RESUMO
Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.
Assuntos
Citocromo P-450 CYP3A , Hepatomegalia , Regeneração Hepática , Fígado , Receptor de Pregnano X , Carbonitrila de Pregnenolona , Animais , Receptor de Pregnano X/metabolismo , Receptor de Pregnano X/genética , Regeneração Hepática/efeitos dos fármacos , Masculino , Citocromo P-450 CYP3A/metabolismo , Carbonitrila de Pregnenolona/farmacologia , Fígado/metabolismo , Fígado/enzimologia , Fígado/efeitos dos fármacos , Ratos , Hepatomegalia/metabolismo , Hepatomegalia/patologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/metabolismo , Família 2 do Citocromo P450/genética , Ratos Sprague-Dawley , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 12-alfa-Hidroxilase/metabolismo , Esteroide 12-alfa-Hidroxilase/genética , HepatectomiaRESUMO
CYP2A5, an enzyme responsible for metabolism of diverse drugs, displays circadian rhythms in its expression and activity. However, the underlying mechanisms are not fully established. Here we aimed to investigate a potential role of CRY1/2 (circadian clock modulators) in circadian regulation of hepatic CYP2A5. Regulatory effects of CRY1/2 on CYP2A5 were determined using Cry1-null and Cry2-null mice, and validated using AML-12, Hepa1-6 and HepG2 cells. CYP2A5 activities both in vivo and in vitro were assessed using coumarin 7-hydroxylation as a probe reaction. mRNA and protein levels were detected by qPCR and western blotting, respectively. Regulatory mechanism was studied using a combination of luciferase reporter assays, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP). We found that ablation of Cry1 or Cry2 in mice reduced hepatic CYP2A5 expression (at both mRNA and protein levels) and blunted its diurnal rhythms. Consistently, these knockouts showed decreased CYP2A5 activity (characterised by coumarin 7-hydroxylation) and a loss of its time-dependency, as well as exacerbated coumarin-induced hepatotoxicity. Cell-based assays confirmed that CRY1/2 positively regulated CYP2A5 expression and rhythms. Based on combined luciferase reporter, ChIP and Co-IP assays, we unraveled that CRY1/2 interacted with E4BP4 protein to repress its inhibitory effect on Cyp2a5 transcription and expression. In conclusion, CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4. These findings advance our understanding of circadian regulation of drug metabolism and pharmacokinetics.
Assuntos
Ritmo Circadiano , Criptocromos , Fatores de Transcrição , Animais , Camundongos , Ritmo Circadiano/genética , Fígado , Luciferases , RNA Mensageiro , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismoRESUMO
Sepsis is a pathological condition that affects the metabolism of administered drugs, leading to changes in the duration and intensity of their intended efficacies. Proinflammatory cytokines downregulate the expression of cytochrome P450s (P450s). The effects of P450 expression under inflammatory conditions have been studied using prophlogistic substances such as lipopolysaccharide; however, few studies have focused on clinical models of sepsis. Here, we show that cecal ligation and puncture (CLP), an approach for the study of human polymicrobial sepsis, leads to the expression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNFα) at 24 h after the CLP operation. Following CLP, IL-6-/- mice exhibited markedly lower survival than WT mice. In addition, CLP led to the significant downregulation of Cyp2c29 and Cyp3a11 gene expression in IL-1α-/-/ß-/- (IL-1-/-) and TNFα-/- mice as well as in WT mice. In contrast, CLP elicited no significant effect on Cyp3a11 expression in IL-6-/- mice. Although CLP reduced the Cyp2c29 expression level in IL-6-/- mice, the expression of Cyp2c29 was lower in CLP-operated WT mice than in CLP-operated IL-6-/- mice. The reduction in the respective P450 protein levels and activities due to CLP-induced sepsis, reflected in the mRNA expression levels, was abolished by IL-6 depletion. Thus, CLP-induced sepsis downregulates P450 gene expression, particularly Cyp2c expression, and this effect is associated with IL-6 without affecting resistance to CLP-induced sepsis. These findings demonstrate the usefulness of CLP for studying the regulation of P450s and highlight IL-6 as a potential indicator of drug-metabolizing capacity under septic conditions.
