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Algae are a large group of photosynthetic organisms responsible for approximately half of the earth's total photosynthesis. In addition to their fundamental ecological roles as oxygen producers and as the food base for almost all aquatic life, algae are also a rich source of bioactive natural products, including several clinical drugs. Cytochrome P450 enzymes (P450s) are a superfamily of biocatalysts that are extensively involved in natural product biosynthesis by mediating various types of reactions. In the post-genome era, a growing number of P450 genes have been discovered from algae, indicating their important roles in algal life-cycle. However, the functional studies of algal P450s remain limited. Benefitting from the recent technical advances in algae cultivation and genetic manipulation, the researches on P450s in algal natural product biosynthesis have been approaching to a new stage. Moreover, some photoautotrophic algae have been developed into "photo-bioreactors" for heterologous P450s to produce high-value added pharmaceuticals and chemicals in a carbon-neutral or carbon-negative manner. Here, we comprehensively review these advances of P450 studies in algae from 2000 to 2021.
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OBJECTIVE To inv estigate the in vitro inhibitory effects of acteoside on cytochrome P 450（CYP）enzymes in liver microsomes of rats. METHODS Using probe substrates method ，acteoside（0.1，0.3，1，3，10，30 μmol/L）was incubated with probe substrates phenacetin ，mephentoin，diclofenac，coumarin，dextromethorphan and testosterone （substrates of CYP 1A2， CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）in liver microsomes of rats. Another blank control group and positive inhibitor group [ α-naphthoflavone，ticlopidine，sulfabendazole，pilocarpine，quinidine and ketoconazole （inhibitors of CYP 1A2，CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）] were set up. Using indapamide as the internal standard ， the contents of corresponding metabolites （acetaminophen， 4-hydroxymephenytoin， 4-hydroxydiclofenac，7-hydroxycoumarin，dextran，6 β-hydroxytestosterone） were detected by ultra high performance liquid chromatography-tandem mass spectrometry . The IC 50 values were calculated by GraphPad v 8.0 software. By computer molecular docking technology ，acteoside and positive inhibitors were molecularly docked with the CYP enzyme ，and the binding mode and strength of the two molecules were analyzed. RESULTS The IC 50 values of acteoside to CYP 1A2 and CYP 2A6 enzymes were more than 30 μmol/L，and those of acteoside to CYP 2D6，CYP2C19，CYP2C9 and CYP 3A4 enzymes were 24.87，21.52，12.56 and 7.55 μmol/L，respectively. The hydrogen bond and hydrophobic force could form between acteoside and CYP 3A4 enzyme，and the hydrogen bond and electrostatic interaction could form between ketoconazole and CYP 3A4 enzyme. The binding free energy of acteoside and ketoconazole to CYP 3A4 enzyme were － 10.2 and － 12.4 kcal/mol （1 kcal/mol＝4.19 kJ），respectively. CONCLUSIONS Acteoside shows moderate inhibitory effect on CYP 3A4 enzyme in liver microsomes of rats ，and its affinity is equivalent to that of positive inhibitor ；the compound shows weak inhibitory effect on other 5 CYP enzymes.
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Cytochrome P450 enzymes participate in the biotransformation of various organic compounds in living organisms. Cytochrome P450 enzymes are present and active in various brain regions, which mediate the synthesis and metabolism of various endogenous substances in the brain, so the enzymes play an important role in maintaining homeostasis of the central nervous system. And cytochrome P450 enzymes in brain are widely involved in metabolism of central drugs such as tramadol, so the changes of their activities or expressions may affect the drug's efficacy in brain. In addition, in view of the important roles of cytochrome P450 enzymes in the biotransformation of endogenous and exogenous substances in brain, the importance of their genetic polymorphism or activity changes in neuropsychiatric diseases is being gradually described. Therefore, in order to provide theoretical basis for the development of related targeted therapeutic drugs and explore whether cytochrome P450 enzymes in the brain can be used as therapeutic targets for central diseases, the development in the function of cytochrome P450 enzymes in brain and their recent advances in neuropsychiatric diseases by reviewing the relevant research in recent years are summarized.
