Tissue-, Region-, and Gene-Specific Induction of Microsomal Epoxide Hydrolase Expression and Activity in the Mouse Intestine by Arsenic in Drinking Water.

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.

In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19.

BACKGROUND: Recent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PURPOSE: Our study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. METHODS: HPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. RESULTS: HPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4'-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. CONCLUSION: HT was mainly metabolized into 4'-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.

A novel system to determine activity of individual uridine 5'-diphospho-glucuronosyltransferase (UGT) isoforms: Recombinant UGT-beads.

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

Novel 1,3,4-oxadiazole derivatives as highly potent microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors.

Selective inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) is implicated as a new therapeutic modality for the development of new-generation anti-inflammatory drugs. Here, we present the discovery of new and potent inhibitors of human mPGES-1, i.e., compounds 13, 15-25, 29-30 with IC50 values in the range of 5.6-82.3 nM in a cell-free assay of prostaglandin (PG)E2 formation. We also demonstrate that 20 (TG554, IC50 = 5.6 nM) suppresses leukotriene (LT) biosynthesis at low µM concentrations, providing a benchmark compound that dually intervenes with inflammatory PGE2 and LT biosynthesis. Comprehensive lipid mediator (LM) metabololipidomics with activated human monocyte-derived macrophages showed that TG554 selectively inhibits inflammatory PGE2 formation over all cyclooxygenase (COX)-derived prostanoids, does not cause substrate shunting towards 5-lipoxygenase (5-LOX) pathway, and does not interfere with the biosynthesis of the specialized pro-resolving mediators as observed with COX inhibitors, providing a new chemotype for effective and safer anti-inflammatory drug development.

Identification and Functional Assessment of Eight CYP3A4 Allelic Variants *39-*46 Detected in the Chinese Han Population.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex polymerase chain reaction (PCR) amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising six previously reported alleles and eight new nonsynonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by coexpressing them with cytochromes P450 oxidoreductase (CYPOR) in Saccharomyces cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was used as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, whereas the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. SIGNIFICANCE STATEMENT: In this study, we established a multiplex polymerase chain reaction (PCR) amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these eight novel CYP3A4 allele variants in vitro. This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.

Computational exploration of microsomal cytochrome P450 3A1 enzyme modulation by phytochemicals of Cichorium intybus L.: Insights into drug metabolism.

Drug metabolism plays a crucial role in drug fate, including therapeutic inactivation or activation, as well as the formation of toxic compounds. This underscores the importance of understanding drug metabolism in drug discovery and development. Considering the substantial costs associated with traditional drug development methods, computational approaches have emerged as valuable tools for predicting the metabolic fate of drug candidates. With this in mind, the present study aimed to investigate the potential mechanisms underlying the modulation of microsomal cytochrome P450 3A1 (CYP3A1) enzyme activity by various phytochemicals found in Cichorium intybus L., commonly known as chicory. To achieve this goal, several in silico methods, including molecular docking and molecular dynamics (MD) simulation, were employed to explore computationally the microsomal CYP3A1 enzyme. Schrodinger software was utilized for the molecular docking study, which involved the interaction analysis between CYP3A1 and 28 phytoconstituents of Cichorium intybus. Virtual screening of 28 compounds from chicory led to the identification of the top five ranked compounds. These compounds were evaluated for drug-likeness properties, pharmacokinetic profiles, and predicted binding affinities to CYP3A1. Caffeoylshikimic acid and cichoric acid emerged as promising candidates due to their favorable characteristics, including good oral bioavailability and high binding affinities to CYP3A1. Molecular dynamics simulations were conducted to assess the stability of caffeoylshikimic acid within the CYP3A1 binding pocket. The results demonstrated that caffeoylshikimic acid maintained stable interactions with the enzyme throughout the simulation, suggesting its potential as an effective modulator of CYP3A1 activity. The findings of this study have the potential to provide valuable insights into the complex molecular mechanisms by which Cichorium intybus L. acts on hepatocytes and modulates CYP3A1 enzyme expression or activity. By elucidating the impact of these phytochemicals on drug metabolism, this research contributes to our understanding of how chicory may interact with drugs and influence their efficacy and safety profiles.

Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform.

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.

Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins.

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 µg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC-MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Synthesis and evaluation of indomethacin prodrugs with a diester structure that are metabolically activated by human carboxylesterases.

1. Carboxylesterase (CES) has been studied extensively, mostly with substrates in the monoester structures. We investigated the relationship between indomethacin diester prodrugs and metabolic activation by microsomes and recombinant human CES.2. Eight indomethacin diester prodrugs were synthesised in two steps. They were used as substrates and hydrolysis rates were calculated.3. As a result, the major hydrolysis enzyme was CES. The hydrolysis rate of recombinant CES2A1 was comparable to that of recombinant CES1A1.4. In this study, by changing the structure of the prodrug to a diester structure, it was found that CES2 activity was equivalent to CES1 activity.5. It should be noted that the use of diester prodrugs in prodrug discovery, where organ-specific hydrolysis reactions are expected, may not yield the expected results.

A multivariate data analysis approach for the investigation of in vitro derived metabolites of ACP-105 in comparison with human in vivo metabolites.

Selective androgen receptor modulators (SARMs) such as ACP-105 are prohibited in sports due to their anabolic properties. ACP-105 has in previous equine studies shown to undergo extensive metabolism, which makes its metabolite profile important to investigate in humans, since the metabolism is unknown in this species. The aims of the study were to systematically optimize in vitro microsome incubations for improved metabolite yield and to utilize a multivariate data analysis (MVDA) approach to aid the metabolite discovery. Microsomes together with S9 fractions were used at optimal conditions, both with and without phase II additives. Furthermore, the relevance of the in vitro derived metabolites was evaluated as analytical targets in doping control by comparison with results from a human post-administration urine sample collected after a single dose of 100 µg ACP-105. All samples were analyzed with liquid chromatography - Orbitrap mass spectrometry. The use of the systematical optimization and MVDA greatly simplified the search and a total of 18 in vitro metabolites were tentatively identified. The yield of the two main monohydroxylated isomers increased by 24 and 10 times, respectively. In the human urine sample, a total of seven metabolites of ACP-105, formed by a combination of hydroxylations and glucuronic acid conjugations, were tentatively identified. The main metabolites were two monohydroxylated forms that are suggested as analytical targets for human doping control after hydrolysis. All the in vivo metabolites could be detected with the MVDA approach on the in vitro models, demonstrating its usefulness for prediction of the in vivo metabolite profile.

Microsome Mediated in Vitro Metabolism: A Convenient Method for the Preparation of the PET Radioligand Metabolite [18F]FE-PE2I-OH for Translational Dopamine Transporter Imaging.

Undesired radiometabolites can be detrimental to the development of positron emission tomography (PET) radioligands. Methods for quantifying radioligand metabolites in brain tissue include ex vivo studies in small animals or labeling and imaging of the radiometabolite(s) of interest. The latter is a time- and resource-demanding process, which often includes multistep organic synthesis. We hypothesized that this process could be replaced by making use of liver microsomes, an in vitro system that mimics metabolism. In this study, rat liver microsomes were used to prepare radiometabolites of the dopamine transporter radioligand [18F]FE-PE2I for in vitro imaging using autoradiography and in vivo imaging using PET in rats and nonhuman primates. The primary investigated hydroxy-metabolite [18F]FE-PE2I-OH ([18F]2) was obtained in a 2% radiochemical yield and >99% radiochemical purity. In vitro and in vivo imaging demonstrated that [18F]2 readily crossed the blood-brain barrier and bound specifically and reversibly to the dopamine transporter. In conclusions, the current study demonstrates the potential of liver microsomes in the production of radiometabolites for translational imaging studies and radioligand discovery.

