RESUMO
Human hepatic UGT2B15 developmental expression changes may alter the metabolism of important drugs and toxicants such as bisphenol A (BPA). Previously, UGT2B15 ontogeny knowledge consisted of transcript data, a dubious surrogate for protein expression. Herein, UGT2B15 protein content was determined in human hepatic microsomes (n = 236, 8 weeks gestation to 18 years). The impact of a common, functional single nucleotide polymorphism (g.253G>T), present in UGT2B15*2 and *5 alleles, was also tested. UGT2B15 expression began during late fetal life, at about 18% of mature values (medians = 48, 267 pmoles/mg of microsomal protein, respectively; p < 0.001). UGT2B15 neonatal (n = 39) and late fetal (≥28 weeks, n = 10) content was similar, but lower than that of infants between 3 and 15 weeks age (n = 46; medians = 38, 48, 404 pmoles/mg microsomal protein, respectively; p < 0.001). Values for the latter group were higher compared with the remaining age group (15 weeks to 18 years; n = 82, p < 0.001). UGT2B15 expression varied 31-fold across the entire sample, and within groups, ranged from 4- to 27-fold. Among postnatal samples, age group, the presence of g.253T and male gender were each significantly associated with greater UGT2B15 expression (p < 0.001, <0.01, and <0.05, respectively; stepwise linear regression). In summary, hepatic UGT2B15 protein onset begins in late gestation; however, the greatest rate of change occurs during the first few weeks after birth. We speculate that the fetus and neonate may have lower clearance of some UGT2B15 substrates, such as BPA, compared with older individuals.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glucuronosiltransferase/genética , Fígado/enzimologia , Organogênese , Adolescente , Criança , Pré-Escolar , Feminino , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Masculino , Microssomos Hepáticos/enzimologia , Organogênese/genética , Polimorfismo de Nucleotídeo Único , Gravidez , Caracteres SexuaisRESUMO
Within the human cytochrome P450 family, specific forms show developmental expression patterns that can affect drug clearance, efficacy, and safety. The objective of this study was to use dextromethorphan O-demethylase activity and quantitative Western blotting to identify CYP2D6 developmental expression patterns in a large (n = 222) and developmentally diverse set of pediatric liver samples. Immunodetectable levels of CYP2D6 protein determined for selected samples across all age categories showed a significant correlation with the corresponding dextromethorphan O-demethylase activity. Of gender, ethnicity, postmortem interval, and genotype, only increasing gestational age was associated with CYP2D6 activity and protein content in prenatal samples. In contrast, both age and genotype were associated with CYP2D6 expression in postnatal samples. CYP2D6 expression in liver samples from neonates less than 7 days of age was higher than that observed in first and second trimester samples, but not significantly higher than third trimester fetal samples. In contrast, expression in postnatal samples greater than 7 days of age was substantially higher than that for any earlier age category. Higher CYP2D6 expression also was observed in liver samples from Caucasians versus African Americans. Finally, using phenotype categories inferred from genotype, CYP2D6 activity was higher in postnatal samples predicted to be extensive or intermediate metabolizers versus poor metabolizers. These results suggest that age and genetic determinants of CYP2D6 expression constitute significant determinants of interindividual variability in CYP2D6-dependent metabolism during ontogeny.
Assuntos
Citocromo P-450 CYP2D6/genética , Regulação da Expressão Gênica no Desenvolvimento , Western Blotting , Criança , Pré-Escolar , Citocromo P-450 CYP2D6/metabolismo , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Microssomos Hepáticos/enzimologiaRESUMO
OBJECTIVES: To test the hypothesis that children taking hydroxyurea who fail codeine therapy have an increase in reduced-functioning cytochrome P450 2D6 (CYP2D6) alleles. STUDY DESIGN: Children with sickle cell disease presenting to an emergency department with a pain crisis unresponsive to codeine were genotyped. The proportion of children with reduced-functioning alleles and CYP2D6 enzyme activity scores < or = 1.5, were compared, by chi2 analysis, in children taking hydroxyurea and those with mild disease. RESULTS: Of the 73 children completing the study, 42 had reduced-functioning alleles; 82% of the 27 children taking hydroxyurea had reduced-functioning alleles, versus 47% of 36 those with mild disease (P < .05). Activity scores were decreased in 78% of the children taking hydroxyurea and in 44% of those with mild disease (P < .05). The odds ratios of children taking hydroxyurea were 4.9 (95% confidence interval [CI] = 1.5 to 15.9) for having reduced-functioning alleles, and 4.4 (95% CI = 1.4 to 13.4) for having a low activity score. CONCLUSIONS: Failing codeine therapy for a pain crisis while taking hydroxyurea is associated with an increase in reduced-functioning CYP2D6 alleles. We recommend genetic analysis or trial of a non-CYP2D6 analgesic for these children.