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Forskolin is a complex labdane plant diterpenoid, which has been used in the treatment of a variety of diseases based on its activity as an activator of adenosine monophosphate(cAMP) cyclase. Natural forskolin exists only in the cork layer of the root of Coleus forskohlii. Due to the complexity of the extraction and chemical synthesis processes, the yield and purity of forskolin cannot meet commercial requirements. In recent years, with the rapid development of synthetic biology and the analysis and interpretation of many diterpene biosynthetic pathways, a new approach has been provided for the green production of forskolin. In this paper, the structure, activity, biosynthetic pathway and the heterologous biosynthesis of forskolin were reviewed. The problems and solutions in the heterologous biosynthesis of forskolin were also discussed and summarized, which will provide references for the construction of high-yielding forskolin engineering strains.
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Biosynthetic Pathways , ColforsinABSTRACT
OBJECTIVE To establish three-dimensional (3D) spherical tissue-like models for HepaRG cells and HepG2 cells and compare Ihe difference in morphology, functional protein expressions and drug hepatotoxicity tests to provide data for the selection of drug hepatotoxicity detection models in vitro. METHODS Two in vitro 3D hepatocyte spheroid models were constructed on HepaRG and HepG2 cells in logarithmic growth phase. The two types of cells were separately seeded in ultra-low attachment surface 96-well plates at a cell density of 100 cells per well, and cultured in a conventional manner without adding any inducer. The morphology of the spheroids on the 3∗ day (D3), D7, D14, D21 after seeding was observed under the microscope, and the average diameters of spheroids were caculated. Differential expressions of cytochrome P450 enzymes and albumin at mRNA and protein levels in the two models were studied with quantitative real-time PCR, immunofluorescence staining and Western blotting. Seven drugs (thiamine, fialuridine, acetaminophen, benzbromarone, cyclophosphamide, isoniazid and nefazodone) were selected for hepatotoxicity detection. After a single dose and repeated dose experiments, the cell inhibitory rate was measured and inhibitory concentration 50 (IC∗) was calculated. RESULTS The average diameters of the two models increased with time. The average diameters of HepaRG cell spheroids on D7, D14 and D21 after seeding were 317.5, 334.3 and 397.8 pm, respectively, which were smaller than those of HepG2 spheroids (P40, 0.87, >20 , 35.74 and 2.57 mmol • L"\ respectively. Under the single administration and partial drug repeated administration, the inhibitory effect on the suivival of 2 spheroid cells did not reach half of the inhibiory level. CONCLUSION HepaRG spheroids are superior to HepG2 spheroids in morphology control and functional protein expressions. They are more sensitive in drug hepatotoxicity detection in vitro. HepaRG spheroids are a better 3D spherical tissue-like model in vitro.
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We investigated the potential hepatoprotective effect of Radix Bupleuri (RB) by inducing acute liver injury (ALI) in an animal model using acetaminophen (APAP) after pretreatment with RB aqueous extract for three consecutive days. Compared to those of the APAP group, the biochemical and histological results of the RB pretreatment group showed lower serumaspartate transaminase (AST) and alanine transaminase (ALT) levels as well as less liver damage. Pharmacokinetic study of the toxicity related marker acetaminophen-cysteine (APC) revealed a lower exposure level in rats, suggesting that RB alleviated APAP-induced liver damage by preventing glutathione (GSH) depletion. The results of cocktail approach showed significant inhibition of CYP2E1 and CYP3A activity. Further investigation revealed the increasing of CYP2E1 and CYP3A protein was significantly inhibited in pretreatment group, while no obvious effect on gene expression was found. Therefore, this study clearly demonstrates that RB exhibited significant protective action against APAP-induced acute live injury via pretreatment, and which is partly through inhibiting the increase of activity and translation of cytochrome P450 enzymes, rather than gene transcription.
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OBJECTIVE:To study the inhibition effect of spinosin on 7 subtypes (CYP2B6,CYP2C8,CYP2C9,CYP2D6, CYP1A1,CYP2C19 and CYP3A4)of cytochrome P450(CYP450)enzymes from human liver microsomes in vitro. METHODS:Tak-ing 200.00,100.00,50.00,25.00,12.50,6.25,3.13,1.56,0.78,0.39 μmol/L spinosin and human liver microsomes for incuba-tion,using daktarin,bupropion,amodiaquine hydrochloride,diclofenac sodium,mephenytoin,dextromethorphan hydrobromide and midazolam as the specific probe drugs for above-mentioned 7 subtypes of CYP450 enzymes. UPLC-Q-TOF-MS was conducted to detect generation amount of 7 probe drug metabolites,and the half inhibitory concentration (IC50) of spinosin on 7 subtypes of CYP450 enzymes from human liver microsomes was calculated. RESULTS:IC50 of spinosin on 7 subtypes of CYP450 enzymes from human liver microsomes were 1714,1158,226.1,2288,80.59,101.1,1119 μmol/L,respectively,which were higher than 50μmol/L. CONCLUSIONS:Spinosin has no inhibition effect on above-mentioned 7 subtypes of CYP450 enzymes from human liver microsomes,with very low probability of inducing metabolic drug interactions.
