Microsomal Preparations Research Articles

Effects of ammonia on gill (Na+, K+)-ATPase kinetics in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum.

Water quality is essential for successful aquaculture. For freshwater shrimp farming, ammonia concentrations can increase considerably, even when culture water is renewed frequently, consequently increasing the risk of ammonia intoxication. We investigated ammonia lethality (LC50-96 h) in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum from the Paraná/Paraguay River basin, including the effects of exposure to 4.93 mg L-1 total ammonia concentration on gill (Na+, K+)-ATPase activity. The mean LC50-96 h was 49.27 mg L-1 total ammonia, corresponding to 1.8 mg L-1 un-ionized ammonia. Except for NH4+ affinity that increased 2.5-fold, that of the gill (Na+, K+)-ATPase for ATP, Mg2+, Na+, K+ and ouabain was unchanged after ammonia exposure. Western blotting of gill microsomal preparations from fresh caught shrimps showed a single immunoreactive band of ≈110 kDa, corresponding to the gill (Na+, K+)-ATPase α-subunit. Ammonia exposure increased (Na+, K+)-ATPase activity by ≈25%, coincident with an additional 130 kDa α-subunit immunoreactive band, and increased K+-stimulated and V(H+)-ATPase activities by ≈2.5-fold. Macrobrachium amazonicum from the Paraná/Paraguay River basin is as tolerant to ammonia as are other Amazon River basins populations, showing toxicity comparable to that of marine crustaceans.

Metabolic interactions of benzodiazepines with oxycodone ex vivo and toxicity depending on usage patterns in an animal model.

Opioids and benzodiazepines are frequently combined in medical as well as in non-medical contexts. At high doses, such combinations often result in serious health complications attributed to pharmacodynamics interactions. Here, we investigate the contribution of the metabolic interactions between oxycodone, diazepam and diclazepam (a designer benzodiazepine) in abuse/overdose conditions through ex vivo, in vivo and in silico approaches. A preparation of pooled human liver microsomes was used to study oxycodone metabolism in the presence or absence of diazepam or diclazepam. In mice, diazepam or diclazepam was concomitantly administered with oxycodone to mimic acute intoxication. Diclazepam was introduced on Day 10 in mice continuously infused with oxycodone for 15 days to mimic chronic intoxication. In silico modelling was used to study the molecular interactions of the three drugs with CYP3A4 and 2D6. In mice, in acute conditions, both diazepam and diclazepam inhibited the metabolism of oxycodone. In chronic conditions and at pharmacologically equivalent doses, diclazepam drastically enhanced the production of oxymorphone. In silico, the affinity of benzodiazepines was higher than oxycodone for CYP3A4, inhibiting oxycodone metabolism through CYP3A4. Oxycodone metabolism is likely to be diverted towards CYP2D6. Acute doses of diazepam or diclazepam result in the accumulation of oxycodone, whereas chronic administration induces the accumulation of oxymorphone, the toxic metabolite. This suggests that overdoses of opioids in the presence of benzodiazepines are partly due to metabolic interactions, which in turn explain the patterns of toxicity dependent on usage. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Formation of an n-oxide metabolite following metabolism of 1-(3-chlorobenzyl)[1,2,4] triazolo[4,3-a]quinoxaline by in vitro rat liver microsomal preparations

Formation of an n-oxide metabolite following metabolism of 1-(3-chlorobenzyl)[1,2,4] triazolo[4,3-a]quinoxaline by in vitro rat liver microsomal preparations

Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations.

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory–inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

Abstract P228: Preclinical pharmacokinetics and pharmacodynamics of KB-0742, a selective, oral CDK9 inhibitor

Abstract Tumorigenesis is driven by the accumulation of adverse genetic changes resulting in dysregulated transcription promoting altered gene expression and the cancer cell state. Thus, tumors can develop dependencies on the transcriptional regulators that promote the reprogrammed gene expression landscape. One such regulator is cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation and promotes activation of transcription factors. We developed a potent, selective, and orally bioavailable CDK9 inhibitor, KB-0742. KB-0742 is highly selective for CDK9 as compared to other CDKs and has shown minimal off-target effects in kinase and receptor panel screens. Here we present the nonclinical pharmacologic characterization of KB-0742. The pharmacokinetics (PK) of KB-0742 in rats and dogs showed rapid absorption and high bioavailability with a %F of ≥84 after oral administration in rats and >100 in dogs. KB-0742 exhibited low turnover in human microsome and hepatocyte preparations and is projected to have low clearance and a high volume of distribution in humans based on allometric scaling from nonclinical species. These properties may facilitate achievement of sustained target engagement in patients using intermittent dosing which may mitigate the toxicity of CDK9 inhibition. To test this hypothesis, mice bearing MV4-11 (acute myeloid leukemia) xenografts were treated with KB-0742 at either 60 mg/kg for 3-days on/4-days off or continuous dosing at 25 mg/kg. Intermittent dosing showed similar tumor growth inhibition (81%) as seen with continuous dosing (74%). Target engagement was assessed in the tumors by measuring the inhibition of RNA polymerase II (pSER2). KB-0742 treatment resulted in an over 50% decrease in pSER2 compared with vehicle-treated controls. To support the phase I clinical trial, 2 pharmacodynamic assays were developed to measure drug target engagement in peripheral blood mononuclear cells (PBMCs). The first used RNA-expression profiling to measure alterations in gene expression with treatment, and the second used MSD protein analysis to measure changes in pSER2 levels. Ex vivo experiments using PBMCs from 3 healthy donors showed an over 80% reduction in pSER2 levels at 4 hours post-exposure to 1 µM of KB-0742. Similar results were observed using the RNA-expression profiling assay, in which decreased gene expression was observed in key genes at 1 µM exposures. KB-0742’s high specificity, oral bioavailability, and favorable PK properties distinguish it from other available CDK9 inhibitors. We are currently testing the hypothesis in the clinic with a phase I trial (NCT04718675) that the ability to dose intermittently will allow for favorable anti-tumor activity while minimizing adverse effects. Citation Format: Melinda A. L. Day, Douglas C. Saffran, Nathalie Rioux, Tom Chen, Christina Lee, David B. Freeman, Crystal MacKenzie, Joseph P. Vacca, Peter B. Rahl, Benjamin Wesley Trotter, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. Preclinical pharmacokinetics and pharmacodynamics of KB-0742, a selective, oral CDK9 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P228.

Effect of non-competitive inhibitors of aminopeptidase N on viability of human and murine tumor cells

Abstract Cancer is the second leading cause of death worldwide. Peptidases participate in tumor development and growth. Mammalian neutral aminopeptidase (APN, EC 3.4.11.2, M1 family) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of substrates. APN expression is dysregulated in several types of cancer, being a target for the development of new anticancer agents. Recently, we identified three new non-competitive inhibitors of soluble porcine APN (pAPN) by virtual screening (BTB11079, JFD00064, BTB07018, from Maybridge). In the present contribution we assayed their effect on the activity of APN in a microsomal preparation of porcine kidney cortex, a model of the physicochemical environment of the enzyme. These classical inhibitors had an IC50 value of 3–5 µM. Additionally, using a kinetic approach and a specific substrate, we quantified APN activity on the cell surface of human and murine lung, colon, prostate, and skin tumor cells. APN inhibitors reduced tumor cells viability, more efficiently in the higher APN activity tumor cell lines, but not in non-tumoral cells. BTB11079, JFD00064, BTB07018 effects on cell viability were stronger than that of bestatin, a positive control. Thus, these non-competitive APN inhibitors may be useful tools for cancer treatment.

Camelina sativa phosphatidylcholine:diacylglycerol cholinephosphotransferase-catalyzed interconversion does not discriminate between substrates.

Phosphatidylcholine:diacylglycerol cholinephosphotransferases (PDCT) regulate the fatty acid composition of seed oil (triacylglycerol, TAG) by interconversion of diacylglycerols (DAG) and phosphatidylcholine (PtdCho). PtdCho is the substrate for polyunsaturated fatty acid biosynthesis, as well as for a number of unusual fatty acids. By the action of PDCT, these fatty acids can be transferred into the DAG pool to be utilized in TAG biosynthesis by the action of acyl-CoA:DAG and phospholipid:diacylglycerol acyltransferases. Despite its importance in regulating seed oil composition, biochemical characterization of PDCT enzymes has been lacking. We characterized Camelina sativa PDCT in microsomal preparations of a yeast strain expressing Camelina PDCT and lacking the capacity of producing TAG. Camelina PDCT was specific for PtdCho and the sn-1,2 enantiomer of DAG and could not utilize ceramide. The interconversion reaches equilibrium within 15 min of incubation, indicating that only distinct pools of DAG and PtdCho were available for exchange. However, the pool sizes of DAG and PtdCho involved in the exchange were not fixed but increased with the amount of exogenous DAG or PtdCho added. Camelina PDCT showed about the same selectivity for di-oleoyl, di-linoleoyl, and di-linolenoyl species in both PtdCho and DAG substrates, suggesting that no unidirectional transfer of particular unsaturated substrates occurred. Camelina PDCT had a good activity with erucoyl-DAG as a substrate despite low erucic acid levels in PtdCho in plant species accumulating a high amount of this fatty acid in the seed oil.

Silencing of CYP6AS160 in Solenopsis invicta Buren by RNA interference enhances worker susceptibility to fipronil.

Cytochrome P450 monooxygenases play a key role in pest resistance to insecticides by detoxification. Four new P450 genes, CYP6AS160, CYP6AS161, CYP4AB73 and CYP4G232 were identified from Solenopsis invicta. CYP6AS160 was highly expressed in the abdomen and its expression could be induced significantly with exposure to fipronil, whereas CYP4AB73 was not highly expressed in the abdomen and its expression could not be significantly induced following exposure to fipronil. Expression levels of CYP6AS160 and CYP4AB73 in workers were significantly higher than that in queens. RNA interference-mediated gene silencing by feeding on double-stranded RNA (dsRNA) found that the levels of this transcript decreased (by maximum to 64.6%) when they fed on CYP6AS160-specific dsRNA. Workers fed dsCYP6AS160 had significantly higher mortality after 24 h of exposure to fipronil compared to controls. Workers fed dsCYP6AS160 had reduced total P450 activity of microsomal preparations toward model substrates p-nitroanisole. However, the knockdown of a non-overexpressed P450 gene, CYP4AB73 did not lead to an increase of mortality or a decrease of total P450 activity. The knockdown effects of CYP6AS160 on worker susceptibility to fipronil, combined with our other findings, indicate that CYP6AS160 is responsible for detoxification of fipronil. Feeding insects dsRNA may be a general strategy to trigger RNA interference and may find applications in entomological research and in the control of insect pests in the field.

Mutagenicity Assessment to Pesticide Adjuvants of Toluene, Chloroform, and Trichloroethylene by Ames Test.

Pesticide adjuvants (PAs) denote the general term for auxiliaries in pesticide preparations except for the active components. Toluene, chloroform, and trichloroethylene are the three most commonly used PAs as organic solvents. The residues of the three chemicals in the process of production and application of pesticides may endanger the ecosystem. In the present study, the mutagenicity of toluene, chloroform, and trichloroethylene as well the mixture of the three chemicals was tested by the Salmonella typhimurium reverse mutation test (Ames test) with TA97, TA98, TA100, and TA102 strains in the system with and without rat liver microsomal preparations (S9). The four tester strains have been used for more than 40 years to detect mutagenic compounds in chemicals, cosmetics, and environmental samples. The mutagenicity was detected on tester strains in the separated experiment from the three chemicals. The addition of S9 decreased the mutation ratios of toluene to four strains, except for the TA100 strain, but increased the mutation ratios of chloroform to four strains except for the TA98 strain. Trichloroethylene caused positive mutagenicity to become negative on the TA102 strain. In the mixed experiment, positive effects were detected only on the TA102 strain in the absence of S9. The addition of S9 increased the mutagenicity except for the TA102 strain. The mixture of toluene, chloroform, and trichloroethylene showed antagonism in mutagenicity to tester strains, except for the TA102 strain without S9. However, the mixture showed a synergistic effect to tester strains after adding S9 except for the TA98 strain.

Morphological and Mechanistic Aspects of Thiourea-Induced Acute Lung Injury and Tolerance in the Rat.

Thiourea-based molecules cause pulmonary edema when administered to rats at relatively low doses. However, rats survive normally lethal doses after prior exposure to a lower, nonlethal dose; this phenomenon is known as tolerance. The present study investigated the morphological and functional aspects of acute lung injury (ALI) induced by methylphenylthiourea (MPTU) in the Wistar rat and the pulmonary response involved in prevention of the injury. We identified pulmonary endothelial cells as the main target of acute MPTU injury; they exhibited ultrastructural alterations that can result in increased vascular permeability. In tolerant rats, the lungs showed only transient endothelial changes, at 24-hour post dosing, and mild type II pneumocyte hyperplasia on day 7 post dosing. They exhibited glutathione levels similar to the controls and increased expression of flavin-containing monooxygenase 1 (FMO1), the enzyme responsible for bioactivation of small thioureas in the laboratory rat. Incubation of rat pulmonary microsomal preparations with MPTU inhibited FMO activity, indicating that tolerance is related to irreversible inhibition of FMOs. The rat model of thiourea-induced pulmonary toxicity and tolerance represents an interesting approach to investigate certain aspects of the pathogenesis of ALI and therapeutic approaches to lung diseases, such as acute respiratory distress syndrome.

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.).

Lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) is an evolutionarily conserved key enzyme in the Lands cycle that catalyzes acylation of lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), the main phospholipid in cellular membranes. In this study, three LPCAT genes from sunflower were identified and the corresponding proteins characterized. These HaLPCAT genes encoded functionally active enzymes that were able to complement a deficient yeast mutant. Moreover, enzymatic assays were carried out using microsomal preparations of the yeast cells. When acyl specificities were measured in the forward reaction, these enzymes exhibited a substrate preference for unsaturated acyl-CoAs, especially for linolenoyl-CoA, while in the reverse reaction, linoleoyl or linolenoyl acyl groups were transferred from PC to acyl-CoA to a similar extent. Expression levels of LPCAT genes were studied revealing distinct tissue-specific expression patterns. In summary, this study suggests that the combined forward and reverse reactions catalyzed by sunflower LPCATs facilitate acyl-exchange between the sn-2 position of PC and the acyl-CoA pool. Sunflower LPCATs displayed different characteristics, which could point to different functionalities, favoring the enrichment of seed triacylglycerols (TAGs) with polyunsaturated fatty acid (PUFA).

The Human UGT2B7 Nanodisc.

The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil and regulate the activity and clearance of scores of small molecule metabolites. Biochemical and biophysical UGT studies have been hampered by the inability to purify these membrane-bound proteins. Here, using cell-free expression and nanodisc technology, we assemble and purify to homogeneity the first UGT nanodisc-the human UGT2B7•nanodisc. The complex is readily isolated in milligram quantities. It is stable and its initial-rate parameters are identical within error to those associated with UGT2B7 in microsomal preparations (i.e., Supersomes). The high purity of the nanodisc preparation simplifies UGT assays, which allows complexities traditionally associated with microsomal assays (latency and the albumin effect) to be circumvented. Each nanodisc is shown to harbor a single UGT2B7 monomer. The methods described herein should be widely applicable to UGTs, and these findings are expected to set the stage for experimentalists to more freely explore the structure, function, and biology of this important area of phase II metabolism. SIGNIFICANCE STATