG1, foram inferidos 3 haplótipos para cada gene, todos presentes na população brasileira de estudo.

Le cytochrome P450 (CYP) 2E1 intervient dans la biotransformation de nombreux composés exogènes dont l’alcool, le paracétamol et le CCl4. Au cours du métabolisme, le CYP2E1 produit des espèces réactives de l’oxygène et des métabolites réactifs pouvant entraîner des dommages cellulaires et mitochondriaux. Il est majoritairement exprimé dans le réticulum endoplasmique mais il a également été purifié dans des mitochondries hépatiques de rat. La présence de CYPs métabolisant les xénobiotiques au sein des mitochondries pose de nombreuses questions sur leur rôle, leur capacité de métabolisation et sur d’éventuels effets délétères. Dans la première partie de mon travail, nous avons étudié in vitro l’impact de la localisation du CYP2E1 sur la toxicité induite par l’alcool et le paracétamol. Les résultats indiquent que la présence de CYP2E1 uniquement dans la mitochondrie est suffisante pour induire une cytotoxicité et un stress oxydant après traitement par ces deux composés. La seconde partie de mon travail réalisée in vivo avait pour but d’étudier le rôle de la peroxydation lipidique induite par le CCl4 dans les altérations précoces de l’ADN mitochondrial. L’utilisation d’un inhibiteur du CYP2E1 et d’antioxydants a permis de démontrer le rôle majeur de cette protéine et de la peroxydation lipidique dans ces phénomènes. Par la suite, il serait intéressant de déterminer le rôle du CYP2E1 mitochondrial en utilisant le modèle développé dans notre première étude. Si l’implication du CYP2E1 mitochondrial dans le développement de pathologies hépatiques se précise, une voie de recherche intéressante serait d’adresser spécifiquement des antioxydants ou des inhibiteurs à la mitochondrie
Cytochrome P450 (CYP) 2E1 is implicated in the metabolism of many exogenous compounds such as ethanol, acetaminophen or CCl4. CYP2E1 is one of the CYP able to produce reactive oxygen species during its catalytic cycle. Furthermore, in some cases, CYP2E1 produces reactive metabolites, which could have deleterious cellular and mitochondrial effects. The main localization of this enzyme is the endoplasmic reticulum but it has also been purified from hepatic liver mitochondria. The presence of CYP2E1 in these organelles raises questions regarding its physiological role, its metabolic capacities but also its possible deleterious effects. In the first part of my thesis, we studied in vitro the role of mitochondrial CYP2E1 localization in the toxicity of ethanol or acetaminophen. Our results indicated that the exclusive localization of CYP2E1 within mitochondria was sufficient to induce cytotoxicity and oxidative stress after ethanol or acetaminophen exposure. The second part of my work was devoted to the in vivo study of the implication of CCl4-dependent lipid peroxidation on early mitochondrial DNA alterations. Utilization of a CYP2E1 inhibitor and antioxydants demonstrated the major role of the protein and lipid peroxidation in the qualitative and quantitative alterations of mitochondrial DNA. Next, it would be interesting to determinate the role of mitochondrial CYP2E1 in these DNA lesions using the cellular model developed in our first work. If further studies confirm the implication of mitochondrial CYP2E1 in the development of hepatic injury, it would be interesting to specifically address antioxidants or inhibitors to mitochondria

L'obésité et les maladies du foie associées (NAFLD) augmentent le risque et la sévérité de l’hépatotoxicité induite par certains xénobiotiques, mais les mécanismes impliqués sont encore mal compris. Pour l'éthanol et le paracétamol (APAP), le rôle du cytochrome P450 2E1 (CYP2E1) hépatique est suspecté car l'activité de cette enzyme est augmentée au cours de ces pathologies dysmétaboliques. Le 1er objectif de notre travail expérimental a été de mettre au point un modèle cellulaire de NAFLD caractérisé non seulement par l'accumulation de triglycérides mais aussi par l’augmentation de l'activité du CYP2E1. Pour cela, des cellules humaines HepaRG différenciées ont été incubées pendant une semaine avec de l'acide stéarique ou de l'acide oléique, en présence de 3 concentrations différentes d'insuline. Les triglycérides cellulaires et l'expression de gènes induits au cours de la stéatose étaient similaires avec les deux acides gras. Cependant, l'activité du CYP2E1 était significativement augmentée uniquement par le stéarate et ceci était associé à une diminution de l'activité du CYP3A4, une autre caractéristique des NAFLD. L’activité du CYP2E1 dans les cellules HepaRG était réduite par l'insuline d'une manière concentration-dépendante et cet effet était reproduit sur des hépatocytes humains en culture primaire. Ainsi, l'activité du CYP2E1 était la plus élevée dans les cellules HepaRG cultivées avec du stéarate et sans insuline. Le 2ème but de notre étude était ensuite d'évaluer la cytotoxicité de l’APAP sur des cellules HepaRG présentant ou non une stéatose et une induction du CYP2E1. Des expériences avec une large gamme de concentrations d’APAP (de 1 à 20 mM) indiquaient que la perte cellulaire d'ATP et du glutathion (GSH) était presque toujours plus forte en présence de stéarate. Dans les cellules prétraitées avec le chlorméthiazole (CMZ, un inhibiteur du CYP2E1), la moindre diminution d’ATP était plus importante en présence de stéarate, avec de faibles (2,5 mM) ou de fortes (20 mM) concentrations d’APAP. Cependant, en l'absence d'insuline, la moindre chute d’ATP induite par le CMZ était significativement plus forte uniquement pour 20 mM d’APAP. Étonnamment, suite au prétraitement par le CMZ, il n'y avait pas de protection vis-à-vis de la diminution du GSH et de la formation des adduits APAP-protéines. Enfin, les concentrations du métabolite APAP-glucuronide étaient significativement augmentées en présence d'insuline. Ainsi, lorsqu’elle est étudiée dans des conditions spécifiques de culture, la lignée cellulaire HepaRG semble être un modèle intéressant de NAFLD, notamment en ce qui concerne les activités du CYP2E1 et du CYP3A4. Nos données suggèrent aussi que l’induction du CYP2E1 observée au cours des NAFLD pourrait être secondaire à l'accumulation de certains acides gras et à la présence d’une faible signalisation insulinique dans le foie. Ainsi, ce modèle cellulaire peut être utilisé pour mettre en évidence les principaux facteurs métaboliques et hormonaux favorisant hépatotoxicité de l’APAP chez les personnes obèses. Cette thèse inclut également une revue de la littérature sur l’hépatotoxicité de l’APAP dans le contexte de l’obésité et des NAFLD (Michaut et al., Liver Int 2014)
Obesity and nonalcoholic fatty liver disease (NAFLD) are able to increase the risk and the severity of hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are still poorly understood. For toxic compounds such as ethanol and acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during obesity and NAFLD. The first aim of our experimental study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, differentiated human HepaRG cells were incubated during one week with stearic acid, or oleic acid, in the presence of 3 different concentrations of insulin. Cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids. However, CYP2E1 activity was significantly increased only by stearate and this was associated with lower CYP3A4 activity, another metabolic feature reported in NAFLD. CYP2E1 activity in HepaRG cells was reduced by insulin in a concentration-dependent manner and this effect was reproduced in cultured primary human hepatocytes. Hence, the highest CYP2E1 activity was observed in HepaRG cells with stearate and without insulin. Next, the second aim of our study was to assess APAP cytotoxicity in HepaRG cells presenting or not lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations (1 to 20 mM) showed that the cellular loss of ATP and glutathione (GSH) was almost always stronger in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole (CMZ), recovery of cellular ATP was significantly higher in the presence of stearic acid with both low (2.5 mM) and high (20 mM) concentrations of APAP. However, in the absence of insulin, CMZ-induced ATP recovery was significantly greater only for 20 mM of APAP. Surprisingly, there was no recovery of cellular GSH and no reduction of APAP-protein adducts following CMZ pretreatment. Finally, levels of APAP-glucuronide were significantly enhanced in the presence of insulin. Hence, when studied in specific conditions of culture, the HepaRG cell line can be a valuable model of human NAFLD, especially regarding CYP2E1 and CYP3A4 activity. Our data also suggest that higher CYP2E1 activity in NAFLD could be secondary to the hepatic accumulation of some fatty acids and to the presence of low insulin signaling. This cellular model can be thus used to unveil the main metabolic and hormonal factors favoring APAP hepatotoxicity in obese individuals. This thesis also includes a review on APAP hepatotoxicity in the context of obesity and NAFLD (Michaut et al., Liver Int 2014)