Assuntos
Interleucina-6 , Sepse , Humanos , Camundongos , Animais , Interleucina-6/genética , Interleucina-6/metabolismo , Regulação para Baixo , Fator de Necrose Tumoral alfa/metabolismo , Punções , Ligadura , Expressão Gênica , Sepse/metabolismo , Ceco/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Proteínas de Membrana/metabolismo , Citocromo P-450 CYP3A/genéticaRESUMO
Aim: Our previous results suggest that phenyl/naphthylacetyl pentanoic acid derivatives may exhibit dual MMP-2 and HDAC8 inhibitory activities and show effective cytotoxic properties. Methodology: Here, 13 new compounds (C1-C13) were synthesized and characterized. Along with these new compounds, 16 previously reported phenyl/napthylacetyl pentanoic acid derivatives (C14-C29) were biologically evaluated. Results: Compounds C6 and C27 showed good cytotoxicity against leukemia cell line Jurkat E6.1. The mechanisms of cytotoxicity of these compounds were confirmed by DNA deformation assay and reactive oxygen species assay. MMP-2 and HDAC8 expression assays suggested the dual inhibiting property of these two compounds. These findings were supported by results of molecular docking studies. In silico pharmacokinetic properties showed compounds C6 and C27 have high gastrointestinal absorption. Conclusion: This study highlights the action of phenyl/naphthylacetyl pentanoic acid derivatives as anticancer agents.
Assuntos
Antineoplásicos/síntese química , Simulação de Acoplamento Molecular , Ácidos Pentanoicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/metabolismo , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Relação Estrutura-AtividadeRESUMO
Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Duocarmicinas/farmacologia , Biomarcadores , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Família 2 do Citocromo P450/metabolismo , Desenho de Fármacos , Humanos , Pró-Fármacos , EstereoisomerismoRESUMO
Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.
Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1B1/antagonistas & inibidores , Família 2 do Citocromo P450/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Estudos de Coortes , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Família 2 do Citocromo P450/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Camundongos , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of N-skatyltryptamines was synthesized and their affinities for serotonin and dopamine receptors were determined. Compounds exhibited activity toward 5-HT1A, 5-HT2A, 5-HT6, and D2 receptors. Substitution patterns resulting in affinity/activity switches were identified and studied using homology modeling. Chosen hits were screened to determine their metabolism, permeability, hepatotoxicity, and CYP inhibition. Several D2 receptor antagonists with additional 5-HT6R antagonist and agonist properties were identified. The former combination resembled known antipsychotic agents, while the latter was particularly interesting due to the fact that it has not been studied before. Selective 5-HT6R antagonists have been shown previously to produce procognitive and promnesic effects in several rodent models. Administration of 5-HT6R agonists was more ambiguous-in naive animals, it did not alter memory or produce slight amnesic effects, while in rodent models of memory impairment, they ameliorated the condition just like antagonists. Using the identified hit compounds 15 and 18, we tried to sort out the difference between ligands exhibiting the D2R antagonist function combined with 5-HT6R agonism, and mixed D2/5-HT6R antagonists in murine models of psychosis.
Assuntos
Antipsicóticos/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Indóis/farmacologia , Nootrópicos/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Triptaminas/farmacologia , Animais , Antipsicóticos/síntese química , Família 2 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Inibidores da Captação de Dopamina/síntese química , Células Hep G2 , Humanos , Indóis/síntese química , Ligantes , Masculino , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Nootrópicos/síntese química , Ligação Proteica , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/metabolismo , Transtornos Psicóticos/fisiopatologia , Receptores de Dopamina D2/metabolismo , Receptores de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Relação Estrutura-Atividade , Triptaminas/síntese químicaRESUMO
Since the discovery of its role in vitamin D metabolism, significant progress has been made in the understanding of gene organisation, protein structure, catalytic function, and genetic polymorphism of cytochrome P450 2R1 (CYP2R1). Located on chromosome 11p15.2, CYP2R1 possesses five exons, unlike most other CYP isoforms that carry nine exons. CYP2R1 crystal structure displays a fold pattern typical of a CYP protein, with 12 a-helices as its structural core, and b-sheets mostly arranged on one side, and the heme buried in the interior part of the protein. Overall, CYP2R1 structure adopts a closed conformation with the B' helix serving as a gate covering the substrate access channel, with the substrate vitamin D3 occupying a position with the side chain pointing toward the heme group. In liver, CYP2R1 25-hydroxylates vitamin D and serves as an important determinant of 25(OH)D level in the tissue and in circulation. While substrate profile has been well studied, inhibitor specificity for CYP2R1 requires further investigation. Both exonic and non-exonic single nucleotide polymorphisms (SNPs) have been reported in CYP2R1, including the CYP2R1*2 carrying Leu99Pro exchange, and a number of non-exonic SNPs with variable functional consequences in gene regulation. A non-exonic SNP, rs10741657, has its causal relationship with diseases established, including that of rickets, ovarian cancer, and multiple sclerosis. The role of other CYP2R1 SNPs in vitamin D deficiency and their causal link to other traits however remain uncertain currently and more studies are warranted to help identify possible physiological mechanisms underlying those complex traits.
