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Habilitation Thesis
DOI
10.11606/T.46.2013.tde-20082013-104343
Document
Author
Full name
Ohara Augusto
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 1983
Committee
Meneghini, Rogerio (President)
Caballero, Nelson Eduardo Duran
Giglio, Jose Roberto
Guralnik, Hernan Chaimovich
Paiva, Antonio Cechelli de Mattos
Title in Portuguese
Metabolismo de xenobióticos: formação de radicais de carbono e suas implicações terapêuticas e toxicológicas
Keywords in Portuguese
Alquilação de hemeproteínas
Bioquímica
Citocromo P450
Metabolismo de xenobióticos
Radicais livres
Ressonância para magnética eletrônica
Abstract in Portuguese
Uma nova abordagem experimental para a aplicação da técnica do captador de spin em sistemas biológicos é apresentada. Nós demonstramos que o POBN (α- (4-piridil-1-óxido) N-tercio-butil nitrona) é o captador de spin mais adequado para radicais de carbono formados no sistema microssomal. Outros captadores, 5,5,-dimetil-1-pirrolidina-N-óxido e α-fenil-N-tercio-butil nitrona, se mostraram inadequados, o último porque inibe fortemente o citocromo P450. Essa constatação nos possibilitou isolar biologicamente e caracterizar, por espectroscopia de massa, o aduto formado durante o metabolismo microssomal de 3,5-bis- (etoxicarbonil)-4-etil-2,6-dimetil-1,4-diidropiridina, como sendo o aduto etil-POBN. Esse isolamento e caracterização de radicais formados metabolicamente, propiciaram uma firme base estrutural para a técnica do captador de spin, tornando-a um instrumento bastante útil para a abordagem mecanística de reações envolvendo radicais livres. Como um exemplo dessas novas potencialidades, nós apresentamos nossos estudos sobre o metabolismo da fenelzina (2-feniletilidrazina). Esse composto é um agente antidepressivo conhecido por vários nomes comerciais como Nardil, Fenelzina, Estinerval, kalgan, etc. O metabolismo da fenelzina por microssomos hepáticos de ratos leva a formação dos seguintes compostos: 2-fenilacetaldeido, 2-feniletanol e etilbenzeno. Durante esse processo oxidativo, ocorre a formação de um radical de carbono que reage com o grupo prostético do citocromo P450 inativando-o irreversivelmente. Esse radical foi captado, isolado e caracterizado como sendo o radical 2-feniletila. A porfirina derivada do grupo heme, inativado pela fenelzina, foi isolada e caracterizada como sendo a N-(2-feniletil) protoporfirina IX Assim, o metabolismo da fenelzina, urna alquilidrazina, forma um radical de carbono que pode inativar o citocromo P450, ser convertido num hidrocarboneto, reagir com captadores de spin e provavelmente também, pode reagir com biomoléculas circundantes. Esses resultados, analisados conjuntamente com outros estudos mecanísticos que realizamos sobre transformações biológicas de derivados de hidrazina, são utilizados para urna discussão geral do possível papel de radicais de carbono na expressão dos efeitos terapêuticos e tóxicos de drogas.
Title in English
Xenobiotic metabolism: generation of carbon-centered radicals and therapeutic and toxicological implications
Keywords in English
Biochemistry
Cytochrome P450
Electron paramagnetic resonance
Free radicals
Hemoprotein alkylation
Xenobiotic metabolism
Abstract in English
A new experimental approach to the biological application of the spin-trapping technique is presented. Our studies showed that POBN (α(- (4-pyridyl-1-oxide) N-tert-butylnitrone) is the most suitable trap for carbon radicals formed in microsomal systems. Other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and α-phenyl-N-tert-butylnitrone were found to be ineffective, the latter because it strongly inhibits cytochrome P450. POBN suitability enabled us to isolate and characterize, by mass spectrometry, the adduct formed during the microsomal metabolism of 3,5-bis( ethoxycarbonyl)-4-ethyl-2,6-dimethyl -1,4-dihydropyridine, as a POBN-ethyl adduct. The isolation and characterization of metabolically formed radicals provided a firm structural basis for the spin-trapping methodology, rendering it a useful tool for the mechanistic approach to reactions involving radical formation. As an example of these potencialities, we present our studies on phenelzine (2-phenylethylhydrazine) metabolism. This compound is a tranquilizing drug known under a number of commercial names as Nardil, Fenelzina, Estinerval, Kalgan and others. The transformation of phenelzine by rat liver microsomes yields 2-phenylacetaldehyde, 2-phenylethanol and ethyl-benzene. A carbon radical is formed during the oxidative metabolism of phenelzine that reacts with the prosthetic heme group of cytochrome P450 and irreversibly inactivates the enzyme. The radical formed has been spin-trapped, isolated and shown by mass spectrometry to be the 2-phenylethyl radical. The metal free porphyrin derived from the prosthetic heme group has been isolated and identified as N-(2-phenylethyl)protoporphyrin IX. The metabolism of phenelzine, an alkylhydrazine, thus yields a carbon radical that inactivates cytochrome P450 is converted to a hydrocarbon by hydrogen atom abstraction and reacts with spin traps or, presumably, with alternative celular targets. These results, together with other mechanistic studies done by us on biological transformations of hydrazine derivatives, are taken into account in the discussion of the possible role of carbon radicals on drug action and toxicity.
 
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Publishing Date
2013-08-20
 
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