Orientador: Carmen Silvia Bertuzzo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O Câncer colorretal (CCR) refere-se a uma neoplasia que atinge o cólon e o reto, apesar das diferenças epidemiológicas e biológicas entre o câncer do cólon e reto, as duas condições são combinadas, pois não se faz uma separação clara dos dois locais anatômicos; assim, o câncer do colón e/ou do reto é classificado como câncer colorretal. Dados epidemiológicos atestam a importância de fatores ambientais na patologia do CCR Esporádico. Os polimorfismos de P450 e a suscetibilidade ao câncer podem estar associados ainda, pelo fato dessas isoenzimas participarem também de transformação de compostos endógenos relevantes durante o processo de diferenciação da célula transformada, até o estágio maligno. O objetivo deste trabalho foi avaliar a prevalência dos polimorfismos m1 e m2 no gene CYP1A1, 2C9*2 e 2C9*3 no gene CYP2C9 e o 1B1*3 no CYP1B1 e correlacionar a presença desses polimorfismos com o hábito tabagista e o estadiamento clínico da doença. A análise dos polimorfismos foi realizada por meio da Reação em Cadeia da Polimerase associada a digestões enzimáticas específicas. Foram avaliados 102 pacientes com CCR esporádico e 230 portadores de sangue sem história familial de CCR. Nossos resultados indicaram que indivíduos sem os alelos M1 e M2 do gene CYP1A1 teriam um efeito protetor com relação ao CCR, enquanto que os indivíduos portadores de um alelo M1 ou M2 e os homozigotos M2 teriam um aumento de risco que seriam respectivamente de 6, quase 3 (2,89) e quase 4 vezes (3.70). Com relação ao gene CYP2C9, o genótipo N/N (sem as mutações *2 e *3) teria um efeito protetor, enquanto que o heterozigoto *3 traria um risco 6 vezes maior (5.90) e o homozigoto *3 , três vezes. A presença dos alelos M1 e M2 parecem estar relacionados a um pior prognóstico da doença, enquanto que *3 do CYP2C9 traria um melhor prognóstico. Maiores estudos são necessários para confirmar esses achados. No caso do polimorfismo 1B1*3 do gene CYP1B1, parece não haver relação com o fenótipo CCR
Abstract: The colorectal cancer (CRC) characterizes a neoplasia which reaches the colon and the rectum tissues. It is classified as so, despites the biological and epidemiological differences between the colon and rectum cancer, because there is not a clear anatomical separation of these areas. The importance of external factors in the pathology of the sporadical CRC has been certified by epidemiological data. Thus, P450 polymorphisms might be associated to cancer due to the fact that these proteins play a role in transforming endogenous compounds during the process of differentiation of transformed cells, until its malignant stage. The objective of this work was to evaluate the prevalence of the polymorphisms M1 and M2 in CYP1A1 gene, 2C9*2 and 2C9*3 in CYP2C9 gene and 1B1*3 in CYP1B1 gene. Also, the correlation among the presence of these polymorphisms, the tobaccoism and the clinical stagnancy of the illness was performed. Polymerase chain reactions (PCRs) followed by specific enzymatic digestions were performed in order to analyze the polymorphisms. We evaluated 102 patients with sporadical CRC and 230 control individuals without familial history of CRC. Our results indicated that individuals that did not carry M1 and M2 variants of CYP1A1 gene presented a protective effect against CRC, while individuals who presented M1 or M2 variants had a 6 and almost 3 (2.89) times increasing of the risk, respectively, while patients with both M2 alleles had an almost 4 times (3.70) increasing risk. Analysis of CYP2C9 gene showed that genotype N/N (without * 2 and * 3 mutations) presented a protective effect, while individuals heterozygotes for * 3 mutation had an increase close to 6 times (5.90) of their risk. Homozygotes for the same mutation would present a 3 times increased risk. M1 and M2 alleles presence seems to be related to a worse prognostic of the illness, while the presence of * 3 mutation in CYP2C9 gene would result in a better prognostic. Other studies are necessary to confirm these findings. Polymorphism 1B1*3 of CYP1B1 gene does not seem to be related to CRC phenotype
Mestrado
Mestre em Farmacologia

Il a été montré que le diabète augmentait l'expression des CYP 2E1 et 2B1 de rat au niveau ARN et protéines. Cette élévation a été attribuée à une stabilisation des ARNm et peut être inversé par un traitement quotidien à l'insuline. En utilisant une lignée d'hépatome de rat, il a été précédemment montré au laboratoire que cette hormone agissait par un mécanisme post-transcriptionnel en diminuant la durée de vie des messagers des CYP2E1 et 2B1 de rat. Nous avons entrepris d'identifier les mécanismes moléculaires mis en jeu dans cette régulation. En utilisant des protéines cytoplasmiques provenant de cellules d'hépatome de rat traitées ou non avec l'insuline (10-7 M) et l'ARNm entier des CYP2E1 ou 2B1 marqué au 32P comme sonde, nous avons, par des expériences de protection à la RNase T1, mis en évidence un complexe majoritaire ARN/protéines. Grâce aà des fragments de ces ARNm puis des expériences de compétition avec des oligonucléotides antisens complémentaires, le site de liaison des protéines a été localisé pour chacun des CYP sur une séquence de 16 nucléotides, présentant entre-elles une homologie de 80%. . . .

Acute lymphoblastic leukemia (ALL) is the most common type of cancer affecting children in the world and in our country. The exact molecular etiology of the disease still remains to be elucidated. This study hypothesized that four genes, namely CYP2E1*5B, *6, and *7B, NQO1*2 SNPs, GSTM1 null and GSTT1 null, alone or in combination, could contribute to the risk of development of childhood ALL. Also interactions of these polymorphisms with non-genetic risk factors were investigated. The genotyping of these polymorphisms were done on 209 healthy subjects, and 185 patients with childhood ALL, in Turkish population. Venous blood samples were collected and genomic DNA was isolated from these samples. Genotyping was done by PCR-RFLP techniques. In the case-control analyses for the risk of development of childhood ALL, only GSTT1 null was found to be associated with the development of disease (OR= 1.8, p=0.01). CYP2E1*5B and *6 combination showed an increased risk of 2.7 fold (p= 0.04). Also co-presence of CYP2E1*6-GSTT1 and CYP2E1*7B-GSTT1 polymorphisms increased the risk significantly above 4.0 fold. The risk increased more to 7.6 fold, when CYP2E1*5B,*6 and GSTT1 null were considered together, with borderline significance (p=0.04). When interaction of exposure to cigarette smoke and genetic polymorphisms were investigated, NQO1*2 and GSTM1 null were turned out to be significant risk factors for the development of disease when the parental or child&rsquo
s postnatal exposure to cigarette smoke was considered. This study presented several new findings to the literature in terms of genetic epidemiology of childhood ALL. The present work would also contribute to public health in determining the susceptibility of the Turkish population to childhood ALL.