Sex- and enantiospecific differences in the formation rate of hydroxyeicosatetraenoic acids in rat organs.

Hydroxyeicosatetraenoic acids (HETEs) are hydroxylated arachidonic acid (AA) metabolites that are classified into midchain, subterminal, and terminal HETEs. Hydroxylation results in the formation of R and S enantiomers for each HETE, except for 20-HETE. HETEs have multiple physiological and pathological effects. Several studies have demonstrated sex-specific differences in AA metabolism in different organs. In this study, microsomes from the heart, liver, kidney, lung, intestine, and brain of adult male and female Sprague-Dawley rats were isolated and incubated with AA. Thereafter, the enantiomers of all HETEs were analyzed by liquid chromatography-tandem mass spectrometry. We found significant sex- and enantiospecific differences in the formation levels of different HETEs in all organs. The majority of HETEs, especially midchain HETEs and 20-HETE, showed significantly higher formation rates in male organs. In the liver, the R enantiomer of several HETEs showed a higher formation rate than the corresponding S enantiomer (e.g., 8-, 9-, and 16-HETE). On the other hand, the brain and small intestine demonstrated a higher abundance of the S enantiomer. 19(S)-HETE was more abundant than 19(R)-HETE in all organs except the kidney. Elucidating sex-specific differences in HETE levels provides interesting insights into their physiological and pathophysiological roles and their possible implications for different diseases.

Resveratrol protects against cadmium-induced cerebrum toxicity through modifications of the cytochrome P450 enzyme system in microsomes.

BACKGROUND: Cadmium (Cd), known as a vital contaminant in the environment, penetrates the blood-brain barrier and accumulates in the cerebrum. Acute toxicosis of Cd, which leads to lethal cerebral edema, intracellular accumulation and cellular dysfunction, remains to be illuminated with regard to the exact molecular mechanism of cerebral toxicity. Resveratrol (RES), present in the edible portions of numerous plants, is a simply acquirable and correspondingly less toxic natural compound with neuroprotective potential, which provides some theoretical bases for antagonizing Cd-induced cerebral toxicity. RESULTS: This work was executed to research the protective effects of RES against Cd-induced toxicity in chicken cerebrum. Markedly, these lesions were increased in the Cd group, which also exhibited a thinner cortex, reduced granule cells, vacuolar degeneration, and an enlarged medullary space in the cerebrum. Furthermore, Cd induced CYP450 enzyme metabolism disorders by disrupting the nuclear xenobiotic receptor response (NXRs), enabling the cerebrum to reduce the ability to metabolize exogenous substances, eventually leading to Cd accumulation. Meanwhile, accumulated Cd promoted oxidative damage and synergistically promoted the damage to neurons and glial cells. CONCLUSION: RES initiated NXRs (especially for aromatic receptor and pregnancy alkane X receptor), decreasing the expression of CYP450 genes, changing the content of CYP450, maintaining CYP450 enzyme normal activities, and exerting antagonistic action against the Cd-induced abnormal response of nuclear receptors. These results suggest that the cerebrum toxicity caused by Cd was reduced by pretreatment with RES. © 2023 Society of Chemical Industry.

Hydrolysis of dibutyl phthalate and di(2-ethylhexyl) phthalate in human liver, small intestine, kidney, and lung: An in vitro analysis using organ subcellular fractions and recombinant carboxylesterases.

