Liver Microsomal Fractions Research Articles

STATE OF THE XENOBIOTICS BIOTRANSFORMATION SYSTEM IN THE LIVER MICROSOMAL FRACTION OF RATS UNDER THE EFFECTS OF SODIUM BENZOATE AND ASCORBIC ACID

The combined effect of food preservatives – sodium benzoate and ascorbic acid on the state of monooxygenase system (MOS) components in the microsomal fraction of rat liver cells are studied in the paper. Changes in the level of cytochrome P450 and cytochrome b5, as well as the rate of reduction-oxidation of these hemoproteins in the microsomal fraction of the rat liver under conditions of administration of sodium benzoate and ascorbic acid were studied. During the experiment, rats were divided into four groups: group I – control (intact animals); group II – rats that were injected with ascorbic acid at a dose of 30 mg per kg of animal weight; group III – rats that were injected with sodium benzoate at a dose of 750 mg per kg of animal weight; group IV – rats that were injected with sodium benzoate 30 minutes before the introduction of ascorbic acid. Sodium benzoate and ascorbic acid were administered per os daily for 21 days. Animals were euthanized under light ether anesthesia on the 21st day after the start of administration of sodium benzoate and ascorbic acid. It was established that the three-week introduction of sodium benzoate into the body of animals leads to a decrease in the level of cytochrome P450, which occurs due to an increase in the rate of transition of cytochrome P450 into its inactive form P420. It was shown that along with the decrease in cytochrome P450 in the microsomal fraction of the liver, the level of cytochrome b5 decreases with a simultaneous increase in the rate of reduction and oxidation of this hemoprotein. Sodium benzoate exhibits a higher destructive effect when it is combined with ascorbic acid, which is expressed by a decrease in the level of MOS hemoproteins and may be a consequence of dangerous metabolites formation in the body - benzoic acid and benzene.

A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1–37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

Phenylpropanoid-enriched broccoli seedling extract can reduce inflammatory markers and pain behavior

BackgroundPain is a worldwide problem requiring an effective, affordable, non-addictive therapy. Using the edible plant broccoli, a growth protocol was developed to induce a concentrated combinatorial of potential anti-inflammatories in seedlings.MethodsA growth method was utilized to produce a phenylpropanoid-rich broccoli sprout extract, referred to as Original Extract (OE). OE was concentrated and then resuspended for study of the effects on inflammation events. A rabbit disc model of inflammation and degeneration, and, a mouse model of pain behavior were used for in vivo and in vitro tests. To address aspects of mammalian metabolic processing, the OE was treated with the S9 liver microsome fraction derived from mouse, for use in a mouse in vivo study. Analytical chemistry was performed to identify major chemical species. Continuous variables were analyzed with a number of methods including ANOVA, and two-tailed t tests, as appropriate.ResultsIn a rabbit spine (disc) injury model, inflammatory markers were reduced, and levels of regenerative markers were increased as a result of OE treatment, both in vivo and in vitro. In a mouse pain behavioral model, after treatment with S9 liver microsome fraction, the resultant extract significantly reduced early and late pain behavior in response to a pain stimulus. The OE itself reduced pain behavior in the mouse pain model, but did not achieve the level of significance observed for S9-treated extract. Analytical chemistry undertaken on the extract constituents revealed identities of the chemical species in OE, and how S9 liver microsome fraction treatment altered species identities and proportions.ConclusionsIn vitro and in vivo results indicate that the OE, and S9-treated OE broccoli extracts are worthwhile materials to develop a non-opiate inflammation and pain-reducing treatment.

In vitro and in vivo studies on a group of chalcones find promising results as potential drugs against fascioliasis

About a third of the world population is infected by helminth parasites implicated in foodborne trematodiasis. Fascioliasis is a worldwide disease caused by trematodes of the genus Fasciola spp. It generates huge economic losses to the agri-food industry and is currently considered an emerging zoonosis by the World Health Organization (WHO). The only available treatment relies on anthelmintic drugs, being triclabendazole (TCBZ) the drug of choice to control human infections. The emergence of TCBZ resistance in several countries and the lack of an effective vaccine to prevent infection highlights the need to develop new drugs to control this parasitosis.We have previously identified a group of benzochalcones as inhibitors of cathepsins, which have fasciolicidal activity in vitro and are potential new drugs for the control of fascioliasis. We selected the four most active compounds of this group to perform further preclinical studies. The compound's stability was determined against a liver microsomal enzyme fraction, obtaining half-lives of 34–169 min and low intrinsic clearance values (<13 μL/min/mg), as desirable for potential new drugs. None of the compounds were mutagenic or genotoxic and no in vitro cytotoxic effects were seen. Compounds C31 and C34 showed the highest selectivity index against liver fluke cathepsins when compared to human cathepsin L. They were selected for in vivo efficacy studies observing a protective effect, similar to TCBZ, in a mouse model of infection. Our findings strongly encourage us to continue the drug development pipeline for these molecules.

