ABSTRACT
Cytochrome P450 2D (CYP2D) is important in psychopharmacology as it is engaged in the metabolism of drugs, neurosteroids and neurotransmitters. An unbalanced maternal diet during pregnancy and lactation can cause neurodevelopmental abnormalities and increases the offspring's predisposition to neuropsychiatric diseases. The aim of the present study was to evaluate the effect of maternal modified types of diet: a high-fat diet (HFD) and high-carbohydrate diet (HCD) during pregnancy and lactation on CYP2D in the liver and brain of male offspring at 28 (adolescent) or 63 postnatal days (young adult). The CYP2D activity and protein level were measured in the liver microsomes and the levels of mRNAs of CYP2D1, 2D2 and 2D4 were investigated both in the liver and brain. In the liver, both HFD and HCD increased the mRNA levels of all the three investigated CYP2D genes in adolescents, but an opposite effect was observed in young adults. The CYP2D protein level increased in adolescents but not in young adults. In contrast, young adults showed significantly decreased CYP2D activity. Similar effect of HFD on the CYP2D mRNAs was observed in the prefrontal cortex, while the effect of HCD was largely different than in the liver (the CYP2D2 expression was not affected, the CYP2D4 expression was decreased in young adults). In conclusion, modified maternal diets influence the expression of individual CYP2D1, CYP2D2 and CYP2D4 genes in the liver and brain of male offspring, which may affect the metabolism of CYP2D endogenous substrates and drugs and alter susceptibility to brain diseases and pharmacotherapy outcome.
Subject(s)
Brain , Diet, High-Fat , Lactation , Liver , Prenatal Exposure Delayed Effects , Animals , Pregnancy , Female , Diet, High-Fat/adverse effects , Liver/metabolism , Brain/metabolism , Rats , Male , Prenatal Exposure Delayed Effects/metabolism , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , Microsomes, Liver/metabolism , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/genetics , Rats, WistarABSTRACT
The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P450 Family 2 , Flavin Mononucleotide , NADPH-Ferrihemoprotein Reductase , NADPH-Ferrihemoprotein Reductase/metabolism , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/genetics , Flavin Mononucleotide/metabolism , Flavin Mononucleotide/chemistry , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/chemistry , Aryl Hydrocarbon Hydroxylases/chemistry , Aryl Hydrocarbon Hydroxylases/metabolism , Aryl Hydrocarbon Hydroxylases/genetics , Humans , Mutagenesis, Site-Directed , Protein Domains , Kinetics , AnimalsABSTRACT
Objectives: To investigate the interaction between tramadol and representative tyrosine kinase inhibitors, and to study the inhibition mode of drug-interaction. Methods: Liver microsomal catalyzing assay was developed. Sprague-Dawley rats were administrated tramadol with or without selected tyrosine kinase inhibitors. Samples were prepared and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for analysis. Besides, liver, kidney, and small intestine were collected and morphology was examined by hematoxyline-eosin (H&E) staining. Meanwhile, liver microsomes were prepared and carbon monoxide differential ultraviolet radiation (UV) spectrophotometric quantification was performed. Results: Among the screened inhibitors, crizotinib takes the highest potency in suppressing the metabolism of tramadol in rat/human liver microsome, following non-competitive inhibitory mechanism. In vivo, when crizotinib was co-administered, the AUC value of tramadol increased compared with the control group. Besides, no obvious pathological changes were observed, including cell morphology, size, arrangement, nuclear morphology with the levels of alanine transaminase (ALT) and aspartate transaminase (AST) increased after multiple administration of crizotinib. Meanwhile, the activities of CYP2D1 and CYP3A2 as well as the total cytochrome P450 abundance were found to be decreased in rat liver of combinational group. Conclusions: Crizotinib can inhibit the metabolism of tramadol. Therefore, this recipe should be vigilant to prevent adverse reactions.
Subject(s)
Crizotinib , Cytochrome P-450 CYP3A , Microsomes, Liver , Rats, Sprague-Dawley , Tramadol , Animals , Tramadol/pharmacology , Crizotinib/pharmacology , Rats , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Cytochrome P-450 CYP3A/metabolism , Male , Drug Interactions , Humans , Tandem Mass Spectrometry , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacokinetics , Analgesics, Opioid/pharmacologyABSTRACT
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.