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Introduction: The xenobiotics are exogenous substances to the organism, as N-nitrosamines, heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs), can which result in DNA adducts formation. Polymorphisms in genes involved in the metabolism of xenobiotics could contribute to this process and modulate the development of cancer. Objectives: To investigate the CYP1A1*2A (rs4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs2031920), CYP1E1*6 (rs6413432), Tyr113His EPHX1 (rs1051740) and His139Arg EPHX1 (rs2234922) polymorphisms related to the metabolism of xenobiotics, the risk of sporadic colorectal (SCRC) cancer, the interaction of these polymorphisms with lifestyle (smoking and drinking) and clinical and histopathological parameters and to evaluate the association of SCRC with socio-demographic factors. Methods: A case-control study was conducted in 641 subjects in the Brazilian population (241 patients with colorectal cancer and 400 controls (individuals without a history of cancer). Real-Time PCR and PCR-RFLP was performed for genotyping. Statistical analysis was performed using the chi-square tet and multiple logistic regression binary. Results: The results showed statistically significant differences between the case and control groups for age greater than 50 years (OR=8.21, 95%CI=5.49-12.28, p<0.01) and male gender (OR=0.50, 95%CI=0.32-0.87, p<0.01) The analysis of polymorphisms revealed an association between the alleles polymorphic CYP2E1*5B (OR=2.84, 95%CI=1.78-4.52, p<0.01, additive model) and CYP2E1*6 (OR=2.78, 95%CI=1.91-4.06, p<0.01, additive model) and the SCRC. Tumor size, lymph node involvement and disease primary site were not associated with polymorphisms. Conclusion: The CYP2E1*5B and CYP2E1*6 polymorphisms are involved in the risk of SCRC and individuals with age ≥ 50 years are more susceptible to this tumor type, of males are less susceptible.
Introducão: Os xenobióticos são substâncias exógenas ao organismo, tais como as N-nitrosaminas, aminas heterocíclicas (HAs) e hidrocarbonetos policíclicos aromáticos (HPAs), que podem formar adutos de DNA. Polimorfismos em genes envolvidos no metabolismo dos xenobióticos podem contribuir com este processo e, consequentemente, modular o desenvolvimento de câncer. Objetivos: Investigar os polimorfismos CYP1A1*2A (rs 4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs 2031920), CYP1E1*6 (rs 6413432), EPHX1 Tyr113His (rs1051740) e EPHX1 His139Arg (rs2234922), relacionados com o metabolismo dos xenobióticos, no risco de câncer de colorretal esporádico (CCRE), a interação desses polimorfismos com os hábitos de vida (tabagismo e etilismo) e parâmetros clínico-histopatológicos e avaliar a associação do CCRE com os fatores sócio-demográficos. Os Métodos: Um estudo caso-controle foi realizado em 641 indivíduos da população brasileira (241 pacientes com câncer de coloretal e 400 controles (indivíduos sem histórico de câncer). As técnicas de PCR em Tempo Real e PCR-RFLP foram realizadas para a genotipagem dos polimorfismos. A análise estatística utilizou os testes de Qui-Quadrado e Regressão Logística Múltipla Binária. Resultados: Os resultados mostraram diferenças estatisticamente significantes entre os grupos caso e controle para idade superior a 50 anos (OR=8,21; IC95%=5,49-12,28, p<0,01) e gênero masculino (OR=0,50; IC95%=0,32-0,87, p<0,01). A análise dos polimorfismos revelou associação entre os alelos polimórficos CYP2E1*5B (OR=2,84; IC95%=1,78-4,52; p<0,01, modelo aditivo) e CYP2E1*6 (OR=2,78; IC95%=1,91-4,06, p<0,01, modelo aditivo) e o CCRE. O tamanho do tumor, envolvimento de linfonodos e sítio primário da doença não foram associados com os polimorfismos. Conclusão: Os polimorfismos CYP2E1*5B e CYP2E1*6 estão envolvidos no risco de CCRE e indivíduos com idade superior ou igual a 50 anos são mais suscetíveis a este tipo tumoral, enquanto aqueles do gênero masculino são menos suscetíveis.

Stroke, a major cause of death and disability, is described as interruption or severe reduction of blood flow in cerebral arteries. Oxidative stress plays an important role in the pathogenesis of atherosclerosis and carotid atherosclerosis is a risk factor for stroke. Combination of multiple environmental and genetic risk factors is thought to increase susceptibility to the development of this disease. Therefore, investigation of the polymorphisms of drug metabolizing enzymes is of crucial importance to determine the molecular etiology of the disease. The main objective of this study was to investigate the possible association between polymorphisms of enzymes causing oxidative stress (CYP2E1, FMO3 and NOS3) and enzymes protecting against oxidative stress (GST and NQO1), and the pathogenesis of atherosclerosis and ischemic stroke risk. The study population consisted of 245 unrelated ischemic stroke patients and 145 healthy control subjects. There was no statistically difference between the patient and control groups in terms of age and gender. Hypertension, diabetes, smoking and obesity were found to be at least 2 times more common in stroke patients than controls. While total cholesterol, triglyceride and LDL-cholesterol level were higher in stroke patients, HDL-cholesterol level was lower in stroke patients when compared to controls. In the case-control analyses for the risk of ischemic stroke, CYP2E1*5B mutant allele, *5B was found to be associated with the development of disease (Odds Ratio
OR=7.876, 95%CI=1.025-60.525, P=0.019). In addition, significant difference was observed between stroke patients and controls with respect to CYP2E1*5B genotype distribution (OR=0.869, 95%CI=1.044-62.339, P=0.017). On the other hand, in the NQO1*2 polymorphism, together with NQO1 heterozygote (*1*2), NQO1 homozygote mutant (*2*2) genotype was found protective against ischemic stroke (OR=0.627, 95%CI=0.414-0.950, P=0.027). The risk of hypertensive individuals having stroke was highest in the FMO3 472GA group (OR=6.110, P=0.000). In diabetics, GSTP1 313AG genotype was found to be the highest risk factor for stroke (OR=3.808 P=0.001). On the other hand, NQO1 *1*2 heterozygote genotype was associated with 5 times increased risk for stroke in smokers (OR=5.000, P=0.000). In addition GSTM1 present genotype constituted 8 times increased stroke risk in obese individuals (OR=8.068, P=0.001). Logistic regression analysis revealed that hypertension, diabetes mellitus, obesity and smoking were significant risk factors for stroke. On the other hand, HDL-cholesterol and having NQO1 *1*2 heterozygote genotype were found to be protective factors against stroke.

Differentiated thyroid carcinoma (DTC) results from complex interactions between genetic and environmental factors. Known etiological factors include exposure to ionizing radiations, previous thyroid diseases, and hormone factors. It has been speculated that dietary acrylamide (AA) formed in diverse foods following the Maillard's reaction could be a contributing factor for DTC in humans. Upon absorption, AA is biotransformed mainly by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA). Considering that polymorphisms within CYP2E1 were found associated with endogenous levels of AA-Hb and GA-Hb adducts in humans, we raised the hypothesis that specific CYP2E1 genotypes could be associated with the risk of DTC. Analysis of four haplotype tagging SNPs (ht- SNPs) within the locus in a discovery case-control study (N=350/350) indicated an association between rs2480258 and DTC risk. This ht-SNP resides within a linkage disequilibrium block spanning intron VIII and the 3'-untranslated region. Extended analysis in a large replication set (2429 controls and 767 cases) confirmed the association, with odds ratios for CT and TT genotypes of 1.24 (95 % confidence interval (CI) 1.03-1.48) and 1.56 (95 % CI, 1.06-2.30), respectively. Functionally, the minor allele was associated with low levels of CYP2E1 mRNA and protein expression as well as lower enzymatic activity in a series of 149 human liver samples. This association suggested that the risk of DTC could, at least in part, be ascribed to AA. We performed an in vivo study to assess the association of AA, GA and their ratio with SNPs of genes encoding for the metabolizing enzymes CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1 in 60 DTC patients and 60 healthy controls. The ratio of GA-Hb/AA-Hb was found associated in a statistically significant way with rs2480258 and the null genotype of GSTM1. The GSTM1 null genotype was also associated with high GA-Hb levels. The multiple regression analysis showed that the joint effect of CYP2E1 and GSTs was significantly associated with the ratio of GA-Hb/AA-Hb levels. Thus, present results not only confirmed previous findings on the role of GSTs in the AA metabolism but reinforced the notion that rs2480258 is a marker of the functional activity of CYP2E1. Since we didn't find any correlation between DTC and AA we therefore extended the human study by performing a screening of 19 other compounds, both identified and unidentified, observed as Hb adducts to N-terminal valine. The adductomics screening of the 19 compounds revealed that the levels of Methyl vinyl ketone and Ethyl vinyl ketone were significantly higher in controls and the levels of 575 m/z and 561_23 m/z were significantly higher in cases. Furthermore, a significant association was also found between the two unknown compounds and Acrylic acid with CYP2E1. Further studies are needed to completely understand the correlation between the different compounds, health status and SNPs.

As enzimas CYP2D6, CYP2C19 e CYP2C9 são responsáveis pelo metabolismo de aproximadamente metade dos 200 medicamentos mais prescritos nos EUA. Padronizamos ensaios de genotipagem baseados na discriminação alélica com o sistema TaqMan® em 198 indivíduos. Para o gene CYP2D6, os alelos *1 e *2 foram os mais freqüentes, seguidos pelos alelos *4, *41, *35, *17, *5, *10, *6, *29 e *9. Desenvolvemos também uma nova metodologia para a determinação do número de cópias do gene CYP2D6. Para o gene CYP2C19, o alelo *1 foi o mais frequente, seguido pelos alelos *17, *2 e *3. Nosso estudo foi o primeiro a determinar a freqüência alélica do gene CYP2C19 no Brasil. Para o gene CYP2C9, o alelo *1 foi o mais frequente, seguido pelos alelos *2 e *3. Desenvolvemos uma metodologia reprodutível e acessível para a genotipagem dos polimorfismos principais dos genes CYP2D6, CYP2C19 e CYP2C9. A identificação precoce de indivíduos suscetíveis a efeitos adversos, bem como de metabolizadores rápidos pode trazer grandes benefícios aos pacientes possibilitando assim uma medicina personalizada.
The enzymes CYP2D6, CYP2C19 and CYP2C9 metabolize approximately half of the 200 most prescribed drugs in the USA. We standardized genotyping tests based on allelic discrimination, using TaqMan® genotyping system in 198 samples. For the CYP2D6 gene, allele *1 and *2 were the most frequent, followed by alleles *4, *4, *35, *17, *5, *10, *6, *29 and *9. We have also developed a new methodology for determining the copy number variations of the CYP2D6 gene. For the CYP2C19 gene, the allele *1 was the most common, followed by the alleles *17, *2 and *3. In our concern, our study was the first to determine the allele frequency of the CYP2C19 gene in Brazil. For CYP2C9 gene, the allele *1 was the most common followed by the alleles *2 and *3. We developed a methodology reproducible and accessible for genotyping the most important polymorphisms of the genes CYP2D6, CYP2C19 and CYP2C9. The previous identification of individuals at risk to develop adverse drug reactions as well as ultrarapid-metabolizers may bring benefits to the patients, leading to a personalized therapy.

