Peroxidase-like activity of uncoupled cytochrome P450: studies with bilirubin and toxicological implications of uncoupling.

The NADPH-dependent consumption of O(2) by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O(2) molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O(2) uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O(2) and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

Isoflavone daidzein possesses no antioxidant activities in cell-free assays but induces the antioxidant enzyme catalase.

Epidemiologic studies have shown that dietary intake of isoflavonones is associated with several properties beneficial to human health. It has been suggested that at least some of these effects are related to the antioxidant activity of isoflavonoids. We analyzed the antioxidant activity of the major isoflavones found in soybeans, but none of these compounds showed prominent antioxidant effects in cell-free assay systems (trolox equivalent antioxidant capacity assay and 2,2-diphenyl-1-picrylhydrazyl assay). Therefore, we examined the hypothesis that the antioxidative effects of isoflavones are caused indirectly by up-regulation of antioxidative enzymes, thereby lowering intracellular concentration of reactive oxygene species. Daidzein shows a significant induction of catalase promoter activity at 100 micromol/L in a reporter gene assay and at 200 micromol/L in Northern blot experiments. Another hypothesis for antioxidant effects caused by isoflavones is due to metabolism by intestinal bacteria. Analyzing the daidzein metabolites 3'-OH-daidzein and 6-OH-daidzein in our cell culture model, we found strong antioxidant effects (2,2-diphenyl-1-picrylhydrazyl and trolox equivalent antioxidant capacity assay). We conclude that isoflavone daidzein up-regulates the antioxidant enzyme catalase but shows only little antioxidant capacity per se. Antioxidant effects of this dietary isoflavonone may also be due to formation of the antioxidant metabolites 6-OH-daidzein and 3'-OH-daidzein.

In vitro investigation of drug metabolism and toxicity in man

El interés de la industria farmacéutica es incrementar la seguridad de los nuevos fármacos, disminuir sus efectos secundarios, conocer sus propiedades farmacocinéticas e identificar las posibles interacciones fármaco-fármaco. El metabolismo de los fármacos es un factor determinante en su aclaramiento, es responsable de la variabilidad interindividual de la farmacocinética y en consecuencia, de la variación en el efecto farmacológico y la toxicidad. Desde la óptica comercial, lo deseable es que los fármacos con propiedades no adecuadas sean descartados en las fases más tempranas del desarrollo, y no en las fases posteriores con mayor coste económico. Como consecuencia de esto, en las últimas décadas se han incorporado estrategias basadas en modelos in vitro en combinación con estudios de ADMET, para la selección de nuevos fármacos cabeza de serie durante fases muy tempranas del desarrollo. Actualmente, la utilización de modelos hepáticos humanos in vitro en las fases preclínicas supone un proceso de selección de nuevas moléculas candidatas a fármacos mucho más racional. Existen varios modelos in vitro para abordar estas cuestiones en las etapas tempranas del desarrollo de fármacos para optimizar la selección de moléculas candidatas y la evaluación del riesgo de hepatotoxicidad

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. From a commercial perspective, it is desirable that poorly behaved compounds are removed early in the discovery phase rather than during the more costly drug development phases. As a consequence, over the past decade, in vitro-based strategies in lead optimization screening in conjunction with ADMET screening studies have been incorporated earlier in the drug discovery phase. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Several in vitro tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment

Papel de los polimorfismos genéticos para enzimas de reparación en el daño en el ADN inducido por el estireno y estireno-7, 8-óxido / Role of genetic polymorphisms of DNA repair enzymes in DNA damage induced by styrene and styrene- 7, 8-oxide

La respuesta individual al daño en el ADN inducido por agentes xenobióticos está condicionada por la eficacia de los sistemas de reparación. Algunos de los polimorfismos genéticos descritos en las enzimas de reparación del ADN pueden afectar a su función, determinando una variación en la susceptibilidad ante la exposición a agentes ambientales. El objetivo de este estudio ha consistido en investigar si las variantes alélicas más frecuentes de las enzimas de reparación XRCC1 ( Arg194Trp y Arg399Gln ), XRCC3 ( Thr241Met ) o APE1 ( Asp148Glu ) pueden condicionar el daño en el ADN inducido por el estireno y su principal metabolito, el estireno-7,8-óxido (EO). Leucocitos periféricos de 30 voluntarios sanos se trataron con estireno o EO, y el daño en el ADN inducido se evaluó mediante el ensayo del cometa. Tras el tratamiento con estireno, los individuos portadores de los alelos XRCC1 399Gln y XRCC3 241Met mostraron mayor nivel de roturas en el ADN, sugiriendo menor eficacia de los sistemas de reparación. Por el contrario, los portadores del alelo APE1 148Glu mostraron daño en el ADN significativamente menor que los individuos 148 Asp/Asp . Sin embargo, no se obtuvo ningún efecto significativo en las células expuestas a EO, debido probablemente a que el daño inducido es inicialmente mayor, y no permite que se pongan de manifiesto pequeñas diferencias en la eficacia de reparación de los genotipos analizados (AU)