Phthalates are widely used plasticizers that are primarily and rapidly metabolized to monoester phthalates in mammals. In the present study, the hydrolysis of dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) in the human liver, small intestine, kidney, and lung was examined by the catalytic, kinetic, and inhibition analyses using organ microsomal and cytosolic fractions and recombinant carboxylesterases (CESs). The Vmax (y-intercept) values based on the Eadie-Hofstee plots of DBP hydrolysis were liver > small intestine > kidney > lung in microsomes, and liver > small intestine > lung > kidney in cytosol, respectively. The CLint values (x-intercept) were small intestine > liver > kidney > lung in both microsomes and cytosol. The Vmax and CLint or CLmax values of DEHP hydrolysis were small intestine > liver > kidney > lung in both microsomes and cytosol. Bis(4-nitrophenyl) phosphate (BNPP) effectively inhibited the activities of DBP and DEHP hydrolysis in the microsomes and cytosol of liver, small intestine, kidney, and lung. Although physostigmine also potently inhibited DBP and DEHP hydrolysis activities in both the microsomes and cytosol of the small intestine and kidney, the inhibitory effects in the liver and lung were weak. In recombinant CESs, the Vmax values of DBP hydrolysis were CES1 (CES1b, CES1c) > CES2, whereas the CLmax values were CES2 > CES1 (CES1b, CES1c). On the other hand, the Vmax and CLmax values of DEHP hydrolysis were CES2 > CES1 (CES1b, CES1c). These results suggest an extensive organ-dependence of DBP and DEHP hydrolysis due to CES expression, and that CESs are responsible for the metabolic activation of phthalates.

Human CYP1B1 enzyme-mediated, AhR enhanced activation of aflatoxin B1 for its genotoxicity in human cells.

Aflatoxin B1 (AFB1) is a human procarcinogen known to be activated by cytochrome P450 (CYP) 1A2 and 3A4. In a previous study AFB1 caused chromosomal rearrangement in a yeast strain genetically engineered for stably expressing human CYP1B1. Yet, further verification of the effect of AFB1 in human cells, a potential role of the aryl hydrocarbon receptor (AhR), and CYP1B1-catalyzed AFB1 metabolism remain unidentified. In this study, a human hepatocyte (L-02) line and a human lymphoblastoid (TK6) cell line were genetically engineered for the expression of human CYP1B1, producing L-02-hCYP1B1 and TK6-hCYP1B1, respectively. They were exposed to AFB1 and analyzed for the formation of micronucleus and elevation of γ-H2AX (indicating double-strand DNA breaks); the metabolites formed by CYP1B1 from AFB1 after incubation of AFB1 with human CYP1B1 isoenzyme microsomes were determined by LC-MS. The results showed significantly more potent induction of micronucleus by AFB1 in L-02-hCYP1B1 and TK6-hCYP1B1 than in the parental (L-02 and TK6) cells, and the effects were reduced by (E)- 2,3',4,5'-tetramethoxystilbene, a specific CYP1B1 inhibitor. In the AFB1- CYP1B1 microsomes incubations AFM1, a known stable metabolite of AFB1, was detected. Moreover, in L-02 and TK6 cells, AFB1 apparently increased the protein levels of AhR, ANRT and CYP1B1, and caused the nuclear translocation of AhR and ARNT, the latter effect being blocked by BAY-218 (an inhibitor of AhR). In conclusion, this study indicates that human CYP1B1 is capable of metabolically activating AFB1 through the AhR signaling pathway.

The dichloroacetamide safener benoxacor is enantioselectively metabolized by monkey liver microsomes and cytosol.

The metabolism and toxicity of current-use herbicide safeners remain understudied. We investigated the enantioselective metabolism of the safener benoxacor in Rhesus monkey subcellular fractions. Benoxacor was incubated with liver microsomes and cytosol from female and male monkeys (≤30 min). Benoxacor levels and enantiomeric fractions were determined with gas chromatography. Benoxacor was metabolized by microsomal cytochrome P450 enzymes (CYPs), cytosolic glutathione-S-transferases (GSTs), and microsomal and cytosolic carboxylesterase (CESs). CES-mediated microsomal metabolism followed the order males > females, whereas the CYP-mediated clearance followed the order females > males. CYP-mediated metabolism initially resulted in an enrichment of the second eluting benoxacor enantiomer (E2-benoxacor), whereas the first eluting benoxacor enantiomer (E1-benoxacor) was enriched after 10 or 30 min in female or male microsomal incubations. Benoxacor metabolism by GSTs was enantiospecific, with a total depletion of E1-benoxacor after approximately 20 min. Thus, the enantioselective metabolism of benoxacor by GSTs and CYPs may affect its toxicity.

Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system.

Cytochrome P450 enzymes (CYPs) are involved in metabolic steps that provide structural diversity during the biosynthesis of carotenoids and their oxidative cleavage products called apocarotenoids. Recent studies on bioactive apocarotenoids in plants revealed the necessity of performing further research to uncover the function of novel CYP enzymes that might be involved in apocarotenoid metabolism. We describe a series of in-vitro methods to characterize plant CYPs that metabolize apocarotenoids, using a specific Saccharomyces cerevisiae strain, WAT11, engineered to express a CYP redox partner, Arabidopsis thaliana NADPH-P450 reductase 1 (ATR1). This chapter provides protocols for construction and transformation of plasmids that express CYPs in yeast, isolation of yeast microsomes, and in-vitro enzymatic assays to validate the final metabolic products using LC-MS.

Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

Functional Analysis of Wild-Type and 27 CYP3A4 Variants on Dronedarone Metabolism In vitro.

BACKGROUND: Cytochrome P450 (P450) is the largest family of enzymatic proteins in the human liver, and its features have been studied in physiology, medicine, biotechnology, and phytoremediation. OBJECTIVE: The aim of this study was to assess the catalytic activities of 28 human CYP3A4 alleles by using dronedarone as a probe drug in vitro, including 7 novel alleles recently found in the Han Chinese population. METHODS: We expressed 28 CYP3A4 alleles in insect microsomes and incubated them with 1-100 µM of dronedarone at 37 °C for 40 minutes to obtain the metabolites of N-debutyl-dronedarone. RESULTS: Compared with the wild type of CYP3A4, the 27 defective alleles can be classified into four categories. Three alleles had no detectable enzyme activity leading to a lack of kinetic parameters of N-debutyl-dronedarone; the other three alleles slightly despaired when it comes to intrinsic clearance values compared with the features of the wild type. Sixteen alleles exhibited 35.91%~79.70% relative values (in comparison to the wild-type) and could be defined as the "moderate decrease group". The rest of the alleles showed a considerable decrease in intrinsic clearance values, ranging from 11.88%~23.34%. Therefore they were classified as a "significantly decreased group". More specifically, 18 CYP3A4 alleles exhibited a substrate inhibition trend toward dronedarone when the concentration rises to 20 µM. CONCLUSION: The outcomes of this novel study on the metabolism of dronedarone by CYP3A4 alleles can be used as experimental data support for the individualized use of this modern drug.

Metabolite Identification of a Novel Anti-Leishmanial Agent OJT007 in Rat Liver Microsomes Using LC-MS/MS.

The purpose of this study was to identify potential metabolic pathways and metabolites of OJT007, a methionine aminopeptidase 1 (MetAP1) inhibitor. OJT007 is a novel drug with potent antiproliferative effects against Leishmania Major. We conducted in vitro Phase I oxidation and Phase II glucuronidation assays on OJT007 using rat liver microsomes. Four unknown metabolites were initially identified using a UPLC-UV system from microsomal incubated samples. LC-MS/MS analysis was then used to identify the structural characteristics of these metabolites via precursor ion scan, neutral loss scan, and product ion scan. A glucuronide metabolite was further confirmed by ß-glucuronidase hydrolysis. The kinetic parameters of OJT007 glucuronidation demonstrated that OJT007 undergoes rapid metabolism. These results demonstrate the liver's microsomal ability to mediate three mono-oxidated metabolites and one mono-glucuronide metabolite. This suggests hepatic glucuronidation metabolism of OJT007 may be the cause of its poor oral bioavailability.