Made available in DSpace on 2016-04-04T12:35:27Z (GMT). No. of bitstreams: 2 thiago_martins_ioc_mest_2008.pdf: 2240776 bytes, checksum: 22f0d88555ae7a38778dfd03f1dd7c9c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os citocromos P450 (CYP) desempenham importantes papéis na biotransformação de xenobióticos e no metabolismo de substâncias endógenas. A subfamília CYP1A foi bem conservada ao longo da evolução dos vertebrados, tendo sido encontrada em todas as espécies de peixes mandibulados estudadas até o momento. Em trabalho anterior, verificamos que algumas espécies de cascudos da família Locariidae não apresentavam atividade da etoxiresorufina-O-desetilase (EROD) em microssomos hepáticos. Como EROD é atividade catalisada predominantemente por CYP1A em diversos vertebrados, esse achado nos motivou a investigar em detalhe os CYP, em especial da subfamília 1A, em cascudos. Este trabalho é um estudo comparativo da capacidade de cascudos (Hyposthomus luetkeni e H. affinis), tilápias do Nilo (Oreochromis niloticus; Cichlidae) e camundongos, controles e tratados com indutores conhecidos de CYP1A [50 mg/kg ip; B-naftoflavona (BNF), ou 7-12 dimetil-benzoantraceno (DMBA)] de: i - catalisar diversas reações químicas sabidamente mediadas por CYP, ii - expressar a proteína CYP1A no tecido hepático, e iii - ativar o pró-mutágeno DMBA. Nos microssomos hepáticos das tilápias e dos camundongos, detectamos atividades constitutivas de EROD e também de desalquilação de outros ésteres da resorufina (MROD, PROD e BROD). Nessas duas espécies, as atividades dessas monooxigenases foram induzidas pelos tratamentos com BNF e DMBA. Nos cascudos, controles e tratados com os indutores de CYP1A, as atividades das alcoxi-resorufina-O-desalquilases não foram detectadas. A atividade da etoxicumarina desetilase (ECOD) foi cerca de cinco vezes maior no fígado de cascudos e de camundongos do que no das tilápias. O CYP1A não parece ter papel importante na catálise de ECOD nessas duas espécies de peixes Os resultados também mostraram que dois anticorpos anti-CYP1A de peixe reconheceram proteínas com massa moleclar compatível com o de CYPs nos microssomos hepáticos de tilápias e camundongos tratados com indutores de CYP1A. Nos cascudos, entretanto, a detecção de CYP1A por immunoblotting com esses anticorpos não foi consistente. Com o emprego de espectrometria de massas, identificamos o CYP1A em microssomos hepáticos de tilápias induzidas, mas não nos cascudos controles ou induzidos. Em conjunto, os resultados sugerem que, em contraste com o observado com tilápias e camundongos, os cascudos não exibem atividade catalítica de CYP1A e não expressam as proteínas correspondentes no fígado, ou as expressam apenas em níveis constitutivos extremamente baixos. Apesar de não apresentarem atividade catalítica de CYP1A, os cascudos foram capazes de ativar o pró-mutágeno DMBA que, em outras espécies, é ativado por enzimas da família CYP1, em especial das subfamílias 1A e 1B / The cytochrome P450 (CYP) superfamily plays im portant roles in the biotransformation of xenobiotic and endogenous compounds. The CYP1A subfamily is well conserved in vertebrates and has been found in all jawed fish studied to date. In a previous study, we had found that suckermouth armored catfishes of the Loricariidae family ( Hyposthomus luetkeni and H.affinis ) did not show ethoxy-resorufin- O -deethylase (EROD) activity in liver microsomes. Since EROD is a marker of CYP1 A catalytic activity in vertebrates, this unexpected finding prompted us to investigate in depth the CYP system of loricariid catfishes, particularly the CYP1A subfamily. In this study, we compared the capacities of suckermouth armored catfishes (Loricariidae), Nile tilapias ( Oreochromis niloticus , Cichlidae) and mice (Swiss Webster), control and treated with CYP1A-inducing agents (50 mg/kg ip ß- naphthoflavone, BNF, and 7-12 dimethyl-benzoanthracene, DMBA) to: i - catalyze several activities that are known to be catalyzed by CYPs, ii - express CYP1A protein in liver tissue and iii – activate the pro-mutagen DMBA. In liver microsomes of tilapias and mice, constitutive activities of EROD, and of other alkoxy-resorufin- O -dealkylases (XROD: MROD, PROD and BROD) were found. In the two species, activities of these monooxygenases were induced after treatment with BNF and DMBA. In suckermouth armored catfishes (control and induced) no XROD activities were noted. In liv er microsomes of suckermouth catfishes and mice, the ethoxycoumarin-deethylase activity (ECOD) was found to be approximately five- fold the ECOD activity recorded in tilapias. CYP1A apparently did not take part in the catalysis of ECOD reaction in the two fish species. Results also showed that two antibodies against- fish CYP1A recognized liver microsomal proteins with a CYP-compatible molecular weight in induced tilapias and mice. These putative CYP1A proteins, however, were not consistently detected in suckermouth catfish liv er microsomes. Taken together, findings from this study suggested that, in contrast to t ilapias and mice, suckermouth catfishes do not show CYP1A catalytic activity and do not express, or do express only tiny amounts of constitutive CYP1A proteins in the liver. Alt hough not showing CYP1A catalytic activity in the liver, armored suckermouth catfishes were able to convert the pro-mutagen DMBA into its genotoxic metabolites, a metabolic activation us ually mediated by CYP1 family enzymes, in particular, CYP1A and 1B.

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The full text will be available at the end of the embargo: 30th July 2026

Cytochrome P4501A1 (CYP1A1) is a phase I bio-transformation enzyme involved in the metabolism of xenobiotics via the oxygenation of polycyclic aromatic hydrocarbons (PAHs) including the carcinogen, benzo(a)pyrene. Induction of the CYP1A1 gene is regulated at the transcriptional level and is ligand dependent with the prototypical 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) being the most potent known inducer of CYP1A1 transcription. This process is mediated by the AHR/ARNT signaling pathway whereby ligand binds AHR in the cytoplasm allowing its translocation to the nucleus where it binds with its hertrodimerization partner, ARNT and subsequently binds DNA at cognate binding sites termed xenobiotic responsive elements (XREs) located in the 5' flanking region of the CYP1A1 and other genes. The zebrafish (Danio rerio) has recently become an important model system for the study of TCDD-mediated developmental toxicity due to their relative ease of maintaining and breeding, external fertilization, abundant transparent embryos, and sensitivity to TCDD similar to mammalian models. It is therefore essential to vii characterize the molecular mechanisms of AHR mediated gene regulation in this organism. The upstream flanking region of a putative CYP1A gene from zebrafish was identified by the screening of a PAC genomic library. Sequencing revealed a region which contains 8 putative core xenobiotic response elements (XREs) organized in two distinct clusters. The region between -580 to -187 contains XRE 1-3 while the region between -2608 to -2100 contains XRE 4-8. Only XRE 1, 3, 4, 7, and 8 exhibited TCDD-dependant association of AHR/ARNT complexes when evaluated by gel shift assays. The use of in vitro mutagenesis and Luciferase reporter assays further showed that only XRE's 4, 7, and 8 were capable of conveying TCDD-mediated gene induction. The role of nucleotides flanking the core XRE was investigated through the use of EMSA and reporter assays. Similar methods were employed on additional transcription factor binding sites identified by in silico analyses revealing two sites conforming to an HNF- 3α and CREB motif, respectively, which demonstrate importance to regulation of the gene.

ABSTRACT MICROARRAY APPLICATIONS FOR DETERMINATION OF THE EFFECTS OF EMODIN ON BREAST CANCER CELL LINES Ekenel Qomi, Emilia M.S., Department of Biotechnology Supervisor: Prof. Dr. Mesude Iscan Co-Supervisor: Assoc. Prof. Dr. Nursen &Ccedil / oruh February 2012, 191 pages Cancer is a genetic disease that is characterized by uncontrolled cells growth. Breast cancer is a type of cancer originating from breast tissue. Some breast cancers are sensitive to hormones such as estrogen which makes it possible to treat them by blocking the effects of these hormones in the target tissues. These require less aggressive treatment than hormone negative cancers. Breast cancers without hormone receptors, are higher-risk, and are treated more aggressively. The aim of our study is to investigate the effect of emodin on MCF-7 which is ER (estrogen receptor) positive, and MDA-MB-231 (ER negative) cancerous cell lines. Emodin which is a phytoestrogen component, extracted from rheum (genus) plant, has been reported to suppress the growth of tumor in some clinical situation, and it&rsquo / s found that emodin induced apoptosis through the decrease of Bcl-2/Bax ratio and the increase of cytoplasm cytochrome c concentration in human breast cancer Bcap-37 cells. Comparing the effect of emodin between ER positive and ER negative cells at the molecular level was investigated by Microarray analysis of gene expressions using Affymetrix Human Genome U133 plus 2.0 Array. The microarray data analysis was performed by using BRB-Array Tools, v.4.2.0. GST and its classes / Alpha, Mu, Pi, Theta, Sigma, Omega, Zeta and Kappa is our interested genes because of its role in regulating susceptibility to cancer, by their ability to metabolize reactive electrophilic intermediates to usually less reactive and more water soluble glutathione conjugates. And also its have a role in detoxifying the damage caused by oxidative stress which is a result of the radiotherapy. v The differentially expressed genes from emodin treated and untreated control breast cancer cell lines were compared after normalization and filtering and annotated, it was shown that the top 10 highly (significantly) varied genes belong to the biological processes such as (namely) cell cycle, cell division, cell proliferation, mitosis and meiosis, this insure the relation of emodin to the cell growth processes in the cancerous cells. The analysis of the change on the cell growth confirmed the anti-tumor effect of emodin. About the effect of emodin treatment on MCF-7 and MDA-MB-231 cancerous cell lines separately / Both cells its significant genes was belong to cell growth biological processes, in MCF-7 cells in-addition other biological processes was shown, for example / stimulus to estradoil response, and the metabolism of xenobiotic by cytochrome p450, so CYP1A1 gene code for a protein which is used in emodin metabolism. The varied gene number was nearly 4400 gene from the scatter plot result in MCF-7 cells while in MDA-MB-231 cells it was nearly 3400 gene, these result insured the effect of emodin as a phytoestrogenic component as MCF-7 cells are ER positive cells, so emodin bind to the ER in MCF-7 cells and affected more gene number than MDA-MB-231. More number of GST enzyme classes changed in MCF-7 cells than MDA-MB-231, and the effect of emodin as anti-cancer showed different change of GST genes between MCF-7 and MDA-MB-231. The results confirmed by network analysis done, to find the most related genes to our top 10 regulated gene list, and these genes were analyzed / most of them where in our gene list, and their regulation after emodin treatment analyzed and the result was supported to emodin as anti-tumor and phytoestrogenic component.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 143 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 123 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 113-123).

Benzo[a]pyrene (BaP), is one of the most potent mutagens and carcinogens known. It requires metabolic activation through cytochrome P450 (CYP)1A1 to yield the ultimate carcinogenic metabolite, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE can bind to DNA and form predominantly covalent (+) trans adducts at the N2 position of guanine causing DNA damage. Rutaecarpine (RTC) is an herbal medicine that has been used to treat several diseases such as headache, hypertension, gastrointestinal disorders, amenorrhea, and anti-inflammation. It has also been reported as a potent inducer of CYP enzymes, including CYP1A1, and CYP1A2. The mechanisms underlying up-regulation of CYP1A1 by RTC is dependent on aryl hydrocarbon receptors. Meanwhile, RTC can inhibit the activity of CYP1A1, CYP1A2 and CYP1B1. To investigate the effect of RTC on the BaP-induced DNA damage, we analyzed the CYP1A1 enzyme activity and DNA damage level in two cell lines, namely mucoepidermoid pulmonary carcinoma cells (H292) and hepatocellular carcinoma cells (Hep3B). The cells either were treated with only 5 μM BaP or 1.25, 2.5, 5 and 10 μM RTC, respectively; or were co-administrated 5 μM BaP and one of the four concentrations of RTC for 24 hours. Ethoxyresorufin-O-deethylase (EROD) assay was used to detect CYP1A1 enzyme activity. The results showed that both BaP and RTC significantly (p<0.05) induced CYP1A1 enzyme activity when administered separately, with RTC induction exhibiting a concentration-dependent manner. Interestingly, co-administration of RTC with BaP, especially at high concentration (10 μM) of RTC, induced less CYP1A1 enzyme activity compared to either only RTC or BaP administration. MuseTM Multi-Color DNA Damage kit was used to evaluate the DNA damage level in cells. The data showed that the DNA damage induced by BaP alone was about 2-fold higher (p&;lt;0.05) than that by concurrent administration of RTC and BaP. In conclusion, our data showed that although both RTC and BaP are inducers of CYP1A1 enzyme, their co-administration will reduce CYP1A1 enzyme activity compared with BaP administration alone. The DNA damage kit results supported that there is a potential protective effect of RTC against BaP-induced DNA damage in both H292 and Hep3B cells.

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No / The identification of an agent that is selectively activated by a cytochrome P450 (CYP) has the potential for tissue specific dose intensification as a means of significantly improving its therapeutic value. Towards this goal, we disclose evidence for the pathway of activation of a duocarmycin analogue, ICT2700, which targets CYP1A1 for biological activity.