Subject(s)
Cytochrome P-450 CYP3A , Hepatomegaly , Liver Regeneration , Liver , Pregnane X Receptor , Pregnenolone Carbonitrile , Animals , Pregnane X Receptor/metabolism , Pregnane X Receptor/genetics , Liver Regeneration/drug effects , Male , Cytochrome P-450 CYP3A/metabolism , Pregnenolone Carbonitrile/pharmacology , Liver/metabolism , Liver/enzymology , Liver/drug effects , Rats , Hepatomegaly/metabolism , Hepatomegaly/pathology , Aryl Hydrocarbon Hydroxylases/metabolism , Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/genetics , Rats, Sprague-Dawley , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1A2/genetics , Steroid 16-alpha-Hydroxylase/metabolism , Steroid 16-alpha-Hydroxylase/genetics , Steroid 12-alpha-Hydroxylase/metabolism , Steroid 12-alpha-Hydroxylase/genetics , HepatectomyABSTRACT
Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET)-producing enzyme, promotes insulin sensitivity, and Cyp2c44-/- mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SCD)-fed male Cyp2c44-/- mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared with wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol (DAG) levels. Cyp2c44-/- mice had impaired glucose tolerance and increased hepatic plasma membrane-associated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SCD and high-fat diet (HFD)-fed Cyp2c44-/- mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SCD-fed Cyp2c44-/- mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44-/- hepatocytes. Finally, Cyp2c44-/- HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, DAG accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.
Subject(s)
Cytochrome P450 Family 2 , Diglycerides , Insulin , Lipid Metabolism , Liver , Mice, Knockout , Protein Kinase C-delta , Signal Transduction , Animals , Male , Mice , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/genetics , Diet, High-Fat , Diglycerides/metabolism , Epoxide Hydrolases , Insulin/metabolism , Insulin Resistance/physiology , Lipid Metabolism/physiology , Liver/metabolism , Mice, Inbred C57BL , Protein Kinase C-delta/metabolism , Protein Kinase C-delta/genetics , Signal Transduction/physiologyABSTRACT
Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3-step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C-C bond scission (removing the 19-oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2 -, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen-18 labeling, i.e., from 18O2 and H2 18O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19-oxo-androstenedione by human P450 19A1 and of a model secosteroid, 3-oxodecaline-4-ene-10-carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non-enzymatic acid-catalyzed deformylation, yielding 19-norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e., Fe3+O2 -) in both P450 19A1 oxidation of 19-oxo-androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016-16025), attributed to several technical modifications.
Subject(s)
Aromatase , Oxidation-Reduction , Aromatase/metabolism , Aromatase/chemistry , Humans , Peroxides/chemistry , Peroxides/metabolism , Animals , Anions/chemistry , Anions/metabolism , Ferric Compounds/chemistry , Ferric Compounds/metabolism , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/chemistry , Rabbits , Steroids/chemistry , Steroids/metabolism , Androstenedione/chemistry , Androstenedione/metabolismABSTRACT
OBJECTIVES: Compromised hepatic fatty acid oxidation (FAO) has been observed in human MASH patients and animal models of MASLD/MASH. It remains poorly understood how and when the hepatic FAO pathway is suppressed during the progression of MASLD towards MASH. Hepatic ChREBP⺠is a classical lipogenic transcription factor that responds to the intake of dietary sugars. METHODS: We examined its role in regulating hepatocyte fatty acid oxidation (FAO) and the impact of hepatic Chrebpa deficiency on sensitivity to diet-induced MASLD/MASH in mice. RESULTS: We discovered that hepatocyte ChREBP⺠is both necessary and sufficient to maintain FAO in a cell-autonomous manner independently of its DNA-binding activity. Supplementation of synthetic PPARâº/δ agonist is sufficient to restore FAO in Chrebp-/- primary mouse hepatocytes. Hepatic ChREBP⺠was decreased in mouse models of diet-induced MAFSLD/MASH and in patients with MASH. Hepatocyte-specific Chrebp⺠knockout impaired FAO, aggravated liver steatosis and inflammation, leading to early-onset fibrosis in response to diet-induced MASH. Conversely, liver overexpression of ChREBPâº-WT or its non-lipogenic mutant enhanced FAO, reduced lipid deposition, and alleviated liver injury, inflammation, and fibrosis. RNA-seq analysis identified the CYP450 epoxygenase (CYP2C50) pathway of arachidonic acid metabolism as a novel target of ChREBPâº. Over-expression of CYP2C50 partially restores hepatic FAO in primary hepatocytes with Chrebp⺠deficiency and attenuates preexisting MASH in the livers of hepatocyte-specific Chrebpâº-deleted mice. CONCLUSIONS: Our findings support the protective role of hepatocyte ChREBPa against diet-induced MASLD/MASH in mouse models in part via promoting CYP2C50-driven FAO.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Fatty Acids , Hepatocytes , Liver , Mice, Inbred C57BL , Oxidation-Reduction , Animals , Female , Humans , Male , Mice , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cytochrome P-450 Enzyme System , Cytochrome P450 Family 2/metabolism , Cytochrome P450 Family 2/genetics , Diet/adverse effects , Disease Models, Animal , Fatty Acids/metabolism , Hepatocytes/metabolism , Lipid Metabolism , Liver/metabolism , Mice, KnockoutABSTRACT