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Objective To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melegueta (A. melengueta) and Dennettia tripetala (D. tripetala) on CYP 3A enzymes. Methods In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay (EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental analysis as implemented in WinNonlin pharmacokinetic program. Results EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly (P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitro dose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pre-treatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin (P < 0.05). Conclusions Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.
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OBJECTIVE@#To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melegueta (A. melengueta) and Dennettia tripetala (D. tripetala) on CYP 3A enzymes.@*METHODS@#In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay (EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental analysis as implemented in WinNonlin pharmacokinetic program.@*RESULTS@#EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly (P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitro dose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pre-treatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin (P < 0.05).@*CONCLUSIONS@#Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.
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OBJECTIVE To investigate the effect of the dosing time on the pharmacokinetics of gefitinib and its potential mechanism. METHODS Female BALB/c nude mice were housed under standardized 12 h light/dark circadian conditions(light on at 7:00,off at 19:00)for two weeks before a non-small cell lung cancer(NSCLC)model was established. Two weeks later,they were divided into 2 groups(8:00,20:00)randomly. Gefitinib was orally administered at the dose of 1 mg · kg-1 to the mice in each group at 8:00 or 20:00,respectively. Blood was collected at 10 different time points after each administration. Livers were collected every 4 h during the 24 h period from the non-administrated nude mice of NSCLC model. The plasma concentration of gefitinib was determined through an HPLC-MS/MS and the parameters were calculated by WinNonlin 6.3. The total RNA was extracted from livers,purified, synthesized to cDNA that was subjected to qRT-PCR analysis for mRNA expression levels of cyto?chrome P450 enzymes(Cyp)3a11,Cyp3a13,pregnane X receptor(PXR)and constitutive androstane receptor (CAR). RESULTS The area under the plasma concentration-time curve (AUC) and mean residence time (MRT) of 8:00 administration group were higher than those of 20:00 administration group(P<0.05). The clearance(Clz/F) of 8:00 administration group was lower than that of 20:00 administration group(P<0.05). The mRNA expression levels of PXR and CAR were consistent with those of Cyp3a11 and Cyp3a13. CONCLUSION Circadian rhythm exists in the pharmacokinetics of gefitinib and it may be closely related to CYP3A and its regulator genes.
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The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.
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Humans , 8,11,14-Eicosatrienoic Acid , Metabolism , Pharmacology , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Apoptosis , Aryl Hydrocarbon Hydroxylases , Genetics , Metabolism , Atherosclerosis , Genetics , Metabolism , Pathology , Catalase , Genetics , Metabolism , Cytochrome P-450 CYP2C8 , Genetics , Metabolism , Gene Expression Regulation , Heme Oxygenase-1 , Genetics , Metabolism , Human Umbilical Vein Endothelial Cells , Cell Biology , Metabolism , Membrane Glycoproteins , Genetics , Metabolism , Models, Biological , NADPH Oxidase 2 , NADPH Oxidases , Genetics , Metabolism , NF-E2-Related Factor 2 , Genetics , Metabolism , Nitric Oxide Synthase Type III , Genetics , Metabolism , RNA, Small Interfering , Genetics , Metabolism , Reactive Oxygen Species , Metabolism , Signal Transduction , Tumor Necrosis Factor-alpha , Metabolism , PharmacologyABSTRACT
Background & objectives: Our previous study showed that cow ghee relative to soybean oil had a protective effect against carcinogen induced mammary cancer in rats. The objective of this study was to elucidate its biochemical mechanism. Methods: Two groups of 21 day old rats (20 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). Five animals from each group were sacrificed at 0 day and at 5, 21 and 44 wk for analysis of phase I and phase II pathways enzymes of carcinogen metabolism. Results: Dietary cow ghee relative to soybean oil decreased the activities of cytochrome P450 (CYP) enzymes, CYP1A1, CYP1A2, CYP1B1 and CYP2B1, responsible for activation of carcinogen in liver. Carcinogen detoxification activities of uridinediphospho-glucuronosyl transferase (UDPGT) and quinone reductase (QR) in liver, and γ-glutamyltranspeptidase (GGTP) and QR in mammary tissue were significantly higher in cow ghee fed rats than in soybean oil fed rats. The hepatic GGTP activity decreased on soybean oil diet; while in cow ghee group it remained unaffected. Interpretation & conclusions: Our findings show that dietary cow ghee compared to soybean oil downregulates the enzyme activities responsible for carcinogen activation in liver and upregulates carcinogen detoxification activities in liver and mammary tissues.