Assuntos
Colestanotriol 26-Mono-Oxigenase , Família 2 do Citocromo P450 , Polimorfismo Genético/genética , Colestanotriol 26-Mono-Oxigenase/química , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Humanos , Conformação ProteicaRESUMO
BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.
Assuntos
Canalículos Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Colestase/genética , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-2/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colagogos e Coleréticos/uso terapêutico , Ácido Cólico , Claudina-1/metabolismo , Família 2 do Citocromo P450/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais , Feminino , Fibrose , Predisposição Genética para Doença , Hepatócitos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Oxazóis/uso terapêutico , Permeabilidade , Fatores de Proteção , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/metabolismo , Junções Íntimas/ultraestrutura , Ácido Ursodesoxicólico/uso terapêutico , Proteína da Zônula de Oclusão-2/deficiênciaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Geissoschizine methyl ether (GM), an indole alkaloid from Uncaria hook, is an active ingredient in the traditional Japanese Kampo medicine yokukansan, which is used to treat neurosis, insomnia, irritability, and night crying in children. AIM OF THE STUDY: Recent our pharmacokinetic studies suggested that there may be gender differences in the plasma concentrations of GM in rats, but not in humans. However, the details of this difference remain unverified. The purpose of this study was to clarify the reasons for the gender differences in rats. MATERIALS AND METHODS: GM plasma pharmacokinetics was compared in male and female rats orally administered yokukansan (4â¯g/kg). To confirm the involvement of cytochrome P450 (CYP) in GM liver metabolism, GM was incubated with male and female rat liver S9 fraction in the absence or presence of 1-aminobenzotriazole (a nonspecific CYP inhibitor). CYP isoforms involved in GM metabolism were estimated using recombinant rat CYP isoforms and anti-rat CYP antibodies. RESULTS: The maximum GM plasma concentrations were significantly higher in female than in male rats. When GM was incubated with rat liver S9 fractions, GM reduction was more striking in male S9 (69.3%) than that in female S9 (10.0%) and was completely blocked with nonspecific CYP inhibitor 1-aminobenzotriazole. Screening experiments using recombinant rat cytochrome P450 (CYP) isoforms showed that CYP1A1, CYP2C6, CYP2C11, CYP2D1, and CYP3A2 were involved in GM metabolism. Of these CYP isoforms, the use of anti-rat CYP antibodies indicated that male-dependent CYP2C11 and CYP3A2 were predominantly involved in the liver microsomal GM metabolism with gender differences. CONCLUSIONS: These results suggest that the cause of gender differences in plasma GM pharmacokinetics in rats is most likely because of male-dependent CYP2C11 and CYP3A2, and provide also useful information to further evaluate the pharmacological and toxicological effects in future. This study is the first to demonstrate that the gender differences in plasma GM pharmacokinetics in rats are caused by the gender-dependent metabolism of GM.