Obesity is strongly associated with disturbances of vitamin D (VD) metabolites in the animal models. However, the related epidemiological evidence is still controversial, especially the different degrees of obesity children. Hence, in this present representative case-control study, 106 obesity school-age children aged 7-12 years were included and divided into different subgroups as degree I (the age- and sex-specific BMI≥95th percentile, n=45), II (BMI ≥120% percentile, n=34) and III (BMI ≥140% percentile, n=27) obesity groups across the ranges of body mass index (BMI). While the age- and sex-matched subjects without obesity were as the control group. Notably, it was significantly different of body composition, anthropological and clinical characteristics among the above four subgroups with the dose-response relationships (P<.05). Moreover, comparing with the control group, the serum VD concentrations were higher, VD metabolites like 25(OH)D, 25(OH)D3 and 1,25(OH)2D, and related hydroxylases as CYP27A1, CYP2R1 and CYP27B1 were lower in the degree I, II, and III obesity subgroups (P<.05), which were more disorder with the anthropological and clinical characteristics as the obesity was worsen in a BMI-independent manner (P<.05). However, there was a significant increase of CYP27B1 in the degree III obesity group than those in the degree I and II obesity subgroups. Furthermore, the methylation patterns on the genome-wide (Methylation/Hydroxymethylation) and VD metabolism genes (CYP27A1, CYP2R1 and CYP27B1) were negatively correlated with the worse obesity and their related expressions (P<.05). In summary, these results indicated that obesity could affect the homeostasis of VD metabolism related genes such as CYP27A1, CYP2R1, CYP27B1 and etc through abnormal DNA methylation, resulting in the disorders of VD related metabolites to decrease VD bio-availability with the BMI-independent manner. In turn, the lower levels of VD metabolites would affect the liver function to exacerbate the progression of obesity, as the Degree II and III obesity subgroups.
Subject(s)
Body Mass Index , DNA Methylation , Pediatric Obesity , Vitamin D , Humans , Child , Male , Female , Vitamin D/blood , Vitamin D/analogs & derivatives , Case-Control Studies , Pediatric Obesity/genetics , Pediatric Obesity/metabolism , Pediatric Obesity/complications , Cholestanetriol 26-Monooxygenase/genetics , Cholestanetriol 26-Monooxygenase/metabolism , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Vitamin D Deficiency/genetics , Obesity/genetics , Obesity/metabolism , Metabolic Diseases/geneticsABSTRACT
OBJECTIVE: Application of Tandem Mass Tags (TMT)-based LC-MS/MS analysis to screen for differentially expressed proteins (DEPs) in traumatic axonal injury (TAI) of the brainstem and to predict potential biomarkers and key molecular mechanisms of brainstem TAI. METHODS: A modified impact acceleration injury model was used to establish a brainstem TAI model in Sprague-Dawley rats, and the model was evaluated in terms of both functional changes (vital sign measurements) andstructural changes (HE staining, silver-plating staining and ß-APP immunohistochemical staining). TMT combined with LC-MS/MS was used to analyse the DEPs in brainstem tissues from TAI and Sham groups. The biological functions of DEPs and potential molecular mechanisms in the hyperacute phase of TAI were analysed by bioinformatics techniques, and candidate biomarkers were validated using western blotting and immunohistochemistry on brainstem tissues from animal models and humans. RESULTS: Based on the successful establishment of the brainstem TAI model in rats, TMT-based proteomics identified 65 DEPs, and bioinformatics analysis indicated that the hyperacute phase of TAI involves multiple stages of biological processes including inflammation, oxidative stress, energy metabolism, neuronal excitotoxicity and apoptosis. Three DEPs, CBR1, EPHX2 and CYP2U1, were selected as candidate biomarkers and all three proteins were found to be significantly expressed in brainstem tissue 30 min-7 days after TAI in both animal models and humans. CONCLUSION: Using TMT combined with LC-MS/MS analysis for proteomic study of early TAI in rat brainstem, we report for the first time that CBR1, EPHX2 and CYP2U1 can be used as biomarkers of early TAI in brainstem by means of western blotting and immunohistochemical staining, compensating for the limitations of silver-plating staining and ß-APP immunohistochemical staining, especially in the case of very short survival time after TAI (shorter than 30 min). A number of other proteins that also have a potential marker role are also presented, providing new insights into the molecular mechanisms, therapeutic targets and forensic identification of early TAI in brainstem.
