RESUMO
The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.
Assuntos
Plaquetas , Megacariócitos , Animais , Humanos , Camundongos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Plaquetas/metabolismo , Diferenciação Celular , Megacariócitos/metabolismo , Mercaptopurina/farmacologia , Mercaptopurina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismoRESUMO
CYP2A6 activity, phenotyped by the nicotine metabolite ratio (NMR), is a predictor of several smoking behaviors, including cessation and smoking-related disease risk. The heritability of the NMR is 60-80%, yet weighted genetic risk scores (wGRSs) based on common variants explain only 30-35%. Rare variants (minor allele frequency <1%) are hypothesized to explain some of this missing heritability. We present two targeted sequencing studies where rare protein-coding variants are functionally characterized in vivo, in silico, and in vitro to examine this hypothesis. In a smoking cessation trial, 1687 individuals were sequenced; characterization measures included the in vivo NMR, in vitro protein expression, and metabolic activity measured from recombinant proteins. In a human liver bank, 312 human liver samples were sequenced; measures included RNA expression, protein expression, and metabolic activity from extracted liver tissue. In total, 38 of 47 rare coding variants identified were novel; characterizations ranged from gain-of-function to loss-of-function. On a population level, the portion of NMR variation explained by the rare coding variants was small (~1%). However, upon incorporation, the accuracy of the wGRS was improved for individuals with rare protein-coding variants (i.e., the residuals were reduced), and approximately one-third of these individuals (12/39) were re-assigned from normal to slow metabolizer status. Rare coding variants can alter an individual's CYP2A6 activity; their integration into wGRSs through precise functional characterization is necessary to accurately assess clinical outcomes and achieve precision medicine for all. Investigation into noncoding variants is warranted to further explain the missing heritability in the NMR.
Assuntos
Citocromo P-450 CYP2A6/genética , Polimorfismo de Nucleotídeo Único , Resultado do Tratamento , Ensaios Clínicos como Assunto , Frequência do Gene , Genótipo , Humanos , Abandono do Hábito de FumarRESUMO
Vitamin D3 (VD3) induces intestinal CYP3A that metabolizes orally administered anti-leukemic chemotherapeutic substrates dexamethasone (DEX) and dasatinib potentially causing a vitamin-drug interaction. To determine the impact of VD3 status on systemic exposure and efficacy of these chemotherapeutic agents, we used VD3 sufficient and deficient mice and performed pharmacokinetic and anti-leukemic efficacy studies. Female C57BL/6J and hCYP3A4 transgenic VD3 deficient mice had significantly lower duodenal (but not hepatic) mouse Cyp3a11 and hCYP3A4 expression compared to VD3 sufficient mice, while duodenal expression of Mdr1a, Bcrp and Mrp4 were significantly higher in deficient mice. When the effect of VD3 status on DEX systemic exposure was compared following a discontinuous oral DEX regimen, similar to that used to treat pediatric acute lymphoblastic leukemia patients, male VD3 deficient mice had significantly higher mean plasma DEX levels (31.7 nM) compared to sufficient mice (12.43 nM) at days 3.5 but not at any later timepoints. Following a single oral gavage of DEX, there was a statistically, but not practically, significant decrease in DEX systemic exposure in VD3 deficient vs. sufficient mice. While VD3 status had no effect on oral dasatinib's area under the plasma drug concentration-time curve, VD3 deficient male mice had significantly higher dasatinib plasma levels at t = 0.25 hr. Dexamethasone was unable to reverse the poorer survival of VD3 sufficient vs. deficient mice to BCR-ABL leukemia. In conclusion, although VD3 levels significantly altered intestinal mouse Cyp3a in female mice, DEX plasma exposure was only transiently different for orally administered DEX and dasatinib in male mice. Likewise, the small effect size of VD3 deficiency on single oral dose DEX clearance suggests that the clinical significance of VD3 levels on DEX systemic exposure are likely to be limited.
Assuntos
Dasatinibe , Vitamina D , Animais , Feminino , Masculino , CamundongosRESUMO
Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Encéfalo/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Histidina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Metabolômica , Purinas/metabolismo , Pirimidinas/metabolismo , Ratos , Ratos TransgênicosRESUMO
Identifying the molecular mechanisms by which genome-wide association study (GWAS) loci influence traits remains challenging. Chromatin accessibility quantitative trait loci (caQTLs) help identify GWAS loci that may alter GWAS traits by modulating chromatin structure, but caQTLs have been identified in a limited set of human tissues. Here we mapped caQTLs in human liver tissue in 20 liver samples and identified 3,123 caQTLs. The caQTL variants are enriched in liver tissue promoter and enhancer states and frequently disrupt binding motifs of transcription factors expressed in liver. We predicted target genes for 861 caQTL peaks using proximity, chromatin interactions, correlation with promoter accessibility or gene expression, and colocalization with expression QTLs. Using GWAS signals for 19 liver function and/or cardiometabolic traits, we identified 110 colocalized caQTLs and GWAS signals, 56 of which contained a predicted caPeak target gene. At the LITAF LDL-cholesterol GWAS locus, we validated that a caQTL variant showed allelic differences in protein binding and transcriptional activity. These caQTLs contribute to the epigenomic characterization of human liver and help identify molecular mechanisms and genes at GWAS loci.
Assuntos
Cromatina/metabolismo , Fígado/metabolismo , Locos de Características Quantitativas , Motivos de Aminoácidos , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The frequencies of genetic variants in the CYP3A4 and CYP3A5 genes differ greatly across global populations, leading to profound differences in the metabolic activity of these enzymes and resulting drug metabolism rates, with important consequences for therapeutic safety and efficacy. Yet, the impact of genetic variants on enzyme activity are incompletely described, particularly in American Indian and Alaska Native (AIAN) populations. To characterize genetic variation in CYP3A4 and CYP3A5 and its effect on enzyme activity, we partnered with AIAN people living in two regions of Alaska: Yup'ik Alaska Native people living in the Yukon-Kuskokwim Delta region of rural southwest Alaska and AIAN people receiving care at the Southcentral Foundation in Anchorage, Alaska. We identified low frequencies of novel and known variation in CYP3A4 and CYP3A5, including low frequencies of the CYP3A4*1G and CYP3A5*1 variants, and linkage disequilibrium patterns that differed from those we previously identified in an American Indian population in western Montana. We also identified increased activity of the CYP3A4*1G allele in vitro and in vivo. We demonstrated that the CYP3A4*1G allele confers increased protein content in human lymphoblastoid cells and both increased protein content and increased activity in human liver microsomes. We confirmed enhanced CYP3A4-mediated 4ß-vitamin D hydroxylation activity in Yup'ik people with the CYP3A4*1G allele. AIAN people in Alaska and Montana who carry the CYP3A4*1G allele-coupled with low frequency of the functional CYP3A5*1 variant-may metabolize CYP3A substrates more rapidly than people with the reference CYP3A4 allele.
Assuntos
/genética , Citocromo P-450 CYP3A/metabolismo , Indígenas Norte-Americanos/genética , Xenobióticos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Linhagem Celular Tumoral , Criança , Pré-Escolar , Citocromo P-450 CYP3A/genética , Ensaios Enzimáticos , Feminino , Humanos , Lactente , Recém-Nascido , Desequilíbrio de Ligação , Masculino , Microssomos Hepáticos , Pessoa de Meia-Idade , Testes Farmacogenômicos , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
The effects of vitamin A and/or vitamin D deficiency were studied in an Arf-/- BCR-ABL acute lymphoblastic leukemia murine model. Vitamin D sufficient mice died earlier (p = 0.003) compared to vitamin D deficient (VDD) mice. Vitamin A deficient (VAD) mice fared worst with more rapid disease progression and decreased survival. Mice deficient for vitamins A and D (VADD) had disease progression similar to VAD mice. Regulatory T cells, previously shown to associate with poor BCR-ABL leukemia control, were present at higher frequencies among CD4+ splenocytes of vitamin A deficient vs. sufficient mice. In vitro studies demonstrated 1,25-dihydroxyvitamin D (1,25(OH)2VD3) increased the number of BCR-ABL ALL cells only when co-cultured with bone marrow stroma. 