Assuntos
Alcaloides Indólicos/sangue , Microssomos Hepáticos/efeitos dos fármacos , Caracteres Sexuais , Uncaria , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP3A/metabolismo , Família 2 do Citocromo P450/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Alcaloides Indólicos/metabolismo , Alcaloides Indólicos/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos Hepáticos/enzimologia , Plasma/efeitos dos fármacos , Plasma/metabolismo , Ratos , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/metabolismoRESUMO
The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2 months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4 weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/genética , Fígado/metabolismo , Esteroide Hidroxilases/genética , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Esteroide Hidroxilases/metabolismoRESUMO
BACKGROUND: Retrorsine is one of the hepatotoxic pyrrolizidine alkaloids, which could be converted into a highly reactive metabolite, dehydroretrorsine, by CYP3A, and to a lesser extent by CYP2C and CYP2B. OBJECTIVE: We employed Cyp3a knockout (3AKO) mice to investigate whether the absence of CYP3A could attenuate dehydroretrorsine formation and the role of CYP2C and CYP2B in the formation. METHODS: Blood and liver samples were collected after intragastrical administration of 35 mg/kg retrorsine or saline for seven days in wild-type (WT) and 3AKO mice. Blood pyrrole-protein adducts were semi quantified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The formations of glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (GSH-DHP) and the activities of CYP3A, CYP2B and CYP2C were evaluated in the liver microsomes of WT and 3AKO mice before and after treatment. The metabolic phenotype of retrorsine was determined in human liver microsomes. The gene and protein expression of retrorsine metabolism-related CYP450s in the liver was measured by quantitative real-time PCR method and western blotting method. The serum cytokine level was detected by the ELISA method to reveal the potential mechanism of Cyp3a, Cyp2b and Cyp2c downregulation. RESULTS: After an oral administration of 35 mg/kg retrorsine for seven days, the blood exposures of DHP adducts between WT and 3AKO mice were similar, consistent with the comparable formation of GSH-DHP in their liver microsomes. The chemical inhibitor experiment in liver microsomes indicated the predominant role of CYP3A and CYP2C in GSH-DHP formation in WT and 3AKO mice, respectively. Real-time qPCR analysis showed that the expressions of Cyp2b10 and Cyp2cs increased 2.3-161-fold in 3AKO mice, which was consistent with protein changes. The increased CYP2B activity in 3AKO mice supported the potential role of CYP2B in GSH-DHP formation. After a seven-day treatment of retrorsine, the yields of GSH-DHP were lower than the untreated ones in both alleles, accompanied by the decreased mRNA of Cyp3a, Cyp2b and Cyp2c. The increased serum IL6 might mediate the retrorsine-induced downregulation of Cyp450s. CONCLUSION: These data demonstrated the increased transcription of Cyp2c and Cyp2b caused by Cyp3a ablation, which played a vital role in the metabolic activation of retrorsine, and long-term exposure of retrorsine can reduce the CYP450 activities.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Alcaloides de Pirrolizidina/farmacocinética , Esteroide Hidroxilases/metabolismo , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Hidrocarboneto de Aril Hidroxilases/genética , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Microssomos Hepáticos , Modelos Animais , Alcaloides de Pirrolizidina/administração & dosagem , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esteroide Hidroxilases/genética , Ativação TranscricionalRESUMO
Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Metabólica , Nicotina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Fumaça/efeitos adversos , Animais , Animais Recém-Nascidos , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Regiões Promotoras GenéticasRESUMO
Cytochrome P450 (CYP) epoxygenases have been considered the main producers of epoxyeicosatrienoic acids (EETs) through the oxidation of arachidonic acid (AA). EETs display various biological properties, notably their powerful anti-inflammatory activities. In the brain, EETs have proven to be neuroprotective and to improve neuroinflammation. However, it is known that inflammation could modify CYP expression. We have previously reported that an inflammatory process in astrocytes is able to down-regulate CYP2J3 and CYP2C11 mRNA, protein levels, and activity (Navarro-Mabarak et al., 2019). In this work, we evaluated the effect of neuroinflammation in protein expression of CYP epoxygenases in the brain. Neuroinflammation was induced by the intraperitoneal administration of LPS (1 mg/kg) to male Wistar rats and was corroborated by IL-6, GFAP, and Iba-1 protein levels in the cortex over time. CYP2J3 and CYP2C11 protein levels were also evaluated in the cortex after 6, 12, 24, 48, and 72 h of LPS treatment. Our results show for the first time that neuroinflammation is able to downregulate CYP2J3 and CYP2C11 protein expression in the brain cortex.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Regulação para Baixo/fisiologia , Mediadores da Inflamação/metabolismo , Esteroide 16-alfa-Hidroxilase/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Encéfalo/efeitos dos fármacos , Família 2 do Citocromo P450/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Masculino , Ratos , Ratos Wistar , Esteroide 16-alfa-Hidroxilase/antagonistas & inibidoresRESUMO
We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.