Polimorfismos nos genes da n-acetiltransferase 2 (NAT2), CYP2E1 e glutationa S-transferase (GST) têm sido associados a diferenças na resposta ao tratamento da tuberculose. O papel de variantes dos genes NAT2, CYP2E1 e GSTM1/GSTT1, no perfil de segurança do tratamento da tuberculose, foi avaliado em 99 pacientes com tuberculose, sem co-infecção por HIV ou vírus da hepatite, tratados por 6 meses. Amostras de sangue foram colhidas antes e durante o tratamento para avaliação de marcadores de lesão hepatocelular (ASLT e AST), colestase (ALP, GGT e bilirrubinas) e função renal (creatinina). O DNA genômico foi extraído de sangue colhido em EDTA pelo método precipitação salina. Os polimorfismos NAT2 foram analisados por PCR-RFLP e seqüenciamento de DNA. Os polimorfismos da região promotora do CYP2E1 foram detectados por PCR-RFLP e para a análise dos genótipos nulos de GSTM1 (GSTM1*0) e GSTT1 (GSTT1*0) foi utilizada a PCR multiplex. Durante o tratamento, 59,6% dos pacientes apresentaram reações adversas aos medicamentos (RAM) e alterações nos marcadores de lesão hepatocelular e colestase, com aumento de 1 a 4 vezes o limite superior de referência. Foi observada forte relação entre RAM e alterações nos marcadores séricos (p< 0,05) e também com o uso de medicação concomitante (p< 0,001). As freqüências dos alelos NAT2*4 e NAT2*6 foram maiores e menores, respectivamente, quando comparadas com outros estudos na população brasileira. O perfil de acetilador lento (alelos NAT2*5, NAT2*6 e NAT2*7) foi associado com manifestação de RAM e hepatotoxicidade. Os portadores dos genótipos NAT2*4/*5 e NAT2*5/*5 apresentaram, respectivamente, risco 2,4 e 5,0 vezes maior de RAM que os portadores dos demais genótipos NAT2 (p< 0,05). O genótipo funcional GSTM1*1/GSTT1*1 foi associado com alterações acentuadas de ALT, AST e ALP (p< 0,05). Enquanto que as variantes da CYP2E1 não foram associadas a alterações no perfil bioquímico ou com risco de RAM ou hepatotoxicidade. Em conclusão, o perfil de acetilação lenta de NAT2 e o genótipo funcional de GSTM1/GSTT1 aumentam a susceptibilidade de lesão hepatocelular e outras RAM induzidas pelos antimicobacterianos utilizados no tratamento da tuberculose.
Polymorphisms in N-acetiltransferase 2 (NAT2), CYP2E1 and glutatione S-transferase (GST) have been associated with differences in response to antituberculosis drugs. The role of the NAT2, CYP2E1 and GSTM1/GSTT1 variants on safety profile of the anti-tuberculosis therapy was evaluated in 99 tuberculosis patients, without co-infection by HIV or hepatitis virus, treated during 6 months. Blood samples were collected before and after the therapy to evaluate serum markers for hepatocelullar damage (ASLT and AST), cholestasis (ALP, GGT and bilirrubin) and kidney function (creatinine). Genomic DNA was extracted from EDTA-blood samples by salting-out method. NAT2 polymorphisms were analyzed by PCR-RFLP and DNA sequencing. CYP2E1 promoter region polymorphisms were detected by PCR-RFLP and for analysis of the null genotypes GSTM1 (GSTM1*0) e GSTT1 (GSTT1*0) PCR multiplex technique was used. During the therapy, 59.6% of the patients had adverse drug reactions (ADR) and alterations on hepatocellular damage and cholestasis serum markers, with increase of 1 to 4 times the upper limit reference level. There was a significant relationship between ADR and serum markers alterations (p< 0,05), as well as, the concomitant medicine (p< 0,001). The frequencies of the NAT2*4 and NAT2*6 alleles were higher and lower, respectively, when compared to other studies in the Brazilian population. The slow acetilator profile (NAT2*5, NAT2*6 and NAT2*7 alleles) was associated with ADR and hepatotoxicity manifestations. The NAT2*4/*5 and NAT2*5/*5 genotypes carriers had, respectively, 2.4 and 5.0 times higher risk for ADR than those carrying the other NAT2 genotypes (p< 0,05). The functional genotype GSTM1*1/GSTT1*1 was associated with enhanced variations on ALT, AST and ALP (p< 0.05). No relationship was found between CYP2E1 variants and variations on biochemical profile or risk for ADR or hepatotoxicity. In conclusion, the NAT2 slow acetilator profile and the GSTM1/GSTT1 functional genotype increase the susceptibility to hepatocellular damage and other ADR induced by antibiotics used in tuberculosis therapy.

Le cancer du col de l'utérus est le second cancer féminin au monde et c'est le plus fréquent dans les pays en voie de développement dans lesquels 80% des cas sont identifiés. Des preuves épidémiologiques incontestables indiquent que les papillomavirus humains à haut risque (HPV HR) sont les agents étiologiques des lésions précancéreuses et cancéreuses du col de l'utérus. Dès la fin des années quatre vingt, le développement d'outils de biologie moléculaire a permis d'étudier ces virus et de les détecter dans les frottis cervico-utérins. Dans le cadre du dépistage, la détection des HPV à haut risque avec des techniques validées s'est révélée plus sensible et plus reproductible que la cytologie pour détecter les lésions de haut grade. - Les techniques de détection des HPV disponibles sont basées sur la réaction de polymérisation en chaîne (PCR) ou sur l'hybridation en phase liquide. Une technique d'amplification de signal d'hybridation, test HCII® (Digene), est la seule trousse commerciale disponible qui soit validée et approuvée pour la détection de 13 génotypes d'HPV HR. La société Roche Diagnostics a récemment mis sur le marché une trousse de PCR standardisée pour la détection des 13 génotypes d'HPV HR (Amplicor® HPV Test). - Dans une première étude, nous avons comparé les performances de l'Amplicor® HPV Test avec celles d'HCII® pour la détection des HPV HR à partir de 470 résidus de cytologie liquide prélevés dans Preservcyt®. La concordance globale entre les deux trousses est excellente (96%) avec un kappa de 0,92. Les échantillons avec un résultat d'HPV discordant ont été génotypés à l'aide de la trousse INNO-LiPA® genotyping v2 test (Innogenetics). Tous ces échantillons se sont révélés positifs pour au moins un HPV. En utilisant un résultat de positivité en HPV consensus (au moins deux tests sur trois positifs), la sensibilité (96,6%) et la spécificité (100%) analytiques des deux trousses étaient identiques. Nos données montrent donc que le test Amplicor® HPV est sensible, spécifique et utilisable en routine. - Parce qu'une infection par un HPV est nécessaire mais pas suffisante pour le développement de lésions précancéreuses et cancéreuses du col de l'utérus, nous avons réalisé une seconde étude visant à étudier le polymorphisme de deux enzymes du métabolisme des xénobiotiques, GSTM1 et CYP2E1, chez des femmes chinoises Uygur et françaises présentant ou non des lésions du col de l'utérus. Nous avons montré que le génotype GSTM1 nul était un marqueur génétique de susceptibilité aux lésions précancéreuses chez les femmes Uygur, mais pas chez les femmes françaises. - Enfin, dans un troisième travail, nous avons étudié la prévalence et les génotypes d'HPV présents au niveau de l'œsophage (tissus sains et tumeurs) de patients kazakh présentant un cancer de l'œsophage. Nous avons détecté de l'ADN d'HPV chez 30% des patients, soit dans le tissu sain, soit dans la tumeur. L'HPV16 est le génotype le plus fréquemment identifié. Aucune association entre HPV et grade histologique ou sexe n'a été trouvée. En revanche, il est vraisemblable que les HPV contribuent à la forte prévalence de cette tumeur dans la population kazakh. - L'oncogenèse est un processus complexe qui se déroule en plusieurs étapes. L'exposition à un carcinogène est généralement nécessaire mais pas suffisante pour expliquer la survenue d'un cancer. Plusieurs cofacteurs liés à l'hôte ou à l'environnement vont participer à la transformation progressive des cellules. Une compréhension des interactions entre ces cofacteurs est essentielle pour identifier des facteurs de susceptibilité au cancer et pour proposer de nouvelles approches en matière de dépistage et de traitement
Cervical cancer is the second most common cancer in women worldwide, and it is the main cancer in women living in developing countries where 80 percent of cases occur. Epidemiologic evidences clearly indicate that certain types of human papillomavirus (HPV) –and in particular 15 high risk genotypes – are the principal cause of cervical intraepithelial neoplasia and invasive cervical cancer. Since the end of the Eighties, the development of molecular biology has allowed the detection and the study of these viruses. Detection of this infection with clinically validated tests has been proven to be a more sensitive and more reproducible methods than cytology for screening. - Several HPV DNA detection methods have been developed during the last two decades. The most widely used include Polymerase Chain Reaction (PCR) and liquid hybridization based methods. A signal amplified hybridization microplate-based assay, HCII® (Digene), is the only commercially available assay approved and validated for the simultaneous detection of 13 HR HPV genotypes. Roche Diagnostics has recently proposed a new standardized PCR-based assay (AmplicorCID HPV Test) permitting the detection of 13 HR HPV. - ln our first study, we compared the performance of the AmplicorCID HPV Test with those of Hybrid Capture II® to detect HR-HPV from 470 cervical samples harvested in Preservcyt®. The concordance between the two assays was 96% with Cohen's kappa=0. 92, demonstrating an excellent agreement level. Samples with discordant results were genotyped with the INNO¬LiPA genotyping v2 test (Innogenetics). Ali these samples harboured HPV DNA. By using a consensus HR-HPV result (at least 2 out of 3 positive tests) as our reference HPV positivity, the analytical sensitivity (96. 6%) and specificity (100%) of AmplicorCID was similar to that of HCII®. Our data show that the AmplicorCID HPV Test is sensitive, specific and suitable for routing use. - Because HR-HPV infection is necessary but not sufficient to induce cervical precancerous lesions, we undertook a second study to assess the genetic polymorphisms of GSTM 1 and CYP2E1 in Uygur, China and French women with or without cervical lesions. Our results demonstrated that the GSTM1 null genotype was a genetic biomarker for precancerous lesion susceptibility in Uygur women but not in French women. - Finally, we investigated in a third study the HPV-specific prevalence in the esophagus of Kazakh patients with ESCC. Our data showed that HPV were present in 30% of patients either in the tumor or in the normal tissue. HPV16 was the most prevalent genotype. No association with histological grade and gender was found, but HPV infection might contribute ta the extremely high incidence of ESCC observed in the Kazakh population of western China. - Oncogenesis is a complex and multiple step process. The exposure to a carcinogen is generally necessary but insufficient to lead to the development of cancers. Numerous cofactors linked to the host or to the environment are needed for efficient cellular transformation. A deep comprehension of interactions between the different factors is a key point to explain cancer susceptibility and to propose new screening and therapeutic strategies

