Em face das recentes exigências das agências regulatórias quanto à aprovação de novos fármacos, os estudos de biotransformação têm-se tornado uma etapa indispensável para a identificação e otimização de compostos bioativos. O objetivo desses estudos é identificar, já nas fases iniciais da descoberta de fármacos, candidatos que apresentam propriedades indesejáveis como a (i) presença de metabólitos ativos ou tóxicos; (ii) inibição de enzimas metabolizadoras; (iii) depuração metabólica inadequada, entre outras. Neste estudo, foi realizada a caracterização metabólica e a identificação de possíveis inibidores das enzimas do citocromo P450 de oito promissores candidatos a fármacos, identificados através de ensaios virtuais como inibidores da TcGAPDH, Cruzaina e TcDHODH, todas do Trypanosoma cruzi, agente causador da doença de Chagas. Esses compostos foram testados contra as três principais isoformas do citrocromo P450: CYP 3A4, CYP 2D6 e CYP2C9. Os valores de IC50 de 1,4 µM e 1,3 µM contra a CYP2C9 foram encontrados para os compostos Nequimed53 e Nequimed125, enquanto o Nequimed42 inibiu a CYP 3A4 com um valor de IC50 de 7,12 µM. Posteriormente foi conduzida a caracterização metabólica dos compostos Nequimed53 e 125 com foco na identificação dos principais metabólitos, sítios de metabolismo e vias de biotransformação através da técnica de LC-ESI-QqTOF-MS. Para ambos os compostos, a biotransformação por microssomas extraídos de fígado de ratos deu-se preferencialmente por uma única via dependente de NADPH. No caso do Nequimed54, o metabólito formado apresentou uma variação da razão m/z de +16, indicando a ocorrência da hidroxilação do composto parental, enquanto que para o composto Nequimed125, o metabólito formado apresentou uma variação da razão m/z de -28, condizente com a perda de um fragmento etila do composto parental. / In the light of recent demands from regulatory agencies for the acceptance of new drugs, the biotransformation studies have become an essential step for the identification and optimization of bioactive compounds. The objective of these studies is to identify compounds that have undesirable properties such as (i) the presence of toxic or active metabolites, (ii) inhibition of metabolizing enzymes, (iii) excessive metabolic clearance, inter alia. In this study we characterized the metabolism and cytochrome P450 inhibition of eight compounds identified by virtual screening as inhibitors of TcGAPDH, Cruzain and TcDHODH which are of interest as targets for intervention in treatment of Chagas Disease. These compounds were tested against cytochrome P450 isoforms 3A4, 2D6 and 2C9. IC50 values of 1.4 µM and 1.3 µM against CYP 2C9 were observed for Nequimed53 and Nequimed125.while Nequimed42 inhibited CYP 3A4 with an IC50 of 7.1 µM. Subsequently, we characterized the in vitro metabolism of Nequimed53 and 125 with a focus on metabolite identification and biotransformation pathways using the LC-ESI-MS-QqTOF technique. For each, the biotransformation by rat liver microsomes occurred by a single NADPH-dependent pathway. For Nequimed54, the observed metabolite [M+16]+ indicated hydroxylation of parent compound. The metabolite [M-28]+ observed for Nequimed125 indicated desethylation of the parent compound.

Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...

Abstract Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerful tool for qualitative and quantitative analytics of organic molecules from various matrices, and the use of this hyphenated technique is very common in bioanalytical laboratories. In this study, LC/MS methods and the required sample preparation applications were developed for plant flavonoid and drug metabolism studies. The main focus was in developing methods to be used during cytochrome P450 (CYP) -specific drug interaction studies. Traditional high performance liquid chromatography (HPLC) and new, more efficient and faster ultra-performance liquid chromatography (UPLC) were utilized together with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry. In the flavonoid study, collision-induced radical cleavage of flavonoid glycosides was tested and observed to be a suitable tool for the structure elucidation of the 15 flavonol glycosides extracted from the medicinal plant Rhodiola rosea. Ten of these glycosides were previously unreported in the plant. Several unreported in vivo bupropion metabolites were identified from human urine when developing the method for the new and more extensive in vitro and in vivo N-in-one interaction cocktail assays. The qualified analysis methods developed here enable faster analysis for the N-in-one cocktail assays, in turn enabling a more efficient screening of drugs that affect CYP-enzyme activities. In the case of the human in vitro cocktail assay, fourteen compounds were analyzed using a single LC/MS/MS run. The method has proven to be very reliable and has been used in several interaction studies utilizing different sample matrices. The in vivo cocktail assay that was developed enables totally non-invasive sample collection from the patients, the urine sample being sufficient for the UPLC/MS/MS analysis of all target compounds. The last part of the study consisted of developing a specific and very sensitive UPLC/MS/MS method for the analysis of one of the in vivo cocktail analytes, the antidiabetic drug repaglinide, from human placenta perfusates. / Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas työväline kvalitatiivisessa ja kvantitatiivisessa analytiikassa, ja tätä tekniikkaa käytetään erityisesti bioalan laboratorioissa. Tässä väitöskirjatyössä kehitettiin ja sovellettiin LC/MS- ja näytteenkäsittelymenetelmiä kasvien flavonoidimetabolian ja lääkeaineiden metaboliatuotteiden tutkimukseen keskittyen erityisesti sytokromi P450 (CYP) -entsyymispesifisten lääkeaineiden interaktiotutkimuksiin tarvittaviin menetelmiin. Työssä hyödynnettiin perinteistä korkean erotuskyvyn nestekromatografiaa (HPLC) ja uutta, suorituskyvyltään vielä tehokkaampaa ja nopeampaa nestekromatografiaa (UPLC) yhdessä lentoaika- (TOF) ja kolmoiskvadrupolimassaspektrometrian (QqQ) kanssa. Tutkimustyön flavonoidimetaboliaan keskittyneessä osuudessa havaittiin törmäyksen aiheuttaman (CID) radikaalipilkkoutumisen soveltuvan lääkinnällisenä kasvina käytetystä ruusujuuresta (Rhodiola rosea) uutettujen viidentoista flavonoliglykosidin rakennemääritykseen. Kymmentä näistä löydetyistä glykosideista ei oltu aiemmin raportoitu ruusujuuresta. Tutkimustyön keskeisimpänä tavoitteena kehitettiin kvalifioidut LC/MS -analyysimenetelmät käytettäväksi aikaisempaa kattavampien in vitro ja in vivo -olosuhteiden N-in-one -tyyppisten CYP-entsyymi-interaktiotutkimusten analyyttisenä työkaluna. Näitä analyysimenetelmiä kehitettäessä löydettiin ja tunnistettiin ihmisen virtsasta aiemmin raportoimattomia metaboliitteja CYP2B6 -entsyymin malliaineena käytetyn bupropionin annostelun jälkeen. Kyseisten kehitettyjen analyysimenetelmien avulla CYP-entsyymien toimintaan vaikuttavien lääkeaineiden tutkiminen on aiempaa nopeampaa ja antaa yhdellä kertaa samasta tutkimuksesta entistä laaja-alaisempaa tietoa. In vitro -tutkimusta varten kehitetty LC/MS/MS -analyysimenetelmä on osoittautunut erittäin käyttökelpoiseksi lukuisissa interaktiotutkimuksissa, ja in vivo -tutkimusta varten kehitetty UPLC/MS/MS -analyysimenetelmä mahdollistaa täysin ei-invasiivisen näytteenoton potilaista. Tutkimustyön viimeisessä vaiheessa kehitettiin erittäin herkkä ja spesifinen UPLC/MS/MS -analyysimenetelmä CYP2C8-entsyymin toiminnan malliaineena käytetyn repaglinidin analysoimiseksi koejärjestelystä, jossa tutkitaan yhdisteiden kulkeutumista raskausaikana äidin ja sikiön verenkierron välillä istukan kautta.

Abstract The evaluation of the effects and levels of contaminants in wildlife is an essential part of assessing risks for chemical exposure in the environment. Although the circumstances are not as controlled as in laboratory, wildlife studies offer the concept of environmental exposure in its entirety, with all the natural variation. In the present study, two wild vole species, bank vole (Myodes glareolus) and field vole (Microtus agrestis), were used in assessing environmental levels of dioxins. The effects of dioxin exposure on tooth and bone development were studied in order to determine whether they could be used as biomarkers for environmental exposure. Xenobiotic metabolism activity after dioxin exposure – both natural and experimental – was studied by quantifying selected cytochrome P-450 (CYP) enzymes. The results confirmed the fact that dioxins are ubiquitous in the environment, also in areas far from contaminant sources and human activity. The development of the third molar in bank vole was found to be a sensitive biomarker for dioxin exposure. The two vole species under study do not respond similarly to environmental concentrations of dioxins; there were significant differences in body burdens and activity levels of xenobiotic metabolizing enzymes. / Tiivistelmä Haitallisten kemikaalien tason ja vaikutusten arviointi ympäristössä on olennainen osa kemikaalien riskin arviointia. Vaikka laboratoriossa olosuhteita kontrolloidaan ja tutkimukseen vaikuttava variaatio on paremmin hallittavissa, luonnonvaraisten lajien tutkiminen luo kokonaisvaltaisen ja todenmukaisen kuvan ympäristön kemikaalialtistuksesta kaikkine todellisine vaihteluineen. Tässä väitöskirjassa tarkastellaan kahden luonnonvaraisen pikkunisäkkään, metsämyyrän (Myodes glareolus) ja peltomyyrän (Microtus agrestis), käyttöä ympäristön kemikaalitason arvioinnissa. Pääpaino on dioksiinien kaltaisissa yhdisteissä. Työssä tutkitaan yhdisteiden kertymistä myyriin kahdessa ympäristössä: voimakkaasti dioksiineilla saastuneella maa-alueella sekä kaukana ihmistoiminnasta sijaitsevassa erämaassa. Herkiksi tiedettyjä vasteita – hampaiden ja luiden kehitystä – käytetään dioksiinialtistuksen indikaattoreina. Vierasainemetaboliasta vastaavien entsyymien (sytokromi P450 eli CYP) aktiivisuutta kartoitetaan molemmilla myyrälajeilla, jotta saadaan tietoa entsyymien indusoinnista luonnonvaraisilla myyrillä yleensä ja selvitetään havaittuja lajien välisiä eroja dioksiinivasteissa. Tulokset vahvistavat, että dioksiinit ovat laajalle levinneitä yhdisteitä, joita löytyy paitsi läheltä päästölähdettä myös kaukana ihmistoiminnasta olevilta alueilta. Metsämyyrällä kolmannen poskihampaan kehitys osoittautuu herkäksi dioksiinialtistuksen biomarkkeriksi. Samasta elinympäristöstä huolimatta tutkituista myyrälajeista mitatut dioksiinipitoisuudet eroavat huomattavasti toisistaan, samoin kuin vierasainemetaboliasta vastaavien entsyymien aktiivisuus ja niiden induktio TCDD-altistuksen jälkeen.

Philosophiae Doctor - PhD / The overall aim of this thesis was to prepare nanostructured more processable heteronuclear sulphonated polyanyline nanocomposites with electroconductive properties suitable for applications in biosensors. The sulphonated self-assembled polyaniline and derivatised polyaniline nanocomposites (SPAHs) were prepared by chemical oxidative polymerisation or electrical decomposition. The SPAHs prepared include those of polyaniline (PANi), poly-o-methoxyaniline (POMA) and poly-2.5 dimethoxyaniline (PDMA). Two types of sulphonic acids of heteronuclear aromatic hydrocarbons were used in the production of sulphonated SPAH composites. These were anthracene sulphonic acid (ASA) and naphthalene sulphonic acids (NSA) wich played both doping and surfactant roles. / South Africa

A doença inflamatória intestinal (DII) e a esclerose sistêmica (ES) são doenças inflamatórias crônicas de difícil diagnóstico e tratamento. A etiologia da DII e da ES ainda não é completamente compreendida, mas sabe-se que fatores genéticos, imunológicos e ambientais estão envolvidos na sua patogênese. A DII possui dois principais subtipos clínicos: a doença de Crohn (DC) e a retocolite ulcerativa (RCU), caracterizados pela inflamação do intestino delgado e/ou cólon. Evidências sugerem que o aumento do estresse oxidativo desempenha um papel importante na fisiopatologia da DII. A ES é uma doença inflamatória autoimune rara, caracterizada pela fibrose progressiva da pele e de órgãos internos. A hipótese de que o aumento do dano oxidativo pode iniciar o dano vascular e desencadear os eventos patológicos observados na ES vem sendo investigada. Genes e enzimas envolvidos na metabolização (Fase I) e detoxificação (Fase II) de xenobióticos são utilizados como marcadores de susceptibilidade para o desenvolvimento de doenças que possuem fatores ambientais como fatores de risco. Em uma reação de Fase I, as enzimas do Citocromo P450 (CYP) inserem um átomo de oxigênio em um substrato deixando-o eletrofílico e reativo, criando um sítio para posterior conjugação pelas enzimas de Fase II. As enzimas Glutationa S-tranferases (GST) de Fase II catalisam a conjugação da glutationa com uma grande variedade de compostos eletrofílicos, detoxificando substâncias endógenas e exógenas. A atividade catalítica aumentada das enzimas CYP, bem como a falha na detoxificação de metabólitos pelas GST pode contribuir para o aumento do estresse oxidativo. O objetivo deste estudo foi investigar o papel de polimorfismos nos genes que codificam enzimas de metabolização (CYP1A*2C e CYP2E1*5B) e detoxificação (GSTT1 nulo, GSTM1 nulo e GSTP1 Ile105Val) na susceptibilidade a estas doenças. O grupo de pacientes com DII era constituído por 235 indivíduos e o grupo controle por 241 indivíduos, todos eurodescendentes. Na ES, 122 pacientes (99 eurodescendentes e 23 afrodescendentes) e 329 controles (241 eurodescendentes e 87 afrodescendentes) foram analisados. Os polimorfismos CYP foram genotipados por PCR-RFLP, enquanto que os polimorfismos em GSTT1 e GSTM1 foram genotipados por PCR multiplex e PCR-RFLP para GSTP1. As frequências alélicas e genotípicas foram comparadas entre pacientes e controles usando o teste de Qui-Quadrado. A respeito dos resultados das análises em DII, as frequências alélicas e genotípicas dos polimorfismos CYP1A1*2C, CYP2E1*5B e GSTP1 Ile105Val, bem como as frequências genotípicas do polimorfismo de presença/ausência de GSTM1, foram similares nos três grupos de pacientes (DII, DC e RCU) quando comparados ao grupo controle (P>0,05). Observouse uma frequência significativamente aumentada do genótipo nulo de GSTT1 no grupo de pacientes com DII quando comparado ao grupo controle [0,28 vs 0,18; χ² com Yates P=0,02; OR=1,71 (IC 95% 1,09 –2,71)]. Quando separamos o grupo de pacientes em DC ou RCU, esta frequência permaneceu significativamente aumentada somente no grupo de pacientes com RCU comparado ao grupo controle [0,29 vs 0,18; χ² com Yates P=0,035; OR=1,84 (IC 95% 1,03 –3,24)]. Com relação aos resultados das análises na ES, uma frequência significativamente aumentada do genótipo *1A/*1A (P=0,03; 0,74 vs. 0,61) e do alelo *1A (P=0,013; 0,86 vs 0,78; OR=0,57, IC 95% 0,36–0,90) do polimorfismo CYP1A1*2C foi observada entre os indivíduos controles eurodescendentes. Em contrapartida, a frequência do alelo *2C estava significativamente aumentada entre os pacientes de mesma etnia (P=0,013; 0,22 vs 0,14; OR=1,75, IC 95% 1,11–2,74). Com relação às frequências alélicas e genotípicas dos polimorfismos CYP2E1*5B e GSTP1 Ile105Val, e as frequências genotípicas do polimorfismo de presença/ausência de GSTM1, nenhuma diferença significativa foi observada quando os grupos de pacientes de ambas as etnias foram comparados aos grupos controle (P>0,05). Uma frequência significativamente aumentada do genótipo nulo de GSTT1 [0,29 vs 0,18; χ² com Yates P=0,035; OR=1,85 (IC 95% 1,03–3,29)], bem como uma alta frequência da dupla deleção de GSTT1/GSTM1 [0,19 vs 0,08; χ² com Yates P=0,007; OR=2,62 (IC 95% 1,25 –5,46)], foi observada no grupo de pacientes comparado aos controles (eurodescendentes). Estas associações não se repetiram entre indivíduos afrodescendentes. Concluindo, nossos resultados sugerem que o genótipo nulo de GSTT1 está associado à susceptibilidade a DII e pode influenciar na definição do curso da doença para a RCU. Além disso, o genótipo nulo de GSTT1 sozinho ou em combinação com o genótipo nulo de GSTM1 é um fator genético de susceptibilidade para a ES, enquanto que o genótipo *1A/*1A ou a presença do alelo *1A do polimorfismo CYP1A1*2C pode exercer um papel protetor contra o desenvolvimento da ES em indivíduos eurodescendentes. / Inflammatory bowel disease (IBD) and systemic sclerosis (SSc) are chronic inflammatory diseases of difficult diagnosis and treatment. The etiology of IBD and SSc is not completely understood but it is known that genetic, immunologic and environmental factors are involved in its pathogenesis. Crohn’s disease (CD) and ulcerative colitis (UC) are the two major subtypes of IBD, characterized by inflammation of the small intestine and/or colon. Evidences suggest that the increase of oxidative stress plays an important role in the pathophysiology of IBD. SSc is a rare autoimmune inflammatory disease of the connective tissue characterized by progressive fibrosis of the skin and internal organs. The hypothesis that the increase of oxidative stress can initiate vascular damage and triggers the pathological events in SSc has been investigated. Genes and enzymes involved in metabolism (Phase I) and detoxification (Phase II) of xenobiotics are used as markers of susceptibility to the development of diseases that have environmental factors as risk factors. In a Phase I reactions, the Cytochrome P450 (CYP) enzymes insert an oxygen atom in a substrate that making it more electrophilic and reactive, and creating a site for subsequent conjugation by Phase II enzymes. Phase II Glutathione S-transferases (GSTs) enzymes catalyze the conjugation of glutathione with a variety of electrophilic compounds, detoxifying endogenous and exogenous substances. A higher catalytic activity of CYP enzymes, as well as the failure in detoxifying of metabolites by GST enzymes may to contribute for the increase of oxidative stress. The aim of this study was investigated the role of polymorphisms in genes coding Phase I enzymes (CYP1A*2C and CYP2E1*5B) and Phase II (GSTT1 null, GSTM1 null and GSTP1 Ile105Val) in susceptibility to these diseases. IBD group was constituted by 235 patients and the control group by 241 individuals, all European-derived. In SSc group, 122 patients (99 European-derived and 23 African-derived) and 329 controls (241 European-derived and 87 African-derived) were analyzed. The CYP polymorphisms were genotyped by PCR-RFLP, whereas polymorphisms in GSTM1 and GSTT1 were genotyped by multiplex PCR and PCRRFLP for GSTP1. Allelic and genotypic frequencies were compared between patients and controls using the Chi-square test. Concerning IBD, allelic and genotypic frequencies of CYP1A1*2C, CYP2E1*5B and GSTP1 Ile105Val polymorphisms, as well as genotypic frequencies of GSTM1 presence/absence polymorphism were similar in all groups patients (IBD, CD, and UC) and controls (P>0.05). We observed a significantly increased frequency of GSTT1 null genotype in IBD group as compared to controls [0.28 vs. 0.18, χ ² with Yates P=0.02, OR=1.71 (95% CI 1.09 – 2.71)]. When patients were classified in CD or UC group, this frequency remained significantly increased only among UC patients [0.29 vs. 0.18, χ ² with Yates P=0,035, OR=1.84 (95% CI 1.03 – 3.24)] as compared to controls. Regarding results in SSc, a frequency significantly increased of *1A/*1A genotype (P=0.03; 0.74 vs. 0.61) and *1A allele (P=0.013; 0.86 vs 0.78; OR=0.57, 95% CI 0.36–0.90) from CYP1A1*2C polymorphism was observed among European-derived controls. On the other hand, the frequency of *2C allele was significantly increased among patients of same ethnic group (P=0.013; 0.22 vs 0.14; OR=1.75, 95% CI 1.11–2.74). The allelic and genotypic frequencies of CYP2E1*5B and GSTP1 Ile105Val polymorphisms, as well as genotypic frequencies of GSTM1 presence/absence polymorphism were similar between SSc patients and controls of both ethnic groups (P>0.05). We observed a significantly increased frequency of GSTT1 null genotype [0.29 vs. 0.18, χ ² with Yates P=0.035, OR=1.85 (95% CI 1.03–3.29)], as well as an increased frequency of GSTT1/GSTM1 double-null in SSc patients as compared to controls [0.19 vs. 0.08; χ ² with Yates P=0.007, OR=2.62 (95% CI 1.25 – 5.46)]. These associations were exclusive to European-derived individuals. In conclusion, our results suggest that the GSTT1 null genotype is associated with susceptibility to IBD and may influence in defining the course of the disease for RCU. Furthermore, the GSTT1 null genotype alone or combined with GSTM1 null genotype is a susceptibility genetic factor to SSc, while the *1A/*1A genotype or the presence of *1A allele from CYP1A1*2C polymorphism may plays a protector role in SSc development in Brazilian Europeanderived individuals.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22080号 / 農博第2372号 / 新制||農||1072(附属図書館) / 学位論文||R1||N5234(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 冨永 達, 教授 奥本 裕, 教授 白岩 立彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...

ABSTRACT Cytochrome b5 (CYB5) is heme protein capable of reduction of cytochromes P450 (CYP) or some other enzymes. However, his regulative capability was also observed by his apo form, i.e. in absence of heme prosthetic group in the active center. CYB5 can accept electron from cytochrome b5 reductase (CYB5R) or from cytochrome P450 reductase (CYPOR). CYPOR by itself is reduced by NADPH and is also able to forward electron to CYP independently of CYB5. CYB5R on the other hand is reduced by NADH. Efficiency of CYB5 to accept and forward an electron was studied in vitro with five different substrates - testosterone, Sudan I, aristolochic acid I (AAI), ellipticine and vandetanib. These substrates were chosen considering their characteristic reactions, which are catalyzed by their respective isoforms of CYP. The experiments with these substrates were carried out in the medium with recombinant CYPs prepared in insect cells or E. coli or in the medium with hepatic microsomes isolated from different organisms. Rats, from which the majority of these microsomes was isolated, were premedicated by different CYP inducers. The experiments were carried out in medium with NADH or NADPH in order to assess the capability of CYB5 to reduce CYP independently of CYPOR. The capability of CYB5 and CYB5R to act as a...

The gene encoding cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt - 149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139- AGTTCAaggccGGGTAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter- transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.