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Biochemical Pharmacology Dec 2022We aimed to investigate the potential role of NPAS2 in controlling diurnal expression and activity of hepatic CYP1A2 and to determine the underlying mechanisms....
We aimed to investigate the potential role of NPAS2 in controlling diurnal expression and activity of hepatic CYP1A2 and to determine the underlying mechanisms. Regulatory effects of NPAS2 on CYP1A2 were determined using Npas2 knockout (Npas2) mice as well as AML-12, Hepa1-6 and HepG2 cells. mRNA and protein levels were detected by reverse transcription-quantitative real-time PCR and western blotting, respectively. In vitro and in vivo CYP1A2 activities were respectively evaluated using the probe substrates phenacetin and theophylline. Transcriptional regulation was investigated using luciferase reporter assays and ChIP-Seq analysis. Loss of Npas2 in mice decreased CYP1A2 expression (at both mRNA and protein levels) and blunted its rhythmicity in the liver. Likewise, Npas2 ablation down-regulated the enzymatic activity of CYP1A2 (probed by metabolism of phenacetin and theophylline) and abrogated its time-dependency. Cell-based assays confirmed that NPAS2 positively regulated CYP1A2 expression. Mechanistic study indicated that NPAS2 trans-activated Cyp1a2 through its specific binding to the -416 bp E-box-like element within the gene promoter. In conclusion, NPAS2 was identified as a key transcriptional regulator of diurnal expression of hepatic CYP1A2 in mice. Our findings have implications for improved understanding of circadian metabolism and chronopharmacokinetics.
Topics: Mice; Animals; Cytochrome P-450 CYP1A2; Circadian Rhythm; Phenacetin; Theophylline; RNA, Messenger; Liver; Basic Helix-Loop-Helix Transcription Factors; Nerve Tissue Proteins
PubMed: 36379250
DOI: 10.1016/j.bcp.2022.115345 -
Environmental Science and Pollution... Feb 2023Phenacetin (PNT) is one of the most frequently detected nonsteroidal anti-inflammatory drugs in the water ecosystems, which poses a potential risk to environmental...
Phenacetin (PNT) is one of the most frequently detected nonsteroidal anti-inflammatory drugs in the water ecosystems, which poses a potential risk to environmental aquatic organisms. Acid-washed zero-valent aluminum (ZVAl) as a highly efficient activator for persulfate (PS) process was investigated to degrade PNT from the aqueous solution. The results indicated that acid-washed pretreatment for ZVAl could efficiently increase the degradation efficiency of PNT in the PS treatment. The degradation efficiency of PNT (50 μM) was up to 90% in 4 hours with the addition of 0.2 g/L acid-washed ZVAl and 8 mM PS at pH 6.8 and 25 °C. The PNT degradation followed pseudo-first order kinetics in the present system. High activator dosage, PS concentration, and reaction temperature could enhance the PNT degradation. The presence of inorganic anions (i.e., NO, HCO) and humic acid (HA) showed inhibitory effects on the PNT degradation. The reuse results illustrated the acid-washed ZVAl material would have continuous and efficient activation performance for PS to degrade the PNT. Radical scavenger experiments and electron paramagnetic resonance indicated that both SO and •OH were major reactive species during the PNT degradation. The possible degradation pathways of PNT mainly included the break of C-N and C-O bonds and further oxidation.
Topics: Aluminum; Phenacetin; Ecosystem; Water Pollutants, Chemical; Oxidation-Reduction; Water
PubMed: 36229732
DOI: 10.1007/s11356-022-23473-z -
Drug Metabolism and Disposition: the... Dec 2022As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that...
As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tissues from human eyes were analyzed for the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes' specific activities. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and diltiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tissues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be detected. Inhibition studies showed that hydrolysis of fluorescein diacetate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue-dependent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacokinetic modeling of the eye. SIGNIFICANCE STATEMENT: Novel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.
Topics: Humans; Carboxylic Ester Hydrolases; Carboxylesterase; Prodrugs; Fluoresceins; Hydrolysis
PubMed: 36195336
DOI: 10.1124/dmd.122.000993 -
Frontiers in Microbiology 2022Mycotoxins, fungal secondary metabolites, are ubiquitously present in food commodities. Acute exposure to high levels or chronic exposure to low levels has an impact on...
Unravelling the pharmacokinetics of aflatoxin B1: determination of Michaelis-Menten constants, intrinsic clearance and the metabolic contribution of CYP1A2 and CYP3A4 in pooled human liver microsomes.