Subject(s)
Proteomics , Tandem Mass Spectrometry , Humans , Rats , Animals , Rats, Sprague-Dawley , Chromatography, Liquid , Proteomics/methods , Brain Stem/metabolism , Biomarkers/metabolism , Cytochrome P450 Family 2/metabolismABSTRACT
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.
Subject(s)
Circadian Rhythm , Cryptochromes , Transcription Factors , Animals , Mice , Circadian Rhythm/genetics , Liver , Luciferases , RNA, Messenger , Transcription Factors/genetics , Transcription Factors/metabolism , Cryptochromes/genetics , Cryptochromes/metabolism , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolismABSTRACT
Background & objectives: Despite being a tropical country, vitamin D deficiency is highly prevalent in India with studies indicating 40-99 per cent prevalence. Apart from calcium and phosphate metabolism, vitamin D is involved in cell cycle regulation, cardiovascular, hepatoprotection. The metabolism of vitamin D is regulated by vitamin D tool genes (CYP2R1/CYP27B1/CYP24A1/VDR). The promoter regions of some of these genes have CpG islands, making them prone to methylation induced gene silencing, which may cause a reduction in circulating vitamin D levels. Epigenetic basis of vitamin D deficiency is yet to be studied in India, and hence, this pilot study was aimed to analyze whether methylation levels of CYP2R1 gene were correlated with the levels of 25(OH)D in healthy, adult individuals in Indian population. Methods: In this cross-sectional study, healthy adults of 18-45 yr of age with no history of malabsorption, thyroidectomy, chronic illness or therapeutic vitamin D supplementation were recruited. DNA methylation analysis was carried out by methylation specific quantitative PCR. Serum calcium, phosphate and vitamin D levels were also quantified. Statistical analysis was done by R 4.0.5 software. Results: A total of 61 apparently healthy adults were analyzed. The serum vitamin D levels did not correlate with CYP2R1 methylation levels in our study population. Significant positive correlation was observed between age and serum vitamin D levels. Significant association of gender was found with CYP2R1 methylation levels. Interpretation & conclusions: This study found no significant correlation between levels of CYP2R1 methylation and circulating 25(OH)D deficiency. Further studies on the Indian population having a larger sample size including entire vitamin D tool genes, among different ethnic groups may be conducted to elucidate molecular etiology of circulating 25(OH)D deficiency. The high prevalence of normal serum calcium and phosphate levels among vitamin D deficient subjects in this study coupled with the strikingly high prevalence of the deficiency at the national level, may suggest the need to revise the cut-off criteria for vitamin D deficiency in the Indian population.