Akrilamid  je toksična hemijska supst anca koja je već dugi niz godina prisutna u životnoj sredini,  jer se kao važan monomer koristi u različite industrijske i laboratorijske svrhe. U poslednjih petnaest godina, akrilamid je postao posebno zanimljiv za šire naučne krugove jer  se pokazalo da  se  nalazi  i u  hrani  biljnog porekla, posebno hrani bogatoj skrobom, koja se priprema pečenjem ili prženjem na temperaturama višim od 120°C.  Do sada ustanovljeni negativni zdravstveni efekti akrilamida su veoma raznovrsni i mogu biti rezultat delovanja samog  akrilamida ili delovanja njegovog metabolita glicidamida koji nastaje  in vivo  kada se jedan deo molekula akrilamida metaboliše oksigenacijom dvostruke veze pomoću enzima citohrom P450 2E1 (CYP2E1). Akrilamid je supstanca koja ima dokazan negativan efekat  na organske sisteme kod ljudi i životinja, i koja je svrstana u moguće humane karcinogene. Negativan efekat akrilamida na egzokrini pankreas je poznat, ali o mogućim efektima akrilamida na endokrini pankreas se i dalje veoma malo zna. Ima puno dokaza koji  ukazuju na to da akrilamid ima citotoksični efekat koji se  manifestuje kroz uticaj na redoks-status ćelija i dovodi do promena u vrednostima biomarkera oksidativnog i nitrozativnog stresa, kao i u aktivnosti antioksidativnih enzima. Pankreas  je  jedan od ciljnih  organa za delovanje akrilamida te je  glavni predmet istraživanja  doktorske teze  bio proučavanje potencijalnog efekta akrilamida na endokrini pankreas pacova.  Ispitivanje je vršeno na 3  eksperimentalne grupe  juvenilnih  mužjaka pacova soja Wistar,  od kojih je  jedna grupa bila kontrolna, dok su dve bile tretirane  sa akrilamidom u dozama od 25 mg/kg tm i 50 mg/kg tm,  5 dana nedeljno,  tokom 3 nedelje. Po isteku tretmana,  nakon dekapitacije, kompletno tkivo pankreasa  je  fiksirano u 10% rastvoru formalina  tokom  24  h i obrađeno prema  standardnoj proceduri za kalupljenje u parafinu.  Parafinski kalupi su sečeni na serijske preseke debljine 5 µm, nakon čega su bojeni  histohemijskom i imunohistohemijskim metodama.  Kod eksperimentalnih grupa posmatrane  su  histološke promene na endokrinom pankreasu, sa akcentom na α-  i β-ćelije.  Takođe, posmatrana je  i  ekspresija  hormona insulina i glukagona, enzima inducibilne azot -oksi d  sintetaze (iNOS) i  CYP2E1,  kao  i ekspresija   antioksidativnih enzima  katalaza  (CAT) i superoksid dismut aza 1 i 2  (SOD1 i SOD2)  u ćelijama Langerhansovih ostrvaca. Potencijalna promena u funkcionalnosti β-ćelija je ispitana i kroz analizu nivoa glukoze u serumu pacova tretiranih sa akrilamidom.Budući da β-ćelije čine 80% ćelija koje grade Langerhansova ostrvca pankreasa,  pored in vivo  eksperimenata, ispitana  je  i toksičnost akrilamida na  Rin-5F ćelijsku liniju insulinoma β-ćelija pacova u in vitro uslovima. Glavni cilj in vitro  istraživanja je bio  da se  ispita  uticaj  rastućih  koncentracija akrilamida na preživljavanje tretiranih  Rin-5F  ćelija, ali i efekat IC₅₀  koncentracije ove supstance primenjene  tokom  različitih vremenskih intervala  (0,5, 1, 3, 6, 12 i 24 h)  na pojavu oksidativnog i nitrozativnog stresa. Redoks-status Rin-5F ćelija tretiranih  sa akrilamidom je ispitan preko analize prisustva biomarkera oksidativnog i nitrozativnog stresa, akrivnosti CAT i ukupne SOD, kao i promene u ekspresiji gena za CAT, SOD1, SOD2   i iNOS.  Pored toga, analiziran je i efekat istog tretmana na  ekspresiju gena za insulin, CYP2E1, Bax i Bcl-2. U okviru teze je pokazano da akrilamid ne dovodi do  značajnih promena u histološkoj građi, dijametru i broju Langerhansovih ostrvaca  kod  tretiranih životinja.  Primena stereoloških metoda  je  ukazala  na mikrostrukturne promene na  endokrinom pankreasu na nivou α-  i β-ćelija. U ovoj tezi je po prvi put pokazano da tretman akrilamidom negativno utiče na broj i površinu β-ćelija pankreasa.  U tezi je, takođe,  pokazan  značajan dozno-zavisni pad u prisustvu insulina u β-ćelijama   pankreasa. Uprkos  tome, kod  akrilamidom tretiranih  životinja  nije konstatovana  promena  u  koncentraciji serumske glukoze.  U  ovoj tezi je pokazano da tretman akrilamidom dovodi do   statistički značajnog porasta  u broju α-ćelija  kod životinja koje su primale nižu dozu tretmana, dok se  broj α-ćelija  kod životinja koje su primale višu dozu tretmana  ne razlikuje značajno od kontrole.  Tretman akrilamidom je doveo do značajnog  porasta u količini   prisutnog glukagona  u α-ćelijama pankreasa.Tretman akrilamidom nije doveo do značajne promene u ekspresiji CAT, SOD1 i SOD2 u ćelijama Langerhansovih ostrvaca.  Kod  tretiranih životinja  došlo do značajnog dozno-zavisnog porasta  u ekspresiji  enzima iNOS,  dok je ekspresija  CYP2E1 značajno dozno-zavisno opala  nakon tretmana. U  tezi je pokazano da tretman akrilamidom negativno utiče na vijabilnost Rin-5F ćelija, i utvrđeno je da IC50  koncentracija akrilamida za Rin-5F ćelije iznosi 10 mM.  Rezultati teze pokazuju da tretman akrilamidom u IC₅₀  koncentraciji u Rin-5F ćelijskoj liniji značajno povećava nivo malondialdehida (MDA) nakon tretmana u trajanju od 1, 12 i 24 h.  Isti tretman  značajno smanjuje nivo redukovanog GSH nakon tretmana od 1, 3, 6, 12 i24 h, kao i nivo slobodnih  –SH grupa nakon tretmana od 3 i 6 h. Tretman akrilamidom u IC₅₀  koncentraciji signifikantno pojačava aktivnost CAT nakon tretmana od 1 h, dok tretman u trajanju od 12 h značajno smanjuje aktivnost ovog enzima. Ovaj tretman smanjuje aktivnost SOD nakon 1, 12 i 24 h, dok  tretman u trajanju od 6 h značajno pojačava aktivnost enzima SOD.  U tezi je, takođe, pokazan i veoma značajan porast  u nivou prisutnih nitrita,  koji  je direktno proporcionalan  sa nivoom azot-oksida i nivoom akivnosti enzima iNOS.  Ovaj  nalaz ukazuje na potencijalnu pojavu nitrozati vnog stresa u akrilamidom-tretiranim Rin-5F ćelijama.  U  tezi je po prvi put pokazano da tretman  akrilamidom dovodi do  značajnih  varijacija  u transkripciji gena za iNOS, SOD1, SOD2,  CAT,  CYP2E1,  Bax i Bcl-2 u tretiranim Rin-5F ćelijama, dok isti tretman ne dovodi do  promene nivoa  transkripcije gena za insulin.  Tretman akrilamidom u koncentraciji od 10mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNKgena za iNOS u svim tačkama tretmana, do porasta  nivoa  iRNK za SOD1 i SOD2 nakon tretmana od 12 i 24 h, kao i do porasta  količine  iRNK za CAT nakon tretmana od 3 h.  U  tezi je pokazano  i  da akrilamid  izaziva  promene  u sintezi  iRNK  za enzim  CYP2E1  koji je  posebno značajan u kontekstu detoksikacije ove toksične supstance.  Porast u transkripciji gena za  CYP2E1  je uočen  nakon tretmana u trajanju od 0,5 i 1 h, dok je  do smanjenja transkripcije  došlo  nakon tretmana od 12  i 24  h.  Tretman akrilamidom u koncentraciji od  10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK  gena za Bax u svim tačkama tretmana, i do porasta u transkripciji gena za Bcl-2 nakon tretmana od 0,5, 1 i 3 h.Sumirajući  sve  rezultate  ove teze,  moze se zaključiti  da je endokrini pankreas  jedno od  ciljnih tkiva, na koje akrilamid ostvaruje višestruki negativni uticaj.
Acrylamide is a toxic chemical used as an important monomer for various industrial and laboratory purposes, which makes it highly present in the environment. In the last fifteen years, acrylamide has become especially interesting for wider scientific circles when it was found in staple foodstuff rich in starch, prepared at temperatures higher than 120°C. The established negative health effects of acrylamide are very diverse and can be the result of the acrylamide action itself or the action of its metabolite glycidamide that occurs in vivo, when acrylamide molecule is metabolized via oxygenation of the double bond by the cytochrome P450 2E1 (CYP2E1). Acrylamide is a substance with a proven adverse effect on humans and animals, and it is classified as a possible human carcinogen. The negative effect of acrylamide on the exocrine pancreas has already been recognized, but the possible effects of acrylamide  on endocrine pancreas are still mostly undetermined. There is a significant amount of evidence to suggest that acrylamide exerts a cytotoxic effect which manifests through the changes in level of oxidative and nitrosative stress biomarkers, as well as in the activity of antioxidant enzymes. Since, pancreas is one of the target organs for acrylamide, the main subject of doctoral thesis was to investigate the potential effect of acrylamide on the rat endocrine pancreas. The investigation was conducted on 3 experimental groups of juvenile male Wistar rats, of which one group was the control group, while two groups were treated with acrylamide at doses of 25 mg/kg bw and 50 mg/kg bw, 5 days a week, during 3 weeks. After termination of the treatment, decapitation was performed, and the complete pancreatic tissue was fixed in a 10% formalin solution for 24 h and treated according to the standard paraffin embedding procedure. Paraffin molds were cut into 5 μm thick serial sections, after which they were stained with histochemical and immunohistochemical methods. Histological changes ofthe endocrine pancreas, with the emphasis on α- and β-cells, were examined in three experimental groups of rats. In addition, the expression of insulin and glucagon hormone, the inducible nitric oxide synthase (iNOS) and CYP2E1 enzymes, and the expression of antioxidative enzymes catalase (CAT) and superoxide dismutases 1 and 2  (SOD1 and SOD2) in the islets of Langerhans were also investigated. A potential change in the functionality of β-cells was also examined by analyzing glucose level in the serum of rats treated with acrylamide. In pancreatic islets of Langerhans the majority of cells (>80%) are β-cells. Therefore, in addition to in vivo experiments, the toxicity of acrylamide was examined in vitro on rat insulinoma Rin-5F cell line.The main goal of in vitro research was to investigate the impact of increasing acrylamide concentrations on the viability of treated Rin-5F cells, and also to examine whether IC50 concentration of this substance, applied at different intervals of time (0.5, 1, 3, 6, 12 and 24 h), induce oxidative and nitrosative stress. Redox-status of Rin-5F cells treated with acrylamide was examined by analyzing oxidative and nitrosative stress biomarkers, CAT and total SOD activity, as well as changes in the expression of the CAT, SOD1, SOD2 and iNOS. In addition, the effect of the same treatment on the transcription of the insulin, CYP2E1, Bax and Bcl-2 gene was analyzed.The results of the thesis showed that acrylamide treatment does not lead to significant changes in the histological structure, diameter and number of islets of Langerhans of treated animals. Application of stereological methods indicated microstructural changes of α- and β-cells ofendocrine pancreas. It has been shown for the first time that treatment with acrylamide negatively affects the number and surface area of pancreatic β-cells. In addition, a significant dose-dependent decline in the amount of insulin in pancreatic β-cells was also demonstrated. However, no change in serum glucose level was observed in treated animals. Acrylamide treatment led to a statistically significant increase in the number of α-cells in animals receiving a lower dose of treatment, while the number of α-cells in animals receiving a higher dose of treatment did not differ significantly from the control. Treatment with acrylamide led to a significant increase in the amount of the glucagon in α-cells. Treatment with acrylamide did not cause a significant change in the expression of CAT, SOD1 and SOD2 in islets of Langerhans. However, there was a significant dosedependent increase in the  expression of iNOS enzyme, whereas expression of CYP2E1 significantly decreased in dose-dependent manner in treated animals. Results of the thesis showed that acrylamide exerts a negative effect on the viability of Rin-5F cell line. It has been established that the IC50 concentration of acrylamide for the Rin-5F cell line is 10 mM. The results of the thesis indicate that treatment of Rin-5F cell line with IC50 concentration of acrylamide for 1, 12, and 24 h significantly increased the level of malondialdehyde (MDA). Exposure to acrylamide for 1, 3, 6, 12 and 24 h significantly decreased the level of reduced GSH, while the level of free -SH groups was reduced after 3 and 6 h of acrylamide treatments. Treatment with IC50 concentration of acrylamide significantly enhanced CAT activity after 1 h of acrylamide exposure, while 12 h exposure significantly reduced the activity of this enzyme. Application of acrylamide reduced SOD activity after 1, 12, and 24 h exposure, while 6 h exposure significantly increased the activity of SOD enzymes. Results of the thesis also showed a very significant increase of the nitrite level, which is directly proportional to the level of nitrogen oxide (NO) and the level of the iNOS activity. This finding points to the potential occurrence of nitrosative stress in acrylamide-treated Rin-5F cells. It has been shown for the first time that acrylamide treatment leads to significant variations in transcription of iNOS, SOD1, SOD2, CAT, CYP2E1, Bax and Bcl-2 genes in treated Rin-5F cells, while the same treatment does not affect transcription of the insulin gene. Treatment with acrylamide at a concentration of 10 mM for increasing periods of time leads to an increase in the relative amount of the iNOS gene iRNA at all treatment points. Twelve and and 24 h of acrylamide exposure increased the transcription of the SOD1 and SOD2 genes. Transcription of CAT gene was increased after 3 h  ofacrylamide exposure. Furthermore, it has been shown that acrylamide treatment leads to variations in the mRNA synthesis of CYP2E1 gene, which is particularly significant in the context of detoxification of this toxic substance. An increase in the transcription ofthe CYP2E1  gene was observed after 0.5 and 1 h of acrylamide exposure, while the reduction of  transcription occurred after 12 and 24 h of acrylamide exposure. The treatment with 10 mM acrylamide has led to an increase of the transcription of the Bax gene at all treatment points, and also to an increase of transcription of the Bcl-2 gene after of 0.5, 1, and 3 h of acrylamide exposure. Summarizing all the results of this thesis, it can be concluded that the endocrine pancreas  is one of the target tissues of acrylamide, to which this substance exerts a multiple adverse effects.

Mehrere Studien wiesen eine Beteiligung der Enzyme CYP2D6, CYP2C19 und CYP2C9 am Metabolismus von trizyklischen Antidepressiva nach. Wir untersuchten die Auswirkungen genetischer Polymorphismen dieser Enzyme auf die Pharmakokinetik von E-, Z-Doxepin und Trimipramin beim Menschen. Eine einzelne orale Dosis von jeweils 75 mg Trimipramin und Doxepin wurde 42 gesunden Probanden verabreicht, die als Schnell- (EM), Intermediär- (IM) und Langsammetabolisierer (PM) von CYP2D6- und CYP2C19-Substraten und als Langsammetabolisierer mit dem CYP2C9-Genotyp *3/*3 genotypisiert worden waren. Die Substrate sowie ihre aktiven Metaboliten wurden mittels HPLC im Plasma gemessen, Daten wurden mit nonparametrischen pharmakokinetischen Methoden analysiert und statistisch ausgewertet. Die mittlere E-Doxepin-Clearance (95%-KI) betrug 406 (390-445), 247 (241-271) and 127 (124-139) l/h bei CYP2D6-EMs, -IMs und –PMs und war auch bei Trägern des CYP2C9*3/*3-Genotyps signifikant niedriger (238 l/h). CYP2C19 beeinflusste die orale Clearance von Z-Doxepin um das 2,5-fache (73 l/h in CYP2C19-PMs verglichen mit 191 l/h bei EMs). Die AUC (0-48h) des aktiven Metaboliten Desmethyldoxepin war vom CYP2D6-Genotyp abhängig mit einem Median von 5,28, 1,35 und 1,28 nmol/l*h bei CYP2D6-PMs, -IMs und –EMs. Die mittlere orale Trimipramin-Clearance betrug 276 l/h (180-444) in der Referenzgruppe aber nur 36 l/h (24-48) bei CYP2D6-PMs. Die AUC von Desmethyltrimipramin war 40-fach höher bei CYP2D6-PMs als bei EMs (1,7 verglichen mit 0,04 mg/l*h bei EMs), aber unter der Nachweisgrenze bei den meisten Probanden mit CYP2C19- oder CYP2C9-Defizienz. Der CYP2D6-Polymorphismus wies eine starke Auswirkung auf die Pharmakokinetik von E-Doxepin und Trimipramin sowie eine ausgeprägte Stereoselektivität bei der Biotransformation von Doxepin auf. CYP2D6-PMs sind möglicherweise einem erhöhten Risiko für unerwünschte Nebenwirkungen ausgesetzt bei der Behandlung mit den EMpfohlenen Dosen dieser Antidepressiva.
Several studies have demonstrated involvement of the enzymes CYP2D6, CYP2C19 and CYP2C9 in the metabolism of tricyclic antidepressants. We studied the effects of genetic polymorphisms in these enzymes on E-,Z-doxepin and trimipramine pharmacokinetics in humans. A single orale dose of each 75 mg timipramine and doxepin was given to 42 healthy volunteers genotyped as extensive (EM), intermediate (IM) and poor (PM) metabolizers of substrates of CYP2D6 and of CYP2C19 and as slow metabolizers with the CYP2C9 genotype *3/*3. E-,Z-doxepin and -desmethyldoxepin as well as trimipramine and desmethyltrimipramine were quantified in plasma by HPLC. Data were analyzed by non-parametric pharmacokinetics and statistics. Mean E-doxepin clearance (95% confidence interval) was 406 (390-445), 247 (241-271) and 127 (124-139) l/h in EMs, IMs and PMs of CYP2D6 and was also significantly lower in carriers of CYP2C9*3/*3 (238 l/h). CYP2C19 was involved in Z-doxepin metabolism with 2.5-fold differences in oral clearances (73 l/h in CYP2C19 PMs compared with 191 l/h in EMs). The AUC (0-48 h) of the active metabolite desmethyldoxepin was dependent on CYP2D6 genotype with a median of 5.28, 1.35 and 1.28 nmol/l*h in PMs, IMs and EMs of CYP2D6. The genetically polymorphic enzymes exhibited highly stereoselective effects on doxepin biotransformation in humans. The median oral clearance of trimipramine was 276 l/h (180-444) in the reference group but only 36 l/h (24-48) in CYP2D6-PMs. The AUC of desmethyltrimipramine was 40-fold greater in CYP2D6 PMs than in the reference group (1.7 vs. 0.04 mg/l*h in EMs), but below the quantification limit in most carriers of deficiencies of CYP2C19 or CYP2C9. The CYP2D6 polymorphism had a strong impact on E-doxepin and trimipramine pharmacokinetics and CYP2D6-PMs might be at an elevated risk for adverse drug effects when treated with common recommended doses of these antidepressants.