Mycotoxins, fungal secondary metabolites, are ubiquitously present in food commodities. Acute exposure to high levels or chronic exposure to low levels has an impact on the human body. The phase I metabolism in the human liver, performed by cytochrome P450 (CYP450) enzymes, is accountable for more than 80% of the overall metabolism of exogenous and endogenous compounds. Mycotoxins are (partially) metabolized by CYP450 enzymes. In this study, research was performed on CYP450 probes and aflatoxin B1 (AFB1), a carcinogenic mycotoxin, to obtain pharmacokinetic data on AFB1, required for further experimental work. The CYP450 probes of choice were a CYP3A4 substrate, midazolam (MDZ) and a CYP1A2 substrate, phenacetin (PH) since these are the main metabolizing phase I enzymes of AFB1. Linearity experiments were performed on the three substrates indicating that linear conditions were achieved at a microsomal protein concentration and incubation time of 0.25 mg/ml and 5 min, 0.50 mg/ml and 20 min and 0.25 mg/ml and 5 min for MDZ, PH and AFB1, respectively. The was determined in human liver microsomes and was estimated at 2.15 μM for MDZ, 40.0 μM for PH and 40.9 μM for AFB1. The associated values were 956 pmol/(mg.min) (MDZ), 856 pmol/(mg.min) (PH) and 11,536 pmol/(mg.min) (AFB1). Recombinant CYP systems were used to determine CYP450-specific Michaelis-Menten values for AFB1, leading to a CYP3A4 of 49.6 μM and an intersystem extrapolation factor (ISEF) corrected of 43.6 pmol/min/pmol P450 and a CYP1A2 of 58.2 μM and an ISEF corrected of 283 pmol/min/pmol P450. An activity adjustment factor (AAF) was calculated to account for differences between microsome batches and was used as a correction factor in the determination of the human hepatic clearance for MDZ, PH and AFB1. The hepatic blood clearance corrected for the AAF CL, CL CL and CL were determined in HLM at 44.1 L/h, 21.7 L/h, 40.0 L/h and 38.5 L/h. Finally, inhibition assays in HLM showed that 45% of the AFB1 metabolism was performed by CYP3A4/3A5 enzymes and 49% by CYP1A2 enzymes.
PubMed: 36110298
DOI: 10.3389/fmicb.2022.988083 -
MethodsX 2022The data presented in this article are related to the research article entitled "Cytochrome P450 inhibition activities of non-standardized botanical products" [1], in...
The data presented in this article are related to the research article entitled "Cytochrome P450 inhibition activities of non-standardized botanical products" [1], in which the possible CYP inhibitory properties of botanical products were investigated. This article describes the optimization and bioanalytical method validation of the CYP (Cytochrome P450 inhibition assay) inhibition assays, namely, phenacetin O-deethylase assay, testosterone 6β-hydroxylase assay, felodipine dehydrogenase assay and midazolam 1'-hydroxylase assay using LC-MS/MS.
PubMed: 36081487
DOI: 10.1016/j.mex.2022.101827 -
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi =... Aug 2022Primary human hepatocytes (PHH) are the gold standard of human liver model for drug screening. However, a problem of culturing PHH is the rapid decline of cytochrome...
Primary human hepatocytes (PHH) are the gold standard of human liver model for drug screening. However, a problem of culturing PHH is the rapid decline of cytochrome P450 (CYP450) activity, which plays an important role in drug metabolism. In this study, thermo-responsive culture dishes were used to explore the conditions for murine embryonic 3T3-J2 fibroblasts to form cell sheet. Based on the cell sheet engineering technology, a three-dimensional (3D) "sandwich" co-culture system of 3T3-J2 cell sheet/PHH/collagen gel was constructed. The tissue structure and protein expression of the model section were observed by hematoxylin eosin staining and immunofluorescence staining respectively. Phenacetin and bupropion were used as substrates to determine the activity of CYP450. The contents of albumin and urea in the system were determined by enzyme linked immunosorbent assay (ELISA). The results showed that the complete 3T3-J2 cell sheet could be obtained when the cell seeding density was 1.5×10 /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D liver model was developed. Compared with the two-dimensional (2D) model, in the 3D model, the cell-cell and cell-matrix connections were tighter, the activities of cytochrome P450 CYP1A2 and cytochrome P450 CYP2B6 were significantly increased, and the secretion levels of albumin and urea were increased. These indexes could be maintained stably for 21 d. Therefore, cell sheet stacking is helpful to improve the level of liver function of 3D liver model. This model is expected to be used to predict the metabolism of low-clearance drugs in preclinical, which is of great significance for drug evaluation and other studies.
Topics: Albumins; Animals; Cytochrome P-450 Enzyme System; Hepatocytes; Humans; Liver; Mice; Urea
PubMed: 36008342
DOI: 10.7507/1001-5515.202108056 -
Annals of Translational Medicine May 2022Tea, the world's second most popular drink, is an essential part of some people's lives. Thus, this study aimed to explore potential tea-drug interactions with a view to...
BACKGROUND
Tea, the world's second most popular drink, is an essential part of some people's lives. Thus, this study aimed to explore potential tea-drug interactions with a view to promoting the rational administration of drugs.
METHODS
A specific and sensitive approach involving high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and a probe cocktail was established and validated to evaluate the inhibitory effects of four teas on five cytochrome P450 (CYP450) enzymes in rats. Metoprolol tartrate (MT), omeprazole (OMP), phenacetin (PNT), tolbutamide (TOL), and testosterone (T) were selected as the probe drugs for CYP2D6, CYP2C19, CYP1A2, CYP2C6, and CYP3A1/2, respectively, and were simultaneously quantified in the multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+) in a single 12-min run.
RESULTS
The extraction recoveries, matrix effect values, as well as intra/interday accuracy and precision met the determination standards. CYP1A2 and CYP2C6 were strongly inhibited by green tea, and CYP2C6 was also strongly inhibited by Pu'er tea. Ti Kuan Yin tea had a weak inhibitory effect, and black tea had only a slight inhibitory effect, on CYP1A2. Furthermore, the four types of tea did not have significantly altered the activity of CYP2D6, CYP2C19, and CYP3A1/2 .
CONCLUSIONS
The method used in the present study was successfully applied to assess the inhibitory effects of aqueous extracts of four types of tea on CYP450 isoforms . The results suggest that different types of tea have different effects on drug metabolism.
PubMed: 35928758
DOI: 10.21037/atm-21-5490 -
Journal of Ethnopharmacology Oct 2022Psoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are...
ETHNOPHARMACOLOGICAL RELEVANCE
Psoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are main bioactive ingredients of PF with anti-tumor, anti-inflammatory, estrogen-like neuroprotection, etc., meanwhile they are also representative hepatotoxic components of PF. Hepatic CYP1A2 has been reported to be the important metabolic enzymes involved in psoralen and isopsoralen-induced hepatotoxicity. However, the relationship between the hepatotoxicity and CYP1A2 expression, and the underlying mechanism of regulating CYP1A2 expression remain unclear.
AIM OF STUDY
The aim of this study was to explore the associated mechanism between psoralen or isopsoralen induced hepatotoxicity and activated aryl hydrocarbon receptor (AhR)-mediated transcriptional induction of CYP1A2 in vitro and in vivo.
MATERIALS AND METHODS
Psoralen and isopsoralen at different doses were treated on HepG2 cells (10, 25, 50, 100, 200 μM for 2, 12, 24, 36, 48 h) and mice (20, 80, 160 mg/kg for 3, 7, 14 days) for different time, to assess the correlation of induced hepatotoxicity and CYP1A2 mRNA and protein expression in vivo and in vitro, as well as the effect on CYP1A2 enzyme activity evaluated by phenacetin metabolism. In addition, the potential mechanism of the regulation of CYP1A2 expression mediated by AhR was explored through nucleocytoplasmic shuttling, immunofluorescence, cellular thermal shift assay and molecular docking, etc. RESULTS: Psoralen and isopsoralen induced cytotoxicity in HepG2 cells, and hepatomegaly, biochemicals disorder and tissue pathological impairment in mice, respectively in dose- and time-dependent manners. Simultaneously accompanied with elevated levels of CYP1A2 mRNA and protein in the same trend, and the CYP1A2 activity was remarkably inhibited in vitro but significantly elevated overall in vivo. Besides, psoralen and isopsoralen bound to AhR and activated translocation of AhR from the cytoplasm to the nucleus, leading to the transcriptional induction of target gene CYP1A2.
CONCLUSIONS
Hepatotoxicities in HepG2 cells and mice aroused by psoralen and isopsoralen were related to the induction of CYP1A2 expression and activity, whose underlying mechanism might be psoralen or isopsoralen activated AhR translocation and induced increase of CYP1A2 transcriptional expression. Hopefully, these finding are conductive to propose an alert about the combined usage of psoralen or isopsoralen and AhR ligands or CYP1A2 substrates in clinical practice.
Topics: Animals; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP1A2; Ficusin; Furocoumarins; Mice; Molecular Docking Simulation; RNA, Messenger; Receptors, Aryl Hydrocarbon
PubMed: 35872289
DOI: 10.1016/j.jep.2022.115577 -
Journal of Ethnopharmacology Oct 2022Scutellaria baicalensis (SB) and Coptis chinensis (CC) are widely used traditional Chinese medicine (TCM) for "heat-clearing and damp-drying" and "purging fire and...