Individual response to DNA damage induced by xenobiotic agents is conditioned by the efficiency of DNA repair systems. Some of the genetic polymorphisms described in DNA repair enzymes may affect the function of these proteins, and thus determine a modified susceptibility to the exposure to environmental agents. The purpose of the present study was to investigate if the most frequent allelic variants of the DNA repair genes XRCC1 ( Arg194Trp and Arg399Gln ), XRCC3 ( Thr241Met ) or APE1 ( Asp148Glu ) might alter the DNA damage induced by styrene and its principal metabolite, styrene-7,8- oxide (SO), in human leukocytes in vitro . Peripheral leukocytes from 30 healthy volunteers were treated with styrene or SO, and induced DNA damage was evaluated by the comet assay. After styrene treatment, carriers of XRCC1 399Gln and XRCC3 241Met alleles displayed higher level of DNA breakage, suggesting lesser efficiency of the DNA repair systems. In contrast, APE1 148Glu carriers showed significantly lower styreneinduced DNA damage than the 148 Asp/Asp individuals. Nevertheless, no significant effect was obtained in cells exposed to SO, probably due to the fact that the initially induced DNA damage is greater and because small differences in the repair efficiency of the selected genotypes are not manifested (AU)

Efectos de la colestásis sobre la toxicidad hepática del acetaminofen en ratas / The effects of cholestasis on the hepatotoxicity of acetaminophen in rats

Existen evidencias clínicas y experimentales de la toxicidad del acetaminofen en altas dosis. La toxicidad ha sido relacionada con un producto metabólico de la droga, a través del citocromo P-450. Pensando que la colestásis, al interferir con la excreción de productos tóxicos o modificar la actividad del P-450 puede incrementar la toxicidad de ciertos medicamentos y en vista del amplio uso del acetaminofen, planificamos un estudio para evaluar su toxicidad en ratas con obstrucción de vías biliares. Se usaron ratas Sprague Dawley, machos, entre 250 y 400 gr. de peso, distribuidas en grupos de 6 animales cada uno: grupo I, considerado control, al cual se le ligó el colédeco y se le administró solución salina al 0,9 por ciento IP (2 dosis de 0,2 ml/100 gr de peso). El grupo II también con ligadura de colédeco, recibió acetaminofen en solución IP, 400 mg/Kg de peso (2 dosis). Este grupo se distribuyó en dos subgrupos de 6 animales cada uno, los cuales fueron sacrificados a las 48 y a las 120 horas respectivamente. A todos se les determinó Bilirrubina total, bilirrubina directa y bilirrubina indirecta, fosfatasas alcalinas, transaminasas (ALT, AST), hematología completa, peso del higado y estudio histológico de hígado y riñón. En los animales con colestásis más acetaminofen hubo un aumento significativo de las transaminasas con respecto a los controles y una elevada incidencia de necrosis hepática en el estudio histológico de hígado (66 por ciento de los casos contra 20 por ciento del control). El tiempo de experimentaciión (48 y 120 horas), no afectó los resultados. La bilirrubina y el peso del hígado no variaron significativamente. Se concluye que la colestásis reciente potencia la toxicidad del acetaminofen a ½ dosis tóxica de la reportada en la literatura para ratas, aparentemente induciendo la actividad del sistema de oxidación mixta del citocromo P-450

The role of CYP enzymes in cocaine-induced liver damage.

Cocaine is hepatotoxic in several species, including man. A high dose of cocaine produces metabolism-dependent, mainly pericentral, liver damage. At 24 h after a single dose of cocaine, mouse hepatic P450 content decreases but CYP2A activities; coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase increase concomitant with prominent diffuse cell necrosis. Repeated administration of cocaine for up to 5 days decreases CYP1A1/2, 2A4/5, 2Cx, and 2E1 related enzymatic activities. However, after five doses of cocaine, CYP2B10 increases in conjunction with the healing process. In the acute phase, the increased CYP2A activities do not participate in cocaine bioactivation. CYP3A enzymes are principally responsible for the cocaine N-demethylation in human and mouse liver microsomes. The hepatic metabolic CYP enzyme profile will change during prolonged cocaine intake, this being accompanied by altered cell morphology. Possible connections to cocaine toxicity in man are discussed.