NADPH-cytochrome P450 reductase (CPR) is a flavoprotein containing both FAD and FMN and functions as the electron donor protein for several oxygenase enzymes found on the endoplasmic reticulum of eukaryotic cells, including cytochrome P450s involved in drug metabolism and cholesterol biosynthesis. As many as three enzymes in the cholesterol biosynthetic pathway have been demonstrated, or proposed, to use CPR as a redox partner: squalene monooxygenase, which converts squalene to 2,3-oxidosqualene; lanosterol demethylase, a cytochrome P450 (CYP51); and 7-dehydrocholesterol reductase, the final step in cholesterol synthesis. In yeast CPR can be replaced by the NADH-cytochrome b5 pathway, but this has not been demonstrated in animals or plants. My studies with hepatic cytochrome P450 reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase that was not previously detected. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol, indicating that lanosterol demethylation, catalyzed by CYP51, is blocked. These results demonstrate that this second reductase plays a significant role in supporting squalene monooxygenase but not cytochrome P450-mediated reactions. 7-Dehydrocholesterol reductase (E.C. 1.3.1.21) catalyzes the reduction of the 7-8 double bond of 7-dehydrocholesterol to yield cholesterol. It has been suggested that cytochrome-P450 reductase is required for this reaction. My studies show that 7-dehydrocholesterol reductase is enzymatically active in CPR-null microsomes, with activity equal to or greater than that found in preparations from wild-type mice. Mammalian cytochrome b5, which can accept electrons from either cytochrome P450 reductase or NADH-cytochrome b5 reductase, is known to be involved in augmenting some P450-dependent monooxygenase reactions. Cytochrome P450 2E1 has been found to exhibit reasonable rates of turnover via an NADHcytochrome b5 pathway in reconstituted enzyme systems and in heterologous hosts. Using microsomes from hepatic CPR-null mice, I have determined that NADH-dependent CYP2E1 activity in the absence of NADPH-dependent activity constituted approximately 10% of CYP2E1 activity observed in microsomal preparations with NADPH from wild-type mice. However, little or no CYP2E1 activity could be detected in primary hepatocytes isolated from CPR-null mice.