Assuntos
Família 2 do Citocromo P450/metabolismo , Microbioma Gastrointestinal , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/prevenção & controle , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Proteínas de Soja/farmacologia , Animais , Carcinogênese/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Elucidating the mechanisms for circadian expression of drug-metabolizing enzymes is essential for a better understanding of dosing time-dependent drug metabolism and pharmacokinetics. CYP2B6 (Cyp2b10 in mice) is an important enzyme responsible for metabolism and detoxification of approximately 10% of drugs. Here, we aimed to investigate a potential role of nuclear receptor co-repressor RIP140 in circadian regulation of Cyp2b10 in mice.We first uncovered diurnal rhythmicity in hepatic RIP140 mRNA and protein with peak values at ZT10 (ZT, zeitgeber time). RIP140 ablation up-regulated Cyp2b10 expression and blunted its rhythm in mice and in AML-12 cells. Consistent with a negative regulatory effect, overexpression of RIP140 inhibited Cyp2b10 promoter activity and reduced cellular Cyp2b10 expression.Furthermore, RIP140 suppressed Car- and Pxr-mediated transactivation of Cyp2b10, and the suppressive effects were attenuated when the RIP140 gene was silenced. Chromatin immunoprecipitation assays revealed that recruitment of RIP140 protein to the Cyp2b10 promoter was circadian time-dependent in wild-type mice. More extensive recruitment was observed at ZT10 than at ZT2 consistent with the rhythmic pattern of RIP140 protein. However, the time-dependency of RIP140 recruitment was lost in RIP140-/- mice.Additionally, we identified a D-box and a RORE cis-element in RIP140 promoter. D-box- and RORE-acting clock components such as Dbp, E4bp4, Rev-erbα/ß and Rorα transcriptionally regulated RIP140, potentially accounting for its rhythmic expression.In conclusion, RIP140 regulates diurnal expression of Cyp2b10 in mouse liver through periodical repression of Car- and Pxr-mediated transactivation. This co-regulator-driven mechanism represents a novel source of diurnal rhythmicity in drug-metabolizing enzymes.
Assuntos
Família 2 do Citocromo P450/metabolismo , Inativação Metabólica/fisiologia , Correpressor 1 de Receptor Nuclear/genética , Animais , Ritmo Circadiano , Sistema Enzimático do Citocromo P-450 , Fígado/metabolismo , Camundongos , Ativação TranscricionalRESUMO
OBJECTIVES: N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA), a derivative of valproic acid (VPA), has been proposed as a potential anticancer agent due to its improved antiproliferative effects in some cancer cell lines. Although there is evidence that VPA is metabolized by cytochrome P450 2C11 rat isoform, HO-AAVPA CYP-mediated metabolism has not yet been fully explored. Therefore, in this work, the biotransformation of HO-AAVPA by CYP2C11 was investigated. METHODS: Kinetic parameters and spectral interaction between HO-AAVPA and CYP were evaluated using rat liver microsomes. The participation of CYP2C11 in metabolism of HO-AAVPA was confirmed by cimetidine (CIM) inhibition assay. Docking and molecular dynamics simulations coupled to MMGBSA methods were used in theoretical study. KEY FINDINGS: HO-AAVPA is metabolized by CYP enzymes (KM = 38.94 µm), yielding a hydroxylated metabolite according to its HPLC retention time (5.4 min) and MS analysis (252.2 m/z). In addition, CIM inhibition in rat liver microsomes (Ki = 59.23 µm) confirmed that CYP2C11 is mainly involved in HO-AAVPA metabolism. Furthermore, HO-AAVPA interacts with CYP2C11 as a type I ligand. HO-AAVPA is stabilized at the CYP2C11 ligand recognition site through a map of interactions similar to other typical CYP2C11 substrates. CONCLUSION: Therefore, rat liver CYP2C11 isoform is able to metabolize HO-AAVPA.
Assuntos
Amidas/farmacocinética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biotransformação , Família 2 do Citocromo P450/metabolismo , Microssomos Hepáticos , Pentanos/farmacocinética , Esteroide 16-alfa-Hidroxilase/metabolismo , Animais , Antineoplásicos/farmacocinética , Proliferação de Células/efeitos dos fármacos , Estabilidade de Medicamentos , Hidroxilação , Isoenzimas/metabolismo , Oxigenases de Função Mista/metabolismo , Simulação de Acoplamento Molecular , Ratos , Ácido Valproico/farmacologiaRESUMO
Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44-/- mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT2c44 mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMTΔ2c44 tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE2). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMTΔ2c44 mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE2 Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE2 The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.