Consideration of vendor-related differences in hepatic metabolic stability data to optimize early ADME screening in drug discovery

Hepatic metabolic stability is a crucial determinant of oral bioavailability and plasma concentrations of a compound, and its measurement is important in early drug discovery. Preliminary metabolic stability estimations are commonly performed in liver microsomal fractions. At the National Center for Advancing Translational Sciences, a single-point assay in rat liver microsomes (RLM) is employed for initial stability assessment (Tier I) and a multi-point detailed stability assay is employed as a Tier II assay for promising compounds. Although the in vitro and in vivo metabolic stability of compounds typically exhibit good correlation, conflicting results may arise in certain cases. While investigating one such instance, we serendipitously found vendor-related RLM differences in metabolic stability and metabolite formation, which had implications for in vitro and in vivo correlations. In this study, we highlight the importance of considering vendor differences in hepatic metabolic stability data and discuss strategies to avoid these pitfalls.

Reduction of four bis‐azo dyes by pig liver microsomal fraction in anaerobic conditions

AbstractThe study of azo dyes degradation products and their harmfulness is an important topic because their diffusion in the environment is still a serious problem. We investigated the reduction by pig liver microsomal fraction of four bis‐azo dyes, Acid Red 73, Acid Red 150, Direct Red 24, and Direct Red 28, in anaerobic condition. These complex molecules, different for position/nature of substituent groups and for the group interposed between the two azo bonds, allowed to investigate the correlation between substrate molecular structure and action of a subcellular fraction representative of the xenobiotic degradation by a living organism and the consequent possible appearance of harmful reaction products. Dyes were first carefully purified to eliminate interferences in the enzymatic assays. The reduction products were identified by gas chromatography/mass spectrometry. Acid Red 73 and Acid Red 150 were markedly reduced with production of harmful aromatic amines, while Direct Red 24 and Direct Red 28 were neither reduced to their synthesis amines nor to other amines. These results suggest that azo dyes with a benzidine derivative group are not degraded by microsomal fraction and therefore molecules with a similar structure could be classified with a lower risk index not producing any harmful catabolic product.

Natural Antioxidant By-Product Mixture Counteracts the Effects of Aflatoxin B1 and Ochratoxin A Exposure of Piglets after Weaning: A Proteomic Survey on Liver Microsomal Fraction.

Mycotoxins are toxic compounds produced by certain strains of fungi that can contaminate raw feed materials. Once ingested, even in small doses, they cause multiple health issues for animals and, downstream, for people consuming meat. It was proposed that inclusion of antioxidant-rich plant-derived feed might diminish the harmful effects of mycotoxins, maintaining the farm animals' health and meat quality for human consumption. This work investigates the large scale proteomic effects on piglets' liver of aflatoxin B1 and ochratoxin A mycotoxins and the potential compensatory effects of grapeseed and sea buckthorn meal administration as dietary byproduct antioxidants against mycotoxins' damage. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three (n = 10) experimental groups (A, M, AM) and one control group (C) and fed with experimental diets for 30 days. After 4 weeks, liver samples were collected, and the microsomal fraction was isolated. Unbiased label-free, library-free, data-independent acquisition (DIA) mass spectrometry SWATH methods were able to relatively quantify 1878 proteins from piglets' liver microsomes, confirming previously reported effects on metabolism of xenobiotics by cytochrome P450, TCA cycle, glutathione synthesis and use, and oxidative phosphorylation. Pathways enrichment revealed that fatty acid metabolism, steroid biosynthesis, regulation of actin cytoskeleton, regulation of gene expression by spliceosomes, membrane trafficking, peroxisome, thermogenesis, retinol, pyruvate, and amino acids metabolism pathways are also affected by the mycotoxins. Antioxidants restored expression level of proteins PRDX3, AGL, PYGL, fatty acids biosynthesis, endoplasmic reticulum, peroxisome, amino acid synthesis pathways, and, partially, OXPHOS mitochondrial subunits. However, excess of antioxidants might cause significant changes in CYP2C301, PPP4R4, COL18A1, UBASH3A, and other proteins expression levels. Future analysis of proteomics data corelated to animals growing performance and meat quality studies are necessary.

