RESUMEN
Quantitative reverse-transcriptase polymerase chain reaction was used to determine the content of mRNA derived from four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in rat liver. CYP3A2 and CYP3A9 gene expression was age- and sex-dependent, whereas CYP3A18 and CYP3A23 mRNA were observed before and after puberty at fairly constant levels that were about 20% higher in males than in females. CYP3A9 mRNA was detected only in adult rats, with a nearly twofold higher expression in females. CYP3A9 induction was different from other CYP3A isoenzymes, since phenobarbital was a more effective inducer than dexamethasone and clotrimazole. The results presented for CYP3A23 are those anticipated for CYP3A1, which may be an allelic variant of CYP3A23 not detected in these experiments. These data show that rat CYP3A genes are variably expressed depending on age, sex, or inducer type.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas N-Desmetilantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citocromo P-450 CYP3A , Inducción Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Factores SexualesRESUMEN
The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5- and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5- and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5- and 6-ring PAH concentration in the soil and the microsomal CYP1A1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.
Asunto(s)
Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxidorreductasas/efectos de los fármacos , Oxidorreductasas/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , Animales , Disponibilidad Biológica , Biomarcadores , Carbono/metabolismo , Citocromo P-450 CYP1A1 , Exposición a Riesgos Ambientales , Isoenzimas , Masculino , Microsomas Hepáticos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Contaminantes del Suelo/toxicidad , Relación Estructura-ActividadRESUMEN
A cDNA library constructed from adult female Sprague Dawley rat liver was screened with polyclonal anti CYP3A-IgG. One of the positive clones, cUT, was found to contain the complete coding sequence of a new gene more similar to hamster gene CYP3A10 (cDNA: 85%, deduced amino acid sequence: 79%) than to the known rat CYP3A genes (cDNA: 75-76%, amino acid sequences: 66-69%). The deduced sequence of the first 28 amino acids is identical to the N-terminus of the testosterone 6 beta-hydroxylase, 6 beta-2 (Nagata, K., Gonzalez, F.J., Yamazoe, Y. and Kato, R. (1990) J. Biochem. 107, 718-725). Northern blots show the presence of cUT mRNA in livers of adult female and male rats. The transcription of the new gene is enhanced in either sex by pregnenolone-alpha-carbonitrile, dexamethasone, phenobarbital, and triacetyloleandomycin, known inducers of CYP3A gene expression.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Oxigenasas de Función Mixta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/química , ADN Complementario , Femenino , Masculino , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
CYP3A proteins are P450 monooxygenases involved in the metabolism of steroids, retinoic acid and several important drugs. In rats, the number of CYP3A genes and proteins, and therefore important aspects of their inducibility, developmental regulation and sex specificity are not known for certain. Using triacetyloleandomycin-metabolite complex formation, testosterone hydroxylase assays and immunoblots from peptide maps, we obtained results suggesting that in liver microsomes from mature rats, at least three, possibly four CYP3A proteins are expressed: one is present in females, another is male-specific, and one or two additional CYP3A proteins are inducible by phenobarbital, steroids, and triacetyloleandomycin.