Subject(s)
Cholestanetriol 26-Monooxygenase , Cytochrome P450 Family 2 , Vitamin D Deficiency , Vitamin D , Adult , Humans , Calcium/metabolism , Cholestanetriol 26-Monooxygenase/genetics , Cholestanetriol 26-Monooxygenase/metabolism , Cross-Sectional Studies , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , Methylation , Pilot Projects , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Vitamin D/blood , Vitamin D Deficiency/epidemiology , Vitamin D Deficiency/genetics , Vitamin D Deficiency/metabolism , VitaminsABSTRACT
The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochromes b5 , Cytochromes b5/chemistry , Cytochromes b5/metabolism , Aryl Hydrocarbon Hydroxylases/chemistry , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P450 Family 2/metabolism , Oxidation-ReductionABSTRACT
Coptisine (COP) is the main active ingredient of Coptis chinensis. In Chinese veterinary clinics, Coptis chinensis is commonly used alongside florfenicol to treat intestinal infections. The goal of this study was to investigate the impact of COP co-administration on the pharmacokinetics of florfenicol in rats.Male Sprague-Dawley rats were orally administered COP (50 mg/kg BW) or sterile water for 7 consecutive days, followed by a single oral dose of florfenicol (25 mg/kg BW) on the 8th day. Pharmacokinetics of florfenicol were analysed using non-compartmental methods, while expression levels of cytochrome P450 (CYP) isoforms in the liver and P-glycoprotein (P-gp) in the jejunum were measured using real-time RT-PCR, Western blot and immunohistochemical analyses.Co-administration of COP and florfenicol significantly increased AUC(0-∞), MRT(0-∞), and Cmax of florfenicol, while CLz/F was significantly decreased. COP down-regulated the expression of CYP1A2, CYP2C11, and CYP3A1 in the liver, as well as P-gp in the jejunum.These findings suggest that co-administration of COP with florfenicol alters the pharmacokinetics of florfenicol in rats. The down-regulation of CYP and P-gp expression may contribute to this effect. Therefore, the co-administration of COP with florfenicol may enhance the prophylactic or therapeutic efficacy of florfenicol in veterinary practice.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP1A2 , Rats , Male , Animals , Cytochrome P-450 CYP1A2/metabolism , Pilot Projects , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Jejunum/metabolism , Rats, Sprague-Dawley , Cytochrome P-450 Enzyme System/metabolism , Liver/metabolism , Cytochrome P-450 CYP3A/metabolism , Cytochrome P450 Family 2/metabolism , Aryl Hydrocarbon Hydroxylases/metabolism , Steroid 16-alpha-Hydroxylase/metabolismABSTRACT
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.
Subject(s)
Interleukin-6 , Sepsis , Humans , Mice , Animals , Interleukin-6/genetics , Interleukin-6/metabolism , Down-Regulation , Tumor Necrosis Factor-alpha/metabolism , Punctures , Ligation , Gene Expression , Sepsis/metabolism , Cecum/metabolism , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , Membrane Proteins/metabolism , Cytochrome P-450 CYP3A/geneticsABSTRACT
Systemic exposure of metronidazole is increased in patients with inflammatory bowel diseases (IBDs), while the underlying mechanism remains unknown. Here, we aim to decipher the mechanisms by which experimental colitis regulates metronidazole disposition in mice. We first confirmed that the systemic exposure of metronidazole was elevated in dextran sulfate sodium (DSS)-induced experimental colitis. Hepatic microsomal incubation with metronidazole revealed that the production rate of 2-hydroxymetronidazole was inhibited, suggestive of a diminished hydroxylation reaction upon colitis. Remarkably, the hydroxylation reaction of metronidazole was selectively catalyzed by CYP2A5, which was downregulated in the liver of colitis mice. In addition, hepatic nuclear factor (NF)-κB (a prototypical and critical signaling pathway in inflammation) was activated in colitis mice. Luciferase reporter and chromatin immunoprecipitation assay indicated that NF-κB downregulated Cyp2a5 transcription through binding to an NF-κB binding site (-1711 to -1720 bp) in the promoter. We further verified that the regulatory effects of colitis on CYP2A5 depended on the disease itself rather than the DSS compound. First, one-day administration of DSS did not alter mRNA and protein levels of CYP2A5. Moreover, CYP2A5 was suppressed in the Il-10-/- spontaneously developing colitis model. Furthermore, Cyp2a5 expression was downregulated in both groups of mice with modest or severe colitis, whereas the expression change was much more significant in severe colitis as compared to modest colitis. Altogether, activated hepatic NF-κB in experimental colitis regulates CYP2A5 and metronidazole disposition, revealing the mechanism of pharmacokinetic instability under IBDs, and providing a theoretical foundation for rational drug use in the future.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Colitis , Animals , Mice , NF-kappa B/metabolism , Metronidazole/pharmacology , Colitis/chemically induced , Colitis/drug therapy , Colitis/metabolism , Signal Transduction , Liver/metabolism , Mice, Inbred C57BL , Disease Models, Animal , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , Aryl Hydrocarbon Hydroxylases/adverse effects , Aryl Hydrocarbon Hydroxylases/metabolismABSTRACT
Cytochrome P450 2U1 (CYP2U1) identified from the human genome remains poorly known since few data are presently available on its physiological function(s) and substrate(s) specificity. CYP2U1 mutations are associated with complicated forms of hereditary spastic paraplegia, alterations of mitochondrial architecture and bioenergetics. In order to better know the biological roles of CYP2U1, we used a bioinformatics approach. The analysis of the data invited us to focus on leukotriene B4 (LTB4), an important inflammatory mediator. Here, we show that CYP2U1 efficiently catalyzes the hydroxylation of LTB4 predominantly on its ω-position. We also report docking experiments of LTB4 in a 3D model of truncated CYP2U1 that are in agreement with this hydroxylation regioselectivity. The involvement of CYP2U1 in the metabolism of LTB4 could have strong physiological consequences in cerebral pathologies including ischemic stroke because CYP2U1 is predominantly expressed in the brain.