1,25(OH)2VD3 induced CXCL12 expression in vivo and in vitro in stromal cells and CXCL12 increased stromal migration and the number of BCR-ABL blasts. Vitamin D plus leukemia reprogrammed the marrow increasing production of collagens, potentially trapping ALL blasts. Vitamin A (all trans retinoic acid, ATRA) treated leukemic cells had increased apoptosis, decreased cells in S-phase, and increased cells in G0/G1. ATRA signaled through the retinoid X receptor to decrease BCR-ABL leukemic cell viability. In conclusion, vitamin A and D deficiencies have opposing effects on mouse survival from BCR-ABL ALL.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Vitamina A/metabolismo , Vitamina D/metabolismo , Animais , Apoptose , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores X de Retinoides/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Vitamina A/genética , Vitamina A/farmacologia , Vitamina D/genética , Vitamina D/farmacologiaRESUMO
We investigated whether pheophorbide A (PhA) could serve as a selective breast cancer resistance protein (BCRP) substrate (victim) to screen in vivo using fluorescent live animal imaging for transporter-mediated interactions with orally administered inhibitors (perpetrators), and whether that could be coupled with serum sampling to measure the systemic concentration of PhA with a fast-throughput in vitro fluorescent assay. PhA is a breakdown product of chlorophyll and is highly fluorescent in the near-infrared (NIR) spectrum. Whole-body NIR fluorescence was greater in the Bcrp KO compared with wild-type (WT) mice fed a regular diet containing chlorophyll and PhA, with fluorescence in WT mice confined to the intestine. PhA intestinal enterocyte fluorescence, after removing lumen contents, was greater in Bcrp knockout (KO) mice versus WT mice due to PhA enterocyte absorption and lack of PhA efflux by Bcrp. This difference was eliminated by maintaining the mice on an alfalfa (chlorophyll/PhA)-free diet. The area under the fluorescence ratio-time curve up to 6 hours (AUCFL 0-6 h) of orally administrated PhA was 3.5 times greater in the Bcrp KO mice compared with WT mice, and the PhA serum concentration was 50-fold higher in KO mice. Pretreatment with known BCRP inhibitors lapatinib, curcumin, elacridar, pantoprazole, and sorafenib, at clinically relevant doses, significantly increased PhA AUCFL 0-6 h by 2.4-, 2.3-, 2.2-, 1.5-, and 1.4-fold, respectively, whereas the area under PhA serum concentration-time curve calculated up to 6 hours (AUCSerum 0-6 h) increased by 13.8-, 7.8-, 5.2-, 2.02-, and 1.45-fold, respectively, and corresponded to their hierarchy as in vitro BCRP inhibitors. Our results demonstrate that live animal imaging using PhA can be used to identify BCRP inhibitors and to assess the potential for BCRP-mediated clinical drug-drug interactions.
Assuntos
Clorofila/análogos & derivados , Interações Medicamentosas/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Clorofila/metabolismo , Cães , Fluorescência , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos KnockoutRESUMO
To understand the systemic impact of breast cancer resistance protein (Bcrp) and P-glycoprotein (Pgp) deletion, untargeted metabolomics was performed on cerebral spinal fluid (CSF) and plasma of wild-type (WT) and Pgp and Bcrp double-knockout (dKO) rats anesthetized with ketamine-xylazine. We unexpectedly found elevated ketamine levels in both CSF and plasma of dKO versus WT rats. Therefore, the effect of these transporters was investigated on the 1) oral and intraperitoneal serum pharmacokinetics (PK) of ketamine, using a liquid chromatography method (high-performance liquid chromatography with ultraviolet detection), and 2) the anesthetic effect of ketamine using a duration of loss-of-righting reflex (dLORR) test in WT, Bcrp knockout (KO), Pgp KO, and Pgp/Bcrp dKO mice. The PK data demonstrated a significantly increased oral bioavailability and serum exposure of ketamine in dKO > Pgp KO > Bcrp KO mice compared with WT mice. Intraperitoneal ketamine-induced dLORR was significantly longer in dKO > Pgp KO > Bcrp KO > WT mice compared with WT mice. Inhibition of Bcrp and Pgp in WT mice using the dual Pgp/Bcrp inhibitor elacridar increased the ketamine-induced dLORR compared with vehicle-treated mice. The ketamine intracellular concentration was significantly decreased in Madin-Darby canine kidney II BCRP/PGP cells compared with the parental cells. In total, these results demonstrate that ketamine appears to be a dual Pgp/Bcrp substrate whose PK and pharmacodynamics are affected by Pgp and Bcrp-mediated efflux.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Ketamina/farmacologia , Ketamina/farmacocinética , Proteínas de Membrana Transportadoras/metabolismo , Animais , Disponibilidade Biológica , Transporte Biológico/fisiologia , Linhagem Celular , Cães , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-DawleyRESUMO
The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (â¼10-fold) with â¼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.
