RESUMEN

We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and apolipoprotein E (ApoE)-/- mice. Mice were fed a high-fat diet (HFD) for 3 mo, together with administration of either cetirizine (4 mg/kg body wt) or fexofenadine (40 mg/kg body wt) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight, and hepatic steatosis in wild-type mice but not in ApoE-/- mice. Lobular inflammation, acute inflammation, and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, whereas lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element-binding protein in wild-type mice, without altering the mRNA expression of sterol regulatory element-binding protein 1c, fatty acid synthase, or ApoB100, in either genotype. Fexofenadine increased both triglycerides and cholesterol ester, whereas cetirizine increased only cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild-type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 and bile salt export pump. These results suggest that H1-antihistamines increase the progression of fatty liver disease in wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE, and increase in hepatic bile acid levels.

Asunto(s)

Apolipoproteínas E/deficiencia , Cetirizina/toxicidad , Dieta Alta en Grasa , Hígado Graso/inducido químicamente , Antagonistas de los Receptores Histamínicos H1/toxicidad , Hígado/efectos de los fármacos , Terfenadina/análogos & derivados , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteínas E/genética , Ácidos y Sales Biliares/metabolismo , Biomarcadores/sangre , Ésteres del Colesterol/metabolismo , Modelos Animales de Enfermedad , Hígado Graso/sangre , Hígado Graso/genética , Hígado Graso/patología , Regulación de la Expresión Génica , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/metabolismo , Hígado/patología , Transportador 1 de Anión Orgánico Específico del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Índice de Severidad de la Enfermedad , Terfenadina/toxicidad , Triglicéridos/metabolismo

RESUMEN

Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a K(i) of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent K(M) and k(cat) of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR increased up to 23-fold with virtually no change in the k(cat) for the reaction, however, at higher concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the k(cat) also decreased by 11-fold. Additionally, CYP2E1 increased the apparent K(M) of CYP2B4 for BNZ by 8-fold and the apparent K(M) did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent K(M) values of CYP2B4 and CYP2E1 for CPR are similar, the apparent K(M) of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Secuencia de Bases , Catálisis , Familia 2 del Citocromo P450 , Cartilla de ADN , Hidroxilación , Especificidad por Sustrato

RESUMEN

The mechanism-based inactivation of human CYP2B6 by tert-butylphenylacetylene (BPA) in the reconstituted system was investigated. The inactivation of CYP2B6 by BPA is time-, concentration-, and NADPH-dependent and exhibits a K(I) of 2.8 µM, a k(inact) of 0.7 min(-1), and a t(1/2) of 1 min. The partition ratio is ∼5. Unlike CYP2B1 and CYP2B4, in addition to the formation of an apoprotein adduct and a glutathione conjugate, a small heme adduct was observed when CYP2B6 was incubated with BPA. The mass increase of the adducted apoprotein and GSH conjugate is 174 Da, equivalent to the mass of one molecule of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B6 was digested with trypsin, and the digest was then analyzed by liquid chromatography-tandem mass spectrometry. A mass shift of 174 Da was used for the SEQUEST database search, and the identity of the modified residue was confirmed by MS/MS fragmentation of the modified peptide. Two residues, Lys274 and Thr302, were identified as having been modified. Further mutagenesis studies have demonstrated that the residue that is modified to result in inactivation is Thr302, not Lys274. Docking studies show that in the enzyme-substrate complex, Thr302 is in close contact with the triple bond of BPA with a distance of 3.8 Å between the terminal carbon of BPA and the oxygen in the hydroxyl group of Thr302. In conclusion, Thr302 of CYP2B6 is covalently modified by a reactive metabolite of BPA, and this modification is responsible for the mechanism-based inactivation.

Asunto(s)

Acetileno/análogos & derivados , Hidrocarburo de Aril Hidroxilasas/metabolismo , Inhibidores Enzimáticos/farmacología , Oxidorreductasas N-Desmetilantes/metabolismo , Acetileno/farmacología , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Bases , Cromatografía Liquida , Citocromo P-450 CYP2B6 , Cartilla de ADN , Humanos , Mutagénesis Sitio-Dirigida , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/genética , Espectrometría de Masas en Tándem

RESUMEN

Transfer RNA genes are distributed throughout eukaryotic genomes, and are frequently found as multicopy families. In Saccharomyces cerevisiae, tRNA gene transcription by RNA polymerase III suppresses nearby transcription by RNA polymerase II, partially because the tRNA genes are clustered near the nucleolus. We have tested whether active transcription of tRNA genes might also suppress recombination, since recombination between identical copies of the repetitive tRNA genes could delete intervening genes and be detrimental to survival. The opposite proved to be the case. Recombination between active tRNA genes was elevated, but only when both genes are transcribed. We also tested the effects of tRNA genes on recombination between the direct terminal repeats of a neighboring retrotransposon, since most Ty retrotransposons reside next to tRNA genes, and the selective advantage of this arrangement is not known.

Asunto(s)

ARN de Hongos/genética , ARN de Transferencia/genética , Recombinación Genética , Retroelementos , Saccharomyces cerevisiae/genética