Evaluation of a Five-Probe Metabolic Control Cocktail in Long-Term Cocultured Human Hepatocytes

Hepatocyte cocultures like HepatoPac have become more frequently used for the assessment of the intrinsic clearance of slowly metabolised drugs during drug discovery due to a superiority in enzymatic activity over time compared to liver microsomal fractions and suspended primary hepatocytes. However, the relatively high cost and practical limitations prevent several quality control compounds to be included in studies and the activities of many important metabolic enzymes are consequently often not monitored. In this study, we have evaluated the possibility for a cocktail approach of quality control compounds in the human HepatoPac system to ensure adequate activity of the major metabolising enzymes. Five reference compounds were selected based on their known metabolic substrate profile in order to capture major CYP and non-CYP metabolic pathways in the incubation cocktail. The intrinsic clearance of the reference compounds when incubated as singlets or in a cocktail was compared and no considerable difference was observed. We show here that a cocktail approach of quality control compounds allows for easy and efficient evaluation of the metabolic competency of the hepatic coculture system over an extended incubation period.

Immobilization of cytochrome P450 enzymes onto magnetic beads: An approach to drug metabolism and biocatalysis

Cytochrome P450 (CYP, P450) presents a wide range of applicability in drug metabolism studies and in biocatalysis as a great alternative to synthesizing compounds. Nonetheless, their lack of stability is one of their major drawbacks. Aiming the possibility of enhancing the catalytic activity and promoting higher stability, liver microsomal fractions have been immobilized on magnetic beads (Mb). The immobilized procedure was modulated by using rat liver microsomal fractions (RLM-Mb). The optimal condition achieved was further employed for the immobilization of the human microsomal fractions on magnetic beads (HLM-Mb). In vitro metabolism assays were conducted using albendazole (ABZ) as a model drug, and the formation of albendazole sulfoxide (ABZSO) was monitored. Biotransformation reactions applying the produced HLM-Mb were examined for the best temperature to increase the production of metabolites and their reuse cycles. A kinetic study was carried out for HLM-Mbs monitoring the production of ABZO by the oxidation reaction of ABZ by CYP3A4. The Km value was 25.6 µmol L−1 and Vmax was 121.0 µmol L−1. Inhibition assays were conducted in the presence of ketoconazole and the production of ABZSO decreased by 46.8 ± 2.5%. Enzymatic activities for CYP2C9 and CYP2D6 on HLM-Mbs were evaluated by monitoring the hydroxylation reactions of diclofenac and bufuralol as substrates. The immobilization of CYP P450 on magnetic beads increased not only the production of ABZSO metabolites but also the stability of CYP. The use of HLM-Mbs jointly with immobilized glucose-6-phosphate dehydrogenase (G6PDH-Mbs) as a unique dual generator system to produce NADPH has established the one-pot conditions for biocatalysis in a greener approach with reuse of the biocatalyst (G6PDH-HLM-Mbs). To this end, the herein reported HLM-Mbs and G6PDH-HLM-Mbs are excellent analytical tools to be explored either in biocatalysis reactions or in in vitro metabolism studies.

Development of a GC/Q-ToF-MS Method Coupled with Headspace Solid-Phase Microextraction to Evaluate the In Vitro Metabolism of β-Caryophyllene.

Sample preparation remains both a challenging and time-consuming process in the field of bioanalytical chemistry. Many traditional techniques often require multi-step processes, which can introduce additional errors to the analytical method. Given the complexity of many biological matrices, thorough analyte extraction presents a major challenge to researchers. In the present study, a headspace solid-phase microextraction (HS-SPME) coupled with a GC/Q-ToF-MS method, was developed to quantify in vitro metabolism of β-caryophyllene by both human liver microsome (HLM) and S9 liver fractions. Validation of the method was demonstrated both in terms of linearity (R2 = 0.9948) and sensitivity with a limit of detection of 3 ng/mL and a limit of quantitation of 10 ng/mL. In addition, the method also demonstrated both inter- and intra-day precision with the relative standard deviation (RSD) being less than 10% with four concentrations ranging from 50-500 ng/mL. Since this method requires no solvents and minimal sample preparation, it provides a rapid and economical alternative to traditional extraction techniques. The method also eliminates the need to remove salts or buffers, which are commonly present in biological matrices. Although this method was developed to quantify in vitro metabolism of one analyte, it could easily be adapted to detect or quantify numerous volatiles and/or semi-volatiles found in biological matrices.