Assuntos
Androgênios/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Fígado/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Variações do Número de Cópias de DNA/genética , Feminino , Genótipo , Humanos , Inativação Metabólica/genética , Lactente , Recém-Nascido , Masculino , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Testosterona/metabolismo , Adulto JovemRESUMO
ATP-binding cassette (ABC) transporters are transmembrane efflux transporters mediating the extrusion of an array of substrates ranging from amino acids and lipids to xenobiotics, and many therapeutic compounds, including anticancer drugs. The ABC transporters are also recognized as important contributors to pharmacokinetics, especially in drug-drug interactions and adverse drug effects. Drugs and xenobiotics, as well as pathologic conditions, can influence the transcription of ABC transporters, or modify their activity or intracellular localization. Kinases can affect the aforementioned processes for ABC transporters as do protein interactions. In this review, we focus on the ABC transporters ABCB1, ABCB11, ABCC1, ABCC4, and ABCG2 and illustrate how kinases and protein-protein interactions affect these transporters. The clinical relevance of these factors is currently unknown; however, these examples suggest that our understanding of drug-drug interactions will benefit from further knowledge of how kinases and protein-protein interactions affect ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , Interações Medicamentosas/fisiologia , Fosfotransferases/metabolismo , Animais , Transporte Biológico/fisiologia , HumanosRESUMO
Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3 In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.
Assuntos
Calcifediol/sangue , Calcifediol/metabolismo , Sulfatos/metabolismo , Sulfotransferases/metabolismo , Vitamina D/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Hidroxilação/fisiologia , Lactente , Cinética , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , Receptores de Esteroides/metabolismo , Adulto JovemRESUMO
OBJECTIVES: Smoking patterns and cessation rates vary widely across smokers and can be influenced by variation in rates of nicotine metabolism [i.e. cytochrome P450 2A6 (CYP2A6), enzyme activity]. There is high heritability of CYP2A6-mediated nicotine metabolism (60-80%) owing to known and unidentified genetic variation in the CYP2A6 gene. We aimed to identify and characterize additional genetic variants at the CYP2A6 gene locus. METHODS: A new CYP2A6-specific sequencing method was used to investigate genetic variation in CYP2A6. Novel variants were characterized in a White human liver bank that has been extensively phenotyped for CYP2A6. Linkage and haplotype structure for the novel single nucleotide polymorphisms (SNPs) were assessed. The association between novel five-SNP diplotypes and nicotine metabolism rate was investigated. RESULTS: Seven high-frequency (minor allele frequencies ≥6%) noncoding SNPs were identified as important contributors to CYP2A6 phenotypes in a White human liver bank (rs57837628, rs7260629, rs7259706, rs150298687 (also denoted rs4803381), rs56113850, rs28399453, and rs8192733), accounting for two times more variation in in-vitro CYP2A6 activity relative to the four established functional CYP2A6 variants that are frequently tested in Whites (CYP2A6*2, *4, *9, and *12). Two pairs of novel SNPs were in high linkage disequilibrium, allowing us to establish five-SNP diplotypes that were associated with CYP2A6 enzyme activity (rate of nicotine metabolism) in-vitro in the liver bank and in-vivo among smokers. CONCLUSION: The novel five-SNP diplotype may be useful to incorporate into CYP2A6 genotype models for personalized prediction of nicotine metabolism rate, cessation success, and response to pharmacotherapies.
Assuntos
Citocromo P-450 CYP2A6/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nicotina/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Frequência do Gene , Estudo de Associação Genômica Ampla , Humanos , Técnicas In Vitro , Desequilíbrio de Ligação , Fígado/química , Bancos de Tecidos , População Branca/genéticaRESUMO
Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. CONCLUSIONS: Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).
Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Bile/metabolismo , Colestase Intra-Hepática/genética , Hepatócitos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Autofagia/genética , Colestase Intra-Hepática/patologia , Feminino , Humanos , Imunossupressores/farmacologia , Lactente , Fígado/patologia , Masculino , Mutação , Sirolimo/farmacologia , Peixe-Zebra/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 (CYP3A5 *3/*3) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA (P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis.
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citocromo P-450 CYP3A/genética , Hepatócitos/metabolismo , Midazolam/metabolismo , Tacrolimo/metabolismo , Linhagem Celular , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação Puntual/genética , RNA Mensageiro/genética , Deleção de Sequência/genéticaRESUMO
Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.
Assuntos
Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , Variação Genética , Fígado/enzimologia , Bancos de Tecidos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Nicotina/farmacologia , Oxirredutases/genética , RNA Mensageiro/genética , Umbeliferonas/farmacologia , Adulto JovemRESUMO
Determining appropriate pharmacotherapy in young children can be challenging due to uncertainties in the development of drug disposition pathways. With knowledge of the ontogeny of drug-metabolizing enzymes and an emerging focus on drug transporters, the developmental pattern of the uptake transporters organic anion transporting polypeptide (OATP) 1B1 and 1B3 was assessed by relative protein quantification using Western blotting in 80 human pediatric liver specimens covering an age range from 9 days to 12 years. OATP1B3 exhibited high expression at birth, which declined over the first months of life, and then increased again in the preadolescent period. In comparison with children 6-12 years of age, the relative protein expression of highly glycosylated (total) OATP1B3 was 235% (357%) in children <3 months of age, 33% (64%) in the age group from 3 months to 2 years, and 50% (59%) in children 2-6 years of age. The fraction of highly glycosylated to total OATP1B3 increased with age, indicating ontogenic processes not only at the transcriptional level but also at the post-translational level. Similar to OATP1B3, OATP1B1 showed high interindividual variability in relative protein expression but no statistically significant difference among the studied age groups.
Assuntos
Envelhecimento/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Fatores Etários , Envelhecimento/genética , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Lactente , Recém-Nascido , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Masculino , Processamento de Proteína Pós-Traducional , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genéticaRESUMO
The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Variação Genética/genética , Inativação Metabólica/genética , Xenobióticos/metabolismo , Animais , Interações Medicamentosas/genética , Humanos , FenótipoRESUMO
P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of â¼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.
Assuntos
Encéfalo/metabolismo , Genes Reporter/fisiologia , Luciferases de Vaga-Lume/biossíntese , Coluna Vertebral/metabolismo , Transcrição Gênica/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Diagnóstico por Imagem , Feminino , Humanos , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos TransgênicosRESUMO
The human pregnane X receptor (hPXR), a member of the nuclear receptor superfamily, senses xenobiotics and controls the transcription of genes encoding drug-metabolizing enzymes and transporters. The regulation of hPXR's transcriptional activation of its target genes is important for xenobiotic detoxification and endobiotic metabolism, and hPXR dysregulation can cause various adverse drug effects. Studies have implicated the putative phosphorylation site serine 350 (Ser(350)) in regulating hPXR transcriptional activity, but the mechanism of regulation remains elusive. Here we investigated the transactivation of hPXR target genes in vitro and in vivo by hPXR with a phosphomimetic mutation at Ser(350) (hPXR(S350D)). The S350D phosphomimetic mutation reduced the endogenous expression of cytochrome P450 3A4 (an hPXR target gene) in HepG2 and LS180 cells. Biochemical assays and structural modeling revealed that Ser(350) of hPXR is crucial for formation of the hPXR-retinoid X receptor alpha (RXRα) heterodimer. The S350D mutation abrogated heterodimerization in a ligand-independent manner, impairing hPXR-mediated transactivation. Further, in a novel humanized transgenic mouse model expressing the hPXR(S350D) transgene, we demonstrated that the S350D mutation alone is sufficient to impair hPXR transcriptional activity in mouse liver. This transgenic mouse model provides a unique tool to investigate the regulation and function of hPXR, including its non-genomic function, in vivo. Our finding that phosphorylation regulates hPXR activity has implications for development of novel hPXR antagonists and for safety evaluation during drug development.