scholarly journals Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase

2019 ◽  
Author(s):  
Maria Natalia Rojas Velazquez ◽  
Shaheena Parween ◽  
Sameer S Udhane ◽  
Amit V Pandey
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Recombinant Proteins ◽  
Correct Diagnosis ◽  
Detailed Knowledge ◽  
Drug Metabolizing Enzymes ◽  
Metabolizing Enzymes ◽  
P450 Oxidoreductase ◽  
Cytochrome P450 Oxidoreductase

AbstractA broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

scholarly journals Sequencing of the Canine Cytochrome P450 CYP2C41 Gene and Genotyping of Its Polymorphic Occurrence in 36 Dog Breeds

2021 ◽  
Vol 8 ◽  
Author(s):  
Emre Karakus ◽  
Clarissa Prinzinger ◽  
Silke Leiting ◽  
Joachim Geyer
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Gene Deletion ◽  
Homologous Sequence ◽  
Pharmacokinetic Studies ◽  
Metabolizing Enzymes ◽  
Genomic Level ◽  
Very High

Cytochrome P450 (CYP) drug metabolizing enzymes play an important role in efficient drug metabolism and elimination. Many CYPs are polymorphic and, thereby, drug metabolism can vary between individuals. In the case of canine CYP2C41, gene polymorphism was identified. However, as the first available canine genome sequences all were CYP2C41 negative, this polymorphism could not be clarified at the genomic level. The present study provides an exact characterization of the CYP2C41 gene deletion polymorphism at the genomic level and presents a PCR-based genotyping method that was used for CYP2C41 genotyping of 1,089 individual subjects from 36 different dog breeds. None of the Bearded Collie, Bernese Mountain, Boxer, Briard, French Bulldog or Irish Wolfhound subjects had the CYP2C41 gene in their genomes. In contrast, in the Chinese Char-Pei, Siberian Husky, Schapendoes and Kangal breeds, the CYP2C41 allele frequency was very high, with values of 67, 57, 43, and 34%, respectively. Interestingly, the site of gene deletion was identical for all CYP2C41 negative dogs, and all CYP2C41 positive dogs showed highly homologous sequence domains upstream and downstream from the CYP2C41 gene. CYP2C41 genotyping can now be routinely used in future pharmacokinetic studies in canines, in order to identify genetically-based poor or extensive drug metabolizers. This, together with more extensive in vitro drug screening for CYP2C41 substrates will help to determine the clinical relevance of CYP2C41, and to optimize drug treatment. Although the relative abundance of the CYP2C41 protein in the canine liver seems to not be very high, this CYP could substantially contribute to hepatic drug metabolism in dogs expressing CYP2C41 from both alleles and, when CYP2C41 shows higher catalytic activity to a given drug than other hepatic metabolic enzymes.

scholarly journals Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells

Mutagenesis ◽  
2019 ◽  
Author(s):  
Lindsay Reed ◽  
Ian W H Jarvis ◽  
David H Phillips ◽  
Volker M Arlt
Keyword(s):  
Cytochrome P450 ◽  
Mouse Model ◽  
Metabolic Activation ◽  
Adduct Formation ◽  
Dna Adduct ◽  
Environmental Carcinogen ◽  
P450 Oxidoreductase ◽  
Cyp Enzymes ◽  
Cytochrome P450 Oxidoreductase

Abstract The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.

scholarly journals Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy

2005 ◽  
Vol 360 (1460) ◽  
pp. 1563-1570 ◽  
Author(s):  
Magnus Ingelman-Sundberg ◽  
Cristina Rodriguez-Antona
Keyword(s):  
Gene Expression ◽  
Cytochrome P450 ◽  
Drug Therapy ◽  
Adverse Effects ◽  
Drug Metabolism ◽  
Drug Metabolizing Enzymes ◽  
Drug Reactions ◽  
Metabolizing Enzymes ◽  
Drug Treatments ◽  
Polymorphic Enzymes

The majority of phase I- and phase II-dependent drug metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively decreased or enhanced drug metabolism. Several examples exist where subjects carrying certain alleles do not benefit from drug therapy due to ultrarapid metabolism caused by multiple genes or by induction of gene expression or, alternatively, suffer from adverse effects of the drug treatment due to the presence of defective alleles. It is likely that future predictive genotyping for such enzymes might benefit 15–25% of drug treatments, and thereby allow prevention of adverse drug reactions and causalities, and thus improve the health of a significant fraction of the patients. However, it will take time before this will be a reality within the clinic. We describe some important aspects in the field with emphasis on cytochrome P450 and discuss also polymorphic aspects of foetal expression of CYP3A5 and CYP3A7 .

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta

Parasitology ◽  
2012 ◽  
Vol 139 (6) ◽  
pp. 809-818 ◽  
Author(s):  
HANA BÁRTÍKOVÁ ◽  
IVAN VOKŘÁL ◽  
LENKA SKÁLOVÁ ◽  
VLADIMÍR KUBÍČEK ◽  
JANA FIRBASOVÁ ◽  
...  
Keyword(s):  
Drug Metabolism ◽  
Ex Vivo ◽  
Mass Spectrometric ◽  
Hymenolepis Diminuta ◽  
Drug Metabolizing Enzymes ◽  
Glutathione S Transferase ◽  
Methyl Derivative ◽  
Metabolizing Enzymes ◽  
Oxidative Metabolites

SUMMARYThe drug-metabolizing enzymes of some helminths can deactivate anthelmintics and therefore partially protect helminths against these drugs' toxic effect. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole) in the rat tapeworm Hymenolepis diminuta, a species often used as a model tapeworm. In vitro and ex vivo experiments were performed. Metabolites of the anthelmintics were detected and identified by HPLC with spectrofluorometric or mass–spectrometric detection. The enzymes of H. diminuta are able to reduce the carbonyl group of flubendazole, mebendazole and several other xenobiotics. Although the activity of a number of oxidation enzymes was determined, no oxidative metabolites of albendazole were detected. Regarding conjugation enzymes, a high activity of glutathione S-transferase was observed. A methyl derivative of reduced flubendazole was the only conjugation metabolite identified in ex vivo incubations of H. diminuta with anthelmintics. The results revealed that H. diminuta metabolized flubendazole and mebendazole, but not albendazole. The biotransformation pathways found in H. diminuta differ from those described in Moniezia expanza and suggest the interspecies differences in drug metabolism not only among classes of helminths, but even among tapeworms.

scholarly journals In Vitro Antioxidant Potential and Effect of a Glutathione-Enhancer Dietary Supplement on Selected Rat Liver Cytochrome P450 Enzyme Activity

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Benoit B. N’guessan ◽  
Seth K. Amponsah ◽  
George J. Dugbartey ◽  
Kwabena D. Awuah ◽  
Eunice Dotse ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Dietary Supplement ◽  
Rat Liver ◽  
Inhibitory Effect ◽  
Antioxidant Potential ◽  
Total Phenolic ◽  
Drug Metabolizing Enzymes ◽  
Metabolizing Enzymes ◽  
Cyp Enzymes

Background. There is considerable evidence that many people take dietary supplements including those of herbal origin as an alternative therapy to improve their health. One such supplement, with an amalgam of constituents, is CellGevity®. However, the effect of this dietary supplement on drug-metabolizing enzymes is poorly understood, as it has not been studied extensively. Therefore, we investigated the effect of CellGevity dietary supplement on selected rat liver microsomal cytochrome P450 (CYP) enzymes, the most common drug-metabolizing enzymes. We also determined the total antioxidant potential of this dietary supplement in vitro. Methods. To determine the antioxidant potential of CellGevity dietary supplement, 2,2-diphenyl-2-picryl-hydrazyl (DPPH), total phenolic, and flavonoid assays were used after initial preparation of a solution form of the supplement (low dose, LD; 4 mg/kg and high dose, HD; 8 mg/kg). Rats received oral administration of these doses of the supplement for 7 days, after which the effect of the supplement on selected liver CYP enzymes was assessed using probe substrates and spectroscopic and high-performance liquid chromatographic methods. Rats which received daily administration of 80 mg/kg of phenobarbitone and distilled water served as positive and negative controls, respectively. Results. The IC50 value of the supplement 0.34 ± 0.07 mg/ml compared to 0.076±0.03 mg/ml of the BHT (positive control). The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. The supplement significantly inhibited rat CYP2B1/2B2 (LDT 92.4%; HDT 100%), CYP3A4 (LDT 81.2%; HDT 71.7%), and CYP2C9 (LDT 21.7%; HDT 28.5%) while it had no significant inhibitory effect on CYPs 1A1/1A2, CYP1A2, and CYP2D6. Conclusion. CellGevity dietary supplement possesses moderate antioxidant activity in vitro and has an inhibitory effect on selected rat liver CYP enzymes, suggesting its potential interaction with drugs metabolized by CYP enzymes.

Polymorphisms in cytochrome P450 oxidoreductase and its effect on drug metabolism and efficacy

2017 ◽  
Vol 27 (9) ◽  
pp. 337-346 ◽  
Author(s):  
Liang Gong ◽  
Cong-Min Zhang ◽  
Jin-Feng Lv ◽  
Hong-Hao Zhou ◽  
Lan Fan
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
P450 Oxidoreductase ◽  
Cytochrome P450 Oxidoreductase

scholarly journals Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

2012 ◽  
Vol 40 (4) ◽  
pp. 754-760 ◽  
Author(s):  
Daniela Moutinho ◽  
Christopher C. Marohnic ◽  
Satya P. Panda ◽  
José Rueff ◽  
Bettie Sue Masters ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cyp3a4 Activity ◽  
In Vitro System ◽  
P450 Oxidoreductase ◽  
Nadph Cytochrome ◽  
Cytochrome P450 Oxidoreductase

scholarly journals Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Shoko Miyata ◽  
Noriaki Saku ◽  
Saeko Akiyama ◽  
Palaksha Kanive Javaregowda ◽  
Kenta Ite ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Liver Injury ◽  
High Expression ◽  
Drug Metabolizing Enzymes ◽  
Drug Induced ◽  
Metabolizing Enzymes ◽  
P450 Gene ◽  
Drug Induced Liver Injury ◽  
Cyp3a4 Gene

Abstract Background Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). Methods To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. Results The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. Conclusions iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

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