INTENSITY OF LIPID PEROXIDATION IN THE MICROSOMAL FRACTION OF RAT LIVER UNDER THE EFFECTS OF SODIUM BENZOATE AND ASCORBIC ACID

The effect of food preservatives on the state of free radical processes in rat liver cells are studied in the paper. The changes of lipid peroxidation (LPO) products (the level of primary products – diene conjugates (DK), secondary products – ketodienes and conjugated trienes (KD + CT), TBA-active products and final products – Schiff bases) in the microsomal fraction of rat liver were studied under conditions of introduction into the body of sodium benzoate and ascorbic acid. Animals were divided into four groups: group I – intact animals (control); group II – rats, which were administra-tion of ascorbic acid; group III – rats, which were administration of sodium benzoate; group IV – rats, which were administration of sodium benzoate 30 min before the introduction of ascorbic acid. Sodium benzoate and ascorbic acid were administered per os daily for 21 days. Euthanasia of animals was performed under light ether anesthesia on the 21st day after the administration of sodium benzoate and ascorbic acid. It was established that the daily introduction of sodium benzoate into the body leads to the initiation of LPO in the microsomal fraction of the liver, which is evidenced by an increase in the levels of primary, secondary and final prod-ucts of LPO compared to the indicators of intact animals. Sodium benzoate exhibits a higher pro-oxidant effect when combined with ascorbic acid. It has been shown that under the conditions of administration of sodium benzoate and ascorbic acid, LPO processes are intensified, as the levels of DK, KD + ST, TBK-active products and Schiff bases significantly exceed the indicators of the group of ani-mals that were administered only sodium benzoate.

Glutathione-S-transferase activity in various organs of Crocodylus siamensis and its attenuation role in aflatoxin B1-induced cell apoptosis in human hepatocarcinoma cells.

Background and Aim:The crocodile is a model for studying relevant sources of environmental contamination. They were determined an appropriate biomonitoring species for various toxins. The cytosolic and microsomal fraction of crocodiles plays a role in detoxifying xenobiotics. Cytochrome P450 1A2 (CYP1A2) metabolizes aflatoxin B1 (AFB1) to aflatoxin M1, while glutathione-S-transferase (GST) catalyzes carcinogenic agents. This study aimed to investigate the GST activity in various organs of Crocodylus siamensis. Further, the fate of microsomal and cytosolic fractions from various crocodile organs against AFB1-induced apoptosis in human hepatocarcinoma (HepG2) cells was investigated.Materials and Methods:The liver, lungs, intestines, and kidneys tissues from a 3-year-old crocodile (C. siamensis) (n=5) were collected. The cytosolic and microsomal fraction of all tissues was extracted, and protein concentrations were measured with a spectrophotometer. Subsequently, a comparison of GST activity from various organs was carried out by spectrophotometry, and the protective effects of CYP450 and GST activity from various crocodile organs were studied. In vitro AFB1-induced apoptosis in HepG2 cells was detected by reverse transcription-quantitative polymerase chain reaction. Comparisons between the metabolisms of the detoxification enzyme in organs were tested using the Kruskal–Wallis one-way analysis of variance and Dunn’s multiple comparison tests. All kinetic parameters were analyzed using GraphPad Prism software version 5.01 (GraphPad Software Inc., San Diego, USA).Results:Total GST activity in the liver was significantly higher than in the kidneys, intestines, and lungs (p<0.05, respectively). The highest GST pi (GSTP) activity was found in the liver, while the highest GST alpha-isoform activity was in the crocodile lung. The kinetics of total GST and GST mu activity in the liver had the highest velocity compared to other organs. In contrast, the kinetics of GSTP enzyme activity was the highest in the intestine. The in vitro study of microsome and cytosol extract against apoptosis induced by AFB1 revealed that the level of messenger RNA expression of the Bax and Bad genes of HepG2 cells decreased in the treatment group in a combination of cytosolic and microsomal fractions of the crocodile liver but not for Bcl-2. Interestingly, the downregulated expression of Bax and Bad genes was also found in the microsome and cytosol of crocodile kidneys.Conclusion:The crocodile liver revealed very effective GST activity and expression of the highest kinetic velocity compared to other organs. The combination of liver microsomal and cytosolic fractions could be used to prevent cell apoptosis induced by AFB1. However, further study of the molecular approaches to enzyme activity and apoptosis prevention mechanisms should be carried out.