En l’absence de structures tridimensionnelles expérimentales des cytochromes P450 CYP26A1, CYP26B1 et CYP26C1, la caractérisation de leur substrats et ligands s’est basée sur l’analyse des modèles structuraux obtenus par modélisation par homologie avec la structure expérimentale du cytochrome P450 CYP120. La justesse des modèles a été validée par l’amarrage de l’acide rétinoïque all-trans dans des configurations compatibles avec les métabolites attendus. L’amarrage d’agonistes et d’antagonistes des récepteurs nucléaires RARs prédirent l’acide tazaroténique (TA) et l’adapalène comme des substrats potentiels. Les expériences in vitro confirmèrent la métabolisation de ces 2 médicaments par les CYP26s. L’analyse de la cinétique de sulfoxidation du TA par CYP26A1 and CYP26B1 a permis d’établir le TA comme la référence contrôle de l’activité de ces enzymes. Puis, la comparaison des modèles des CYP26s avec la structure cristalline de CYP2C8 a permis d’identifier des similarités structurales de leurs inhibiteurs. Une corrélation entre l’inhibition de CYP26A1 et de CYP2C8 par des inhibiteurs connus de CYP2C8 a été démontrée après détermination de leurs IC50 pour CYP26A1 et CYP26B1 en utilisant le TA comme substrat de référence. La mesure de l’inhibition in vitro fut ensuite utilisée pour évaluer la possibilité que les CYP26s soient impliquées dans des interactions médicamenteuses observées pour certaines molécules. Cette thèse caractérise et appuie le rôle encore mal connu des CYP26s dans la métabolisation in vivo de certains xénobiotiques ainsi que l’effet potentiel de leur inhibition qui favoriserait la survenue d'effets indésirables
Without crystal structures to study the CYP26 family of drug metabolizing enzymes, homology models were used to characterize CYP26A1, CYP26B1 and CYP26C1 and to identify substrates and inhibitors of the enzymes. Computational models of each isoform based on structural homology to CYP120 were validated by docking all-trans retinoic acid, an endogenous ligand of CYP26. Docking of retinoic acid receptor agonists and antagonists suggested that tazarotenic acid (TA) and adapalene may be metabolic substrates for CYP26, data which was confirmed using in vitro metabolite identification assays. Phenotyping experiments determined that CYP26s played a major role in the metabolism of these compounds in vitro. The kinetics of TA sulfoxidation by CYP26A1 and CYP26B1 were characterized and the compound was proposed as an in vitro probe of CYP26 activity in single enzyme expression systems. Structural characterization efforts identified similarities between the CYP26 homology models and the known crystal structure of CYP2C8, in agreement with previously published reports. Using TA as a probe, the IC50’s of known CYP2C8 inhibitors was measured against CYP26A1 and CYP26B1, with a statistically significant correlation observed between CYP26A1 and CYP2C8. Additional in vitro and computational experiments were used to characterize the inhibition mechanism for the most potent inhibitors. The observed in vitro inhibition was then used to predict the likelihood of CYP26 inhibition being involved in clinically relevant drug interactions. As a whole, the results presented support the role of the CYP26s in the metabolism of xenobiotic compounds as well as in potential in vivo drug interactions

Abstract Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of energy metabolism and mitochondrial biology in high-energy cell types and tissues. The regulation of PGC-1α is versatile, and both transcriptional and post-transcriptional mechanisms play major roles. External stimuli affect PGC-1α-regulation which in turn adapts cellular signals to meet them. For example, conditions like fasting and diabetes mellitus (DM) are known to activate PGC-1α expression in the liver, resulting in enhanced de novo glucose production, gluconeogenesis. In the present study, the mechanisms of hepatic PGC-1α regulation and PGC-1α-regulated functions were elucidated. We found that PGC-1α was induced by oral type 2 diabetes therapeutic metformin, via AMPK and SIRT1, regulating the mitochondrial gene response, against previous assumptions. Simultaneously, gluconeogenesis was repressed by other means. Furthermore, PGC-1α upregulated the anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). PGC-1α also diminished interleukin 1β-mediated inflammatory response in hepatocytes. Novel, xenobiotic and endobiotic metabolizing Cytochrome P450 enzymes regulated by PGC-1α were also identified in this thesis. CYP2A5 was induced by PGC-1α through hepatocyte nuclear factor 4α (HNF-4α) coactivation. Also, vitamin D metabolizing CYP2R1 and CYP24A1 were identified as novel genes regulated by PGC-1α, suggesting a role for PGC-1α in the regulation of active vitamin D levels. The findings presented in this thesis provide insight into the pathology of glucose perturbations such as type 2 diabetes, and stimulate discovery of therapeutic agents to treat this disease. Furthermore, the findings suggest that vitamin D metabolism and energy metabolism are tightly linked, with PGC-1α emerging as a novel mediator
Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori γ:n koaktivaattori 1α (PGC-1α) on merkittävä glukoosiaineenvaihdunnan ja mitokondrioiden toiminnan säätelijä korkeaenergisissä soluissa ja kudoksissa. PGC-1α:a säädellään monin tavoin: sekä transkriptionaalisella säätelyllä että transkription jälkeisellä muokkauksella on merkittävä rooli. Monet ulkoiset tekijät säätelevät PGC-1α:n aktiivisuutta, joka puolestaan säätelee solunsisäisiä signaalireittejä vastaamaan tähän signaaliin. Esimerkiksi paasto ja diabetes mellitus (DM) ovat fysiologisia tiloja, jotka lisäävät voimakkaasti PGC-1α:n ilmentymistä maksassa, jolloin glukoosin uudistuotanto eli glukoneogeneesi kiihtyy. Tässä väitöskirjassa tutkittiin PGC-1α:n säätelyä sekä PGC-1α -säädeltyjä signaalireittejä maksassa. Osoitimme, että tyypin 2 diabeteslääke metformiini indusoi PGC-1α:n ilmentymistä maksassa, vastoin aikaisempia käsityksiä. PGC-1α indusoitui AMPK:n ja SIRT1:n välityksellä, säädelleen edelleen mitokondriaalisten geenien aktiivisuutta. Samalla glukoneogeneesi kuitenkin repressoitui muilla mekanismeilla. Lisäksi osoitimme, että PGC-1α indusoi tulehdusreaktiota vaimentavaa interleukiini 1 reseptorin antagonistia (IL1Rn). PGC-1α esti interleukiini 1β:n aiheuttamaa tulehdusvastetta hepatosyyteissä. Lisäksi väitöskirjassa tunnistettiin uusia, PGC-1α -säädeltyjä lääkeaineita ja elimistön sisäisiä yhdisteitä metaboloivia sytokromi P450 -entsyymejä (CYP). Hiiren CYP2A5:n ilmentymisen osoitettiin olevan PGC-1α- ja HNF4α-välitteistä. Lisäksi osoitettiin, että D-vitamiinia metaboloivat CYP2R1 ja CYP24A1 ovat uusia PGC-1α -säädeltyjä geenejä. Tämä löydös viittaa siihen, että PGC-1α:lla on rooli aktiivisen D-vitamiinin säätelyssä. Tämän väitöskirjan löydökset lisäävät tietoa glukoosiaineenvaihdunnan häiriöiden kuten tyypin 2 diabeteksen molekulaarisista mekanismeista, joita voidaan hyödyntää mahdollisten uusien lääkeaineiden kehittämisessä. Lisäksi väitöskirjassa osoitettiin, että D-vitamiinimetabolia on kytköksissä energia-aineenvaihduntaan ja että PGC-1α:lla on tässä rooli, jota ei aiemmin ole tunnettu

Drugs and other foreign compounds must often be metabolised before they can be excreted from the body. One enzyme system that is responsible for this is the cytochrome P450 gene family (CYP). In this thesis, new sensitive molecular techniques have been used to study the human gene expression of some CYP enzymes, as well as the P-glycoprotein transporter (P-gp). The aim was to evaluate whether tissues other than the liver, e.g. the blood, could be used to assess an individual's drug metabolic capacity. Another aim was to investigate the gene expression in relation to the liver transplant process and a third aim was to evaluate the expression in gastrointestinal mucosa in both normal and inflamed mucosa.

We evaluated the CYP gene expression in paired specimens of liver and blood but found no correlation in the expression patterns of these two tissues. Instead, we found the opposite pattern, where, for example, CYP1B1 had the highest expression in the blood but the lowest in the liver and CYP2E1 was the enzyme with the highest expression in the liver. In an investigation of the expression of four different CYP enzymes and P-gp in liver transplants before and during the first year after transplantation, we found that the levels of all the CYP enzymes but not P-gp increased with time. We also found that the expression of CYP3A4 was inversely related to the normalised plasma levels of the immunosuppressive drugs cyclosporine and tacrolimus.

In the gastrointestinal tract, CYP2E1 was the enzyme with the highest mRNA expression compared with CYP3A4, CYP3A5 and the transporter P-gp. CYP3A4 has its highest expression in the duodenum compared with the expression in the stomach and the colon. CYP3A5 is expressed at a higher level than CYP3A4 in the colon. P-gp expression levels increase through the gastrointestinal tract to the left colon. Gene expression levels of CYP2E1 and CYP3A4 decrease in severely inflamed rectal mucosa.

In conclusion, this is a sensitive method for studying gene activity in a clinical situation, even though at this point we are not able to use blood or gastrointestinal mucosa as “surrogate” tissue to estimate an individual’s drug metabolic capacity. The studies in liver transplants and gastrointestinal mucosa are unique in that the gene expression is investigated during a clinical course of events.