Toxicological significance of dog liver cytochrome P-450: examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid.

A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)). The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein. P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2). P-450(Dah1) activated 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo[a]pyrene.

Carcinogenésis renal experimental: función de la inducción del citocromo P-450 renal y del receptor estrogénico en la iniciación y la promoción del proceso neoplásico / Experimental renal carcinogenesis: role of renal P-450 cytochrome induction and of the estrogenic receptor on the iniciation and promotion of the neoplastic

Los tumores renales humanos son relativamente ratos. Sin embargo, la administración continua de estrógenos induce la formación de neoplasias renales en 100% de los cricetos machos (Mesocricetus auratus, llamdo también "hámster dorado"). Este modelo animal representa una gran oportunidad para conocer los mecanismos de carcinogénesis producidos bajo control hormonal para el estudio de enfermedad humana. Esta información sustenta las bases para la terapia hormonal con progestágenos y antiestrógenos, y estos hechos sugieren la posible génesis del tumor e indicación que, cuando menos en el hámster, el modelo puede probar su valor extrapolando el estudio a los carcinomas humanos. En el momento actual no existen pruebas que favorezcan la participación de los virus en la inducción del cáncer renal, o de que se incremente la importancia de la carcinogénesis química renal con la posible contribución de una gran variedad de carcinogénos claramente identificados, como los derivados de las nitrodietilaminas y las nitrosaminas. Muchos de los tumores animales espontáneos o experimentales muestran una estrecha semejanza morfológica con las características humanas, y ya que el hombre es otra especie animal, se pone de manifiesto que el análisis y el examen de los factores que participan en el origen de los tumores renales en animales de experimentación es de gran importancia para continuar con la búsqueda de los diversos factores etiológicos que participan en el cáncer humano. Cabe esperar que la información y los datos de la carcinogénesis experimental renal ayuden a estimular nuevas ideas y pensamientos sobre la naturaleza de las neoplasias renales humanas. Berenblum y Shubik (1947) demostraron claramente que en las carcinogénesis de la piel, se pueden distinguir dos etapas durante el desarrollo del tumor denominados eventos de iniciación y promoción; la primera se caracteriza por una unión covalente específica e irreversible del DNA con el agente carcinógejno o con algunos de sus, metabolitos, o bien por alteración de la conformación o la estructura del DNA (acción genotótica) en el segundo estadio;...

Drug metabolism by the human fetus.

A review of the literature that pertains to drug biotransformation in human fetal tissues reveals that, in spite of several publications in this comparatively new area of research, only very limited definitive information is currently available. The large majority of the studies performed have dealt with the cytochrome P-450-dependent microsomal mono-oxygenase systems and for several of the common drug metabolising reactions, very little or no data are available at this time. Some of the more important data that have emerged include observations that important bioactivation reactions can be demonstrated in human fetal tissues obtained during the period of late embryogenesis (high susceptibility to chemical dysmorphogenesis) and that the human fetal adrenal gland possesses considerable capacity to catalyse several important oxidation-reduction reactions. From the data available to date, it would appear that, in most instances, the biotransformation of drugs in the human embryo and fetus would not affect maternal plasma concentrations significantly. From the viewpoint of parameters of the pharmacokinetics of parent drug (or other xenobiotic) substrates under steady-state conditions, human fetal drug metabolism probably is of little consequence in most cases, although exceptions may exist. Pharmacokinetic parameters observed after isolated exposure, however, are very likely to be affected, perhaps markedly, in some instances. The demonstrated capacity of human prenatal tissues and cells to generate reactive intermediary metabolites, including those that produce mutations, has attracted the greatest attention recently. This capacity may be associated with extremely important adverse reactions to drugs and other environmental chemicals. Such adverse responses include transplacental mutagenesis, carcinogenesis, dysmorphogenesis, and perhaps several other undesirable effects. Although far from conclusive, the data tend to suggest that humans and subhuman primates may be more vulnerable than the smaller common experimental animals to the toxic effects of foreign organic chemicals during prenatal life. These factors should be weighed whenever exposure of pregnant women to such agents (e.g. via drug administration) is contemplated.