Evaluation of species differences in the metabolism of the selective NaV1.7 inhibitor DS-1971a, a mixed substrate of cytochrome P450 and aldehyde oxidase

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites. After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite. Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450. When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

Mitochondrial Biotransformation of Drugs and other Xenobiotics.

In vivo biotransformation of exposed chemicals is one of the major factors that determine the concentration and the duration of a substance at the systemic site of effect. Given that toxicity is expressed as a function of two factors, namely dose and time, the type and intensity of the toxicity are directly dependent on the chemical transformation of the exposed parent substance. This dependency involves two different situations. The amount of the chemical reaching the target will be decreased with the extent of metabolism if the parent chemical is toxic, and the opposite is true if the metabolite(s) is toxic instead. To date, the liver microsomal fraction in mammals has been justifiably considered as the center of biotransformation reactions because the liver and microsomes (i.e., endoplasmic reticulum component of the cell) possess the most abundant types and quantities of xenobiotic-metabolizing enzymes, especially the cytochrome P450 supergene enzyme family. These enzymes are common in all kingdoms of life, which strongly suggests that the origin of life is common. It is already known that various drugs enter mitochondria by different mechanisms, and this translocation is believed to be responsible for mitochondrial effects that are part of the therapeutic actions of various drugs such as lipid-lowering statins or antidiabetogenic thiazolidindiones. However, the discovery of mitochondrial forms of the xenobiotic-metabolizing enzymes provoked discussions about whether mitochondria metabolize drugs and other chemicals to some extent. This possibility may particularly be important as mitochondria have various critical cellular structures and functions. In the case of in situ generated metabolite(s), when there are adverse interactions with either these structures or functions, various toxic outcomes may appear. In this review, we compiled studies in the literature regarding biotransformation of drugs and other chemicals catalyzed by mitochondria where it is both an initiator and target of toxicity.

Metabolic characterization of a potent natural neuroprotective agent dendrobine in vitro and in rats.

Dendrobine is the main sesquiterpene alkaloid of Dendrobium nobile Lindl, which exhibits potent neuroprotective activity. However, its metabolism and disposition are little known. In this study, we investigated the metabolic characteristics of dendrobine in vitro and in rats. The metabolic stability and temporal profile of metabolites formation of dendrobine were assayed in human/rat liver microsomal and S9 fractions. Dendrobine metabolites were separated and identified mainly by UPLC-Q/Orbitrap MS. After oral administration of dendrobine (50 mg/kg) to rats, the accumulative excretion rate of dendrobine in feces, urine, and bile was 0.27%, 0.52%, and 0.031%, respectively, and low systematic exposure of dendrobine (AUC0-∞ = 629.2 ± 56.4 ng·h/mL) was observed. We demonstrated that the elimination of dendrobine was very rapid in liver microsomal incubation (the in vitro elimination t1/2 in rat and human liver microsomes was 1.35 and 5.61 min, respectively). Dendrobine underwent rapid and extensive metabolism; cytochrome P450, especially CYP3A4, CYP2B6, and CYP2C19, were mainly responsible for its metabolism. Aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde oxidase were involved in the formation of carboxylic acid metabolites. By the aid of in-source fragmentation screening, hydrogen/deuterium exchange experiment, post-acquisition processing software, and available reference standards, 50 metabolites were identified and characterized in liver microsomal incubation and in rats. The major metabolic pathways of dendrobine were N-demethylation, N-oxidation, and dehydrogenation, followed by hydroxylation and glucuronidation. Collectively, the metabolic fate of dendrobine elucidated in this study not only yields benefits for its subsequent metabolism study but also facilitates to better understanding the mode of action of dendrobine and evaluating the pharmacologic efficiency of the high exposure metabolites.

NOX2 inhibitor GSK2795039 metabolite identification towards drug optimization

Overproduction of reactive oxygen species (ROS) can lead to several disease states, such as diabetic nephropathy and amyotrophic lateral sclerosis. One of the most studied mechanisms to inhibit the over production of ROS is the inhibition of NADPH oxidase (NOX) enzymes, which catalyze the conversion of cytoplasmic NADPH to NADP+, resulting in the formation of superoxide anions. GSK2795039 has been shown to selectively inhibit the NOX2 isoform, however, clearance of the compound was high in rats and mice. Therefore, identifying metabolic soft spots would be crucial in guiding the optimization process to improve its pharmacokinetic properties. GSK2795039 (10 μM) was incubated in the presence of mouse, rat and human liver microsomal (1 mg/mL) and cytosolic (2 mg/mL) fractions and appropriate co-factors, followed by MSe fragment analysis to identify metabolic soft spots. GSK2795039 showed marked species differences in its metabolism. The alkyl side chains and indoline moiety were the most common sites of biotransformation. The compound was identified to be an aldehyde oxidase substrate. Additionally, unique human metabolites were observed in vitro. Our study sheds light on structure optimization opportunities for developing improved NOX2 inhibitors, and it will help overcome the challenges involved in preclinical species selection for its safety evaluations.

Comprehensive characterization of the peroxisome proliferator activated receptor-δ agonist GW501516 for horse doping control analysis.

According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organism's metabolism. Even if urinary metabolism of G501516 has been extensively studied in human subjects, knowledge on GW501516 metabolism in horses remains limited. To fight against doping practices in horses' races, GW501516 metabolism has to be studied in horse urine to identify and characterize the most relevant target metabolites to ensure an efficient doping control. In this article, in vitro and in vivo experiments have been conducted using horse S9 liver microsome fractions and horse oral administration route, respectively. These investigations determined that the detection of GW501516 must be performed in urine on its metabolites because the parent molecule was extremely metabolized. To maximize analytical method sensitivity, the extraction conditions have been optimized. In accordance with these results, a qualitative analytical method was validated to detect the abuse of GW501516 based on its most relevant metabolites in urine. This work enabled the Laboratoire des Courses Hippiques (LCH) to highlight two cases of illicit administration of this forbidden molecule in post-race samples.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17β-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.

In vivo ESR imaging of redox status in mice after X-ray irradiation, measured by acyl-protected hydroxylamine probe, ACP

More detailed investigations on the in vivo redox status are needed to elucidate the mechanisms contributing to damage caused by ionizing radiation. In the present study, the in vivo redox status of mice was examined using in vivo electron spin resonance (ESR) imaging after an intraperitoneal injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) as a probe. ACP is easily hydrolyzed to its hydroxylamine form in the mouse body, and the interconversion between hydroxylamine and the corresponding nitroxyl radical reflects the biological redox status. Liver damage, based on changes in liver weight and plasma aspartate aminotransferase levels, was detected in mice 4 days after X-ray irradiation at 7.5 Gy. ESR imaging showed that the signal intensity of the nitroxyl radical was high at the liver area in both damaged and healthy mice after administration of ACP. Whereas the signal decayed at the liver area for healthy mouse, the decay was negligible in damaged mice. Unlike healthy mouse, signal in the chest for damaged mouse increased with time. The distribution of the sum of hydroxylamine and the nitroxyl radical was similar in damaged and healthy mice. X-ray irradiation slightly lowered the reduction activity of the liver microsomal fraction for the nitroxyl radical. Thiobarbituric acid reactive substances in the liver were higher in damaged mice than in healthy mice; however, no significant differences were noted in reduced glutathione. The present results indicate that the redox status of mice exposed to X-ray irradiation is more oxidative than that in healthy mice.

Quantification of CYP2E1 in rat liver by UPLC-MS/MS-based targeted proteomics assay: a novel approach for enzyme activity assessment.

CYP2E1 is one of the most crucial isozymes of CYP450. It is responsible for metabolizing and activating a large number of toxicants and carcinogens, but the correlation between its abundance and activity has not been widely studied. With the flourishing of modern mass spectrometry technology, quantifying complex biological proteins and studying the relationship between their abundance and activity have become practicable. In our study, an accurate, sensitive, and stable LC-MS/MS-based method was developed and validated. The method can accurately quantify the abundance of CYP2E1 in the rat liver microsome and S9 fraction. The quantitative linearity of the method is between 2 and 320 ng/mL, and the run time is 16.5 minutes. Meanwhile, we used the probe substrate method (with chlorzoxazone as the substrate) as a reference to analyze the correlation between its activity and abundance. The result illustrated that the abundance of CYP2E1 by LC-MS/MS has a strong positive correlation with its activity. This is a relationship worth studying, which has not been reported before. We also explored the correlation between quantitative results by traditional methods (western blot and RT-PCR) and activity, and the positive correlation was not obvious. Therefore, when testing the correlation between metabolic enzyme abundance and activity, the LC-MS/MS-based method is confirmed to be more accurate than conventional methods. It will provide a meaningful way of researching the metabolic enzymes in drug interactions. Furthermore, we found that the S9 fraction can also be used for mass spectrometry quantitative analysis, which is helpful for promoting the practical application of targeted protein technology.