Assuntos
Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Citocromo P-450 CYP2D6/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Analgésicos Opioides/uso terapêutico , Anemia Falciforme/complicações , Antidrepanocíticos/uso terapêutico , Criança , Pré-Escolar , Codeína/uso terapêutico , Deleção de Genes , Genótipo , Humanos , Hidroxiureia/uso terapêutico , Dor/tratamento farmacológico , FenótipoRESUMO
Both transcriptional and post-transcriptional CYP2E1 regulatory mechanisms are known, resulting in 20-fold or greater variation in CYP2E1 expression. To evaluate functional regulatory elements controlling transcription, CYP2E1 promoter constructs were used to make adenovirus vectors containing CYP2E1 promoter-driven luciferase reporters for analyses in both primary human hepatocytes and HepG2 cells. A 1.2-kilobase pair portion of the CYP2E1 promoter was associated with 5- to 10-fold greater luciferase activity. This upstream region contained five direct repeats of 59 base pairs (bp) that increased thymidine kinase-driven luciferase reporter activity in HepG2 cells more than 5-fold, regardless of orientation. Electrophoretic mobility shift assays (EMSAs) identified sequence-specific nuclear protein binding to the 59-bp repeats that was dependent on a 17-bp sequence containing a canonical GATA binding site (WGATAR). Competitive and supershift EMSA identified the participation of GATA4, another GATA family member or GATA-like factor, and a third factor unrelated to the GATA family. Involvement of the tricho-rhino-phalangeal syndrome-1 factor, which also binds a GATA sequence, was eliminated. Rather, competitive EMSA using known binding sequences for the orphan nuclear receptors, steroidogenic factor-1 (or NR5A1), and fetoprotein transcription factor (or NR5A2) implicated an NR5A member in binding a sequence overlapping the canonical GATA. Chromatin immunoprecipitation assay demonstrated in vivo binding of NR5A2 to the enhancer sequence in human hepatocytes. The enhancer sequence is conserved within the human population but seems species-specific. The identification of this novel enhancer and its putative mechanism adds to the complexities of human CYP2E1 regulation.
Assuntos
Citocromo P-450 CYP2E1/genética , Elementos Facilitadores Genéticos/fisiologia , Fator de Transcrição GATA4/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/fisiologia , Sequências Repetitivas de Ácido Nucleico/fisiologia , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Citocromo P-450 CYP2E1/metabolismo , Variação Genética/fisiologia , Hepatócitos/metabolismo , Humanos , Dados de Sequência Molecular , Fator Esteroidogênico 1 , Transativadores , Ativação TranscricionalRESUMO
Human hepatic CYP2E1 expression developmental changes likely have an impact on the effects of xenobiotics metabolized by the encoded enzyme. To resolve previous conflicting results, CYP2E1 content was determined in human hepatic microsomes from samples spanning fetal (n = 73, 8-37 weeks) and postnatal (n = 165, 1 day-18 years) ages. Measurable immunodetectable CYP2E1 was seen in 18 of 49 second-trimester (93-186 gestational days) and 12 of 15 third-trimester (>186 days) fetal samples (medians = 0.35 and 6.7 pmol/mg microsomal protein, respectively). CYP2E1 in neonatal samples was low and less than that of infants 31 to 90 days of age, which was less than that of older infants, children, and young adults [median (range) = 8.8 (0-70); 23.8 (10-43); 41.4 (18-95) pmol/mg microsomal protein, respectively; each P < 0.001, analysis of variance, post hoc]. Among those older than 90 days of age, CYP2E1 content was similar. A 4-fold or greater intersubject variation was observed among samples from each age group, with the greatest variation, 80-fold, seen among neonatal samples. Among subjects of known gestational and postnatal age (n = 29) increasing protein content was associated with increasing postnatal age (P < 0.001, linear regression), but only equivocally with increasing gestational age (P = 0.07). Individuals from the third trimester through 90 days postnatal age with one or more CYP2E1*1D alleles had lower CYP2E1 protein content than similar-aged subjects who were homozygous CYP2E1*1C. In summary, CYP2E1 was clearly expressed in human fetal liver. Furthermore, the postnatal data suggest that infants less than 90 days old would have decreased clearance of CYP2E1 substrates compared with older infants, children, and adults.