Subject(s)
Cytochrome P-450 Enzyme System , Leukotriene B4 , Humans , Leukotriene B4/metabolism , Hydroxylation , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Fatty Acids , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolismABSTRACT
Chiral polychlorinated biphenyls (PCB) are environmentally relevant developmental neurotoxicants. Because their hydroxylated metabolites (OH-PCBs) are also neurotoxic, it is necessary to determine how PCB metabolism affects the developing brain, for example, in mouse models. Because the cytochrome P450 isoforms involved in the metabolism of chiral PCBs remain unexplored, we investigated the metabolism of PCB 91 (2,2',3,4',6-pentachlorobiphenyl), PCB 95 (2,2',3,5',6-pentachlorobiphenyl), PCB 132 (2,2',3,3',4,6'-hexachlorobiphenyl), and PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) using liver microsomes from male and female Cyp2a(4/5)bgs-null, Cyp2f2-null, and wild-type mice. Microsomes, pooled by sex, were incubated with 50 µM PCB for 30 min, and the levels and enantiomeric fractions of the OH-PCBs were determined gas chromatographically. All four PCB congeners appear to be atropselectively metabolized by CYP2A(4/5)BGS and CYP2F2 enzymes in a congener- and sex-dependent manner. The OH-PCB metabolite profiles of PCB 91 and PCB 132, PCB congeners with one para-chlorine substituent, differed between null and wild-type mice. No differences in the metabolite profiles were observed for PCB 95 and PCB 136, PCB congeners without a para-chlorine group. These findings suggest that Cyp2a(4/5)bgs-null and Cyp2f2-null mice can be used to study how a loss of a specific metabolic function (e.g., deletion of Cyp2a(4/5)bgs or Cyp2f2) affects the toxicity of chiral PCB congeners.
Subject(s)
Polychlorinated Biphenyls , Male , Female , Mice , Animals , Polychlorinated Biphenyls/metabolism , Microsomes, Liver/metabolism , Cytochrome P450 Family 2/metabolism , Mice, Transgenic , Chlorine/metabolism , Hydroxylation , Mice, KnockoutABSTRACT
BACKGROUND: The intake of Gynura segetum, a traditional Chinese medicine, may be induce hepatic sinusoidal obstruction syndrome (HSOS). It has a high mortality rate based on the severity of the disease and the absence of therapeutic effectiveness. Therefore, the current study was designed to investigate the effects of bicyclol on HSOS induced by Gynura segetum and the potential molecular mechanisms. METHODS: Gynura segetum (30 g/kg) was administered for 4 weeks in the model group, while the bicyclol pretreatment group received bicyclol (200 mg/kg) administration. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol (CHO), triglyceride (TG), and liver histological assays were detected to assess HSOS. The gene expressions of cytochrome P450 (CYP450) isozymes were quantified by real-time PCR. Moreover, hepatocellular apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, then apoptosis and autophagy-related markers were determined using Western blot. RESULTS: As a result, bicyclol pretreatment is notably protected against Gynura segetum-induced HSOS, as observed by reducing serum ALT levels, inhibiting the reduction in CHO and TG levels, and alleviating the histopathological changes. Bicyclol pretreatment inhibited the changes in mRNA levels of CYP450 isozymes (including the increase in CYP2a5 and decrease in CYP2b10, 2c29, 2c37, 3a11, and 7b1). In addition, the upregulation of Bcl-2 and the downregulation of LC3-II/LC3-I proteins expression in HSOS were inhibited with bicyclol pretreatment. CONCLUSION: Bicyclol exerted a protective effect against HSOS induced by Gynura segetum, which could be attributed to the regulated expressions of CYP450 isozymes and alleviated the downregulation of autophagy.
Subject(s)
Biphenyl Compounds , Hepatic Veno-Occlusive Disease , Humans , Cholesterol , Hepatic Veno-Occlusive Disease/chemically induced , Hepatic Veno-Occlusive Disease/drug therapy , Hepatic Veno-Occlusive Disease/metabolism , Isoenzymes/metabolism , Liver/metabolism , Biphenyl Compounds/therapeutic use , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , Drugs, Chinese Herbal/adverse effectsABSTRACT
To evaluate the effect of imrecoxib on CYP2C11 enzyme activity, mRNA, and protein expression, a UPLC method was established. Tolbutamide was selected as the CYP2C11 enzyme-specific probe drug and incubated with imrecoxib in rat liver microsomes. The yield of 4-hydroxytolbutamide was measured using UPLC to investigate the effect of imrecoxib on CYP2C11 enzyme activity. Imrecoxib (10 mg/kg) was administered intragastrically twice daily. After 1, 7, and 14 days of administration, the liver tissues were analyzed. The expression of CYP2C11 enzyme mRNA was determined using reverse transcription-polymerase chain reaction, and its protein expression was determined using Western blot analysis. Imrecoxib concentration was inversely proportional to the production of 4-hydroxytolbutamide in liver microsomes. Imrecoxib demonstrated a dose-dependent inhibitory effect on CYP2C11 activity with IC50 = 74.77 µM. After administration, reverse transcription-polymerase chain reaction showed CYP2C11 enzyme mRNA expressions were 65% (P < 0.05), 35%, and 34% of the control group, respectively (P < 0.01). Western blot analysis showed CYP2C11 enzyme protein expressions were 80, 37, and 34% of the control group, respectively (P < 0.01). Imrecoxib can reduce mRNA and protein expression of CYP2C11 enzyme in rat liver and inhibit the activity of CYP2C11 enzyme in a dose-dependent manner. However, it does not produce clinically significant drug interactions.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Pyrroles , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , Microsomes, Liver/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Messenger/pharmacology , Rats , Steroid 16-alpha-Hydroxylase/genetics , Steroid 16-alpha-Hydroxylase/metabolism , SulfidesABSTRACT
Copper is a trace element that necessary for plant growth in the soil. However, in recent years, due to human activities, the content of copper in soil exceeds the standard seriously, which is threatening the safety of soil animals, plants and even human beings. In this study, we investigated the effects and molecular mechanisms of 60 days long-term copper exposure on earthworms (Eisenia fetida) at 67.58 mg/kg, 168.96 mg/kg and 337.92 mg/kg concentration by using transcriptome and metabolomics. Transcriptome analysis showed that the expression of energy metabolism related genes (LDH, GYS, ATP6N, GAPDH, COX17), immune system related genes (E3.2.1.14) and detoxification related genes (UGT, CYP2U1, CYP1A1) were down-regulated, the expression of antioxidant system related genes (GCLC, HPGDS) were up-regulated in copper exposure experiment of earthworms. Similarly, metabolomics analysis revealed that the expression of energy metabolism related metabolites (Glucose-1-phosphate, Glucose-6-phosphate), TCA cycle related metabolites (fumaric acid, allantoic acid, malate, malic acid) were down-regulated, digestion and immune system related metabolites (Trehalose-6-phosphate) were up-regulated. Integrating transcriptome and metabolomics data, it was found that higher antioxidant capacity and accelerated TCA cycle metabolism may be an adaptive strategy for earthworms to adapt to long-term copper stress. Collectively, the results of this study will greatly contribute to incrementally understand the stress responses on copper exposure to earthworms and supply molecular level support for evaluating the environmental effects of copper on soil organisms.