ETHNOPHARMACOLOGICAL RELEVANCE
Scutellaria baicalensis (SB) and Coptis chinensis (CC) are widely used traditional Chinese medicine (TCM) for "heat-clearing and damp-drying" and "purging fire and detoxifying". SB-CC are commonly used as a herbal pair for synergistic treatment of various diseases such as bacteria-related infections, metabolic syndromes, and some inflammatory disorders. This herbal pair is commonly used in many famous TCM formula, like Huang-Lian-Jie-Du, Gegen-Qinlian, Banxia Xiexin decoction. Aryl hydrocarbon receptor (AHR) plays an essential role in the disposition of both xenobiotics and endogenous substances through the induction of cytochrome P450 1A (CYP1A) enzymes. Regulation of the AHR-CYP1A axis is increasingly implicated in drug-drug and drug-herb interactions. Research on SB-CC for regulatory effect on the AHR-CYP1A axis is only limited to few compounds.
AIM OF THE STUDY
This study aimed to systematically investigate the regulatory effect of SB-CC and its main constitutes on the AHR-CYP1A axis in vitro and in vivo.
MATERIALS AND METHODS
The livers of mice treated with SB-CC extract were subjected to RNA-sequencing (RNA-seq). The key target genes related to drug metabolism were screened, and the differential expression genes (DEGs) were validated by qRT-PCR, Western blot, and enzyme activity assay. Luciferase reporter gene, qRT-PCR, and Western blot assays were used to determine whether SB-CC and their main constituents could activate AHR and regulate CYP1A expression in HepG2 cells. The effect of SB-CC on the pharmacokinetics of phenacetin, a CYP1A substrate, were further observed in mice to test the net effect of SB-CC on CYP1A functions. The potential CYP1A inhibitors in SB-CC were screened and their inhibitory mechanisms were also studied using human liver microsomes.
RESULTS
AHR and drug metabolism system, especially CYP1A1 and CYP1A2, were strongly affected in the liver of SB-CC-treated mice. These results were further validated by the findings that SB-CC increased CYP1A's mRNA, protein expression and activity in mouse liver. In HepG2 cells, SB, CC, baicalin, baicalein, chrysin, oroxylin A, berberine, coptisine and epiberberine increased CYP1A1 mRNA expression in an AHR-dependent way. Interestingly, SB-CC treatment for 14 days only slightly increased the systemic exposure of paracetamol in mice. In the CYP1A inhibition assay, SB, CC, baicalin, baicalein, wogonoside, wogonin, chrysin, oroxylin A, scutellarein, columbamine, coptisine, palmatine, epiberberine, and berberrubine inhibited CYP1A activity in different degree.
CONCLUSIONS
These results suggested that SB-CC exerted dual regulatory effect on the AHR-CYP1A axis by increasing CYP1A expression but simultaneously inhibiting CYP1A activity, which may contribute to a tight modulation of AHR signaling for homeostatic control.
Topics: Animals; Coptis chinensis; Cytochrome P-450 CYP1A1; Humans; Mice; Plant Extracts; RNA, Messenger; Receptors, Aryl Hydrocarbon; Scutellaria baicalensis
PubMed: 35843411
DOI: 10.1016/j.jep.2022.115545 -
Heliyon Jun 2022Direct evidence of Triphala-drug interactions has not been provided to date.
CONTEXT
Direct evidence of Triphala-drug interactions has not been provided to date.
OBJECTIVE
This study was aimed to determine the effects of Triphala on cytochrome P450 (CYP) isoforms and P-glycoprotein (P-gp) and to investigate pharmacokinetic interactions of Triphala with CYP-probes in rats.
MATERIALS AND METHODS
Effects of Triphala on the activities of CYP isoforms and P-gp were examined using human liver microsomes (HLMs) and Caco-2 cells, respectively. Pharmacokinetic interactions between Triphala and CYP-probes (i.e., phenacetin and midazolam) were further examined in rats.
RESULTS
Triphala extract inhibited the activities of CYP isoforms in the order of CYP1A2>3A4>2C9>2D6 with the IC values of 23.6 ± 9.2, 28.1 ± 9.8, 30.41 ± 16.7 and 93.9 ± 27.5 μg/mL, respectively in HLMs. It exhibited a non-competitive inhibition of CYP1A2 and 2C9 with the values of 23.6 and 30.4 μg/mL, respectively, while its inhibition on CYP3A4 was competitive manner with the i values of 64.9 μg/mL. The inhibitory effects of Triphala on CYP1A2 and 3A4 were not time-dependent. Moreover, Triphala did not affect the P-gp activity in Caco-2 cells. Triphala, after its oral co-administration at 500 mg/kg, increased the bioavailabilities of phenacetin and midazolam by about 61.2% and 40.7%, respectively, in rats.
DISCUSSION AND CONCLUSIONS
Increases observed in the bioavailabilities of phenacetin and midazolam after oral co-administration of Triphala in rats provided a direct line of evidence to show Triphala-drug interactions inhibition of CYP1A and CYP3A activities, respectively. These results, together with the lack of time-dependency of CYP 1A2 and 3A4 inhibition , suggested that the inhibitory effect of Triphala is primarily reversible.
PubMed: 35785236
DOI: 10.1016/j.heliyon.2022.e09764