Assuntos
Neoplasias da Mama/metabolismo , Família 2 do Citocromo P450/metabolismo , Linfangiogênese/fisiologia , Prostaglandinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Família 2 do Citocromo P450/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Ácidos Graxos Insaturados/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfangiogênese/genética , Macrófagos , Camundongos , Camundongos Knockout , Monócitos , Processos Neoplásicos , Proteômica , Transdução de Sinais , Receptores Toll-Like , Regulação para CimaRESUMO
The CYP2D subfamily catalyses the metabolism of about 25% of prescribed drugs, including the majority of antidepressants and antipsychotics. At present, the mechanism of hepatic CYP2D regulation remains largely unknown. This study investigated the role of sex steroid hormones in CYP2D regulation. For this purpose, Cyp2d22 expression was assessed in the distinct phases of the estrous cycle of normocyclic C57BL/6J (WT) female mice. Cyp2d22 was also evaluated in ovariectomised WT and CYP2D6-humanized (hCYP2D6) mice that received hormonal supplementation with either 17ß-estradiol (E2) and/or progesterone. Comparisons were also made to male mice. The data revealed that hepatic Cyp2d22 mRNA, protein and activity levels were higher at estrous compared to the other phases of the estrous cycle and that ovariectomy repressed Cyp2d22 expression in WT mice. Tamoxifen, an anti-estrogenic compound, also repressed hepatic Cyp2d22 via activation of GH/STAT5b and PI3k/AKT signaling pathways. Both hormones prevented the ovariectomy-mediated Cyp2d22 repression. In case of progesterone, this may be mediated by inhibition of the PI3k/AKT/FOX01 pathway. Notably, Cyp2d22 mRNA levels in WT males were similar to those in ovariectomised mice and were markedly lower compared to females at estrous, a differentiation potentially regulated by the GH/STAT5b pathway. Sex steroid hormone-related alterations in Cyp2d22 mRNA expression were highly correlated with Hnf1a mRNA. Interestingly, fluctuations in Cyp2d22 in hippocampus and cerebellum followed those in liver. In contrast to WT mice, ovariectomy induced hepatic CYP2D6 expression in hCYP2D6 mice, whereas E2 and/or progesterone prevented this induction. Apparently, sex steroid hormones display a significant gender- and species-specific role in the regulation of CYP2D.
Assuntos
Família 2 do Citocromo P450/metabolismo , Regulação da Expressão Gênica/fisiologia , Hormônios Esteroides Gonadais/fisiologia , Transdução de Sinais/genética , Animais , Citocromo P-450 CYP2D6/metabolismo , Estradiol/administração & dosagem , Ciclo Estral/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Progesterona/administração & dosagem , RNA Mensageiro/metabolismoRESUMO
Tuberculosis (TB) poses an important health challenge and a significant economic burden for Kazakhstan and in Central Asia. Recent findings show a number of immunological related processes and host Mycobacterium tuberculosis defense are impacted by a variety of genes of the human host including those that play a part in the vitamin D metabolism. We investigated the genetic variation of genes in the vitamin D metabolic pathway of a cohort 50 TB cases in Kazakhstan and compared them to 34 controls living in the same household with someone infected with TB. We specifically analyzed 11 SNPs belonging to the following genes: DHCR7, CYP2R1, GC-1, CYP24A1, CYP27A1, CYP27B1, VDR and TNFα. These genes play a number of different roles including synthesis, activation, delivery and binding of the activated vitamin D. Our preliminary results indicate significant association of VDR (vitamin D receptor) SNPs (rs1544410, BsmI, with OR = 0.425, CI 0.221-0.816, p = 0.009 and rs731236, TaqI with OR = 0.443, CI 0.228-0.859, p = 0.015) and CYP24A1 (rs6013897 with OR = 0.436, CI 0.191-0.996, p = 0.045) with TB. Interaction of genetic variation of VDR and CYP24A1 may impact susceptibility to TB. The findings provided initial clues to understand individual genetic differences in relation to susceptibility and protection to TB.