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Animals , Cytochrome P-450 Enzyme System/analysis , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/physiology , Dietary Fats/administration & dosage , Dietary Fats/physiology , Disease Models, Animal , Rats , Soybean Oil/administration & dosage , Soybean Oil/physiologyABSTRACT
OBJECTIVE: To establish a HPLC-MS/MS method for determing 5-hydroxylansoprazole/lansoprazole sulfone, and optimize the incubated conditions for rat liver microsomes. METHODS: 5-Hydroxylansoprazole/lansoprazole were determined by HPLC-MS/MS. Single factor design was used to optimize the incubated conditions and the enzymes kinetics value was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots. RESULTS: The linear range of 5-hydroxy lansoprazole and lansoprazole sulphone in liver microsome incubation system were 5.57-2 520 and 5.42-2 480 ng · mL-1, respectively. The optimal incubated conditions were 10 μmol · L-1 lansoprazole, 0.16 mg liver microsomes, and 10 min incubation, respectively. CONCLUSION: The HPLC-MS/MS method is accurate and suitable for the determination of 5-hydroxy lansoprazole and lansoprazole sulfone in rat liver microsomes. The incubated condition can be applied for study in the drug interaction with lansoprazole.
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OBJECTIVE:To study the enzyme kinetics of six specific substrates of cytochrome P450 enzymes. METHODS: The transforming amount of the corresponding metabolites of six substrates was determined by LC-MS/MS. The incubation conditions of substrates in human liver microsomes were optimized and the parameters of enzyme kinetics were calculated based on substrate concentration-reaction velocity curve. RESULTS: The optimal incubation time was 20 min and the optimal enzyme concentration was 0.25 mg protein?mL-1. The Km(?mol?L-1) values of phenacetin, tolbutamide, omeprazole, dextromethorphan,chlorzoxazone and nifedipine were 21.6,163.4,10.4,4.6,41.9 and 9.4 respectively. The Vmax (nmol?min-1?mg protein-1) values were 2.08, 0.52, 1.02, 0.40, 2.36 and 1.00 respectively. CONCLUSION: The enzyme kinetics of six specific substrates of cytochrome P450 enzymes has been investigated in this study, and the study serves as a reference and support for the study and application of substrates in CYP.
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The pharmacotherapy of schizophrenia exhibit wide inter-individual variabilities in clinical efficacy and adverse effects. Recently. human genetic diversity has been known as one of the essential factors to the variation in human drug response. This suggests that drug therapy should be tailored to the genetic characteristics of the individual. Pharmacogenetics is the field of investigation that attempts to elucidate genetic basis of an individual's responses to pharmacotherapy, considering drug effects divided into two categories as pharmacokinetics and pharmacodynamics. The emerging field of pharmacogenomics. which focuses on genetic determinants of drug response at the level of the entire human genome, is important for development and prescription of safer and more effective individually tailored drugs and will aid in understanding how genetics influence drug response. In schizophrenia, pharmacogenetic studies have shown the role of genetic variants of the cytochrome P450 enzymes such as CYP2D6, CYP2C19, and CYP2A1 in the metabolism of antipsychotic drugs. At the level of drug targets, variants of the dopamine D_(2), D_(3) and D_(4), and 5-HT_(2A) and 5-HT(2C) receptors have been examined. The pharmacogenetic studies in schizophrenia presently shows controversial findings which may be related to the multiple involvement of genes with relatively small effects and to the lack of standardized phenotypes. For further development in the pharmacogenomics of schizophrenia, there would be required the extensive outcome measures and definitious, and the powerful new tools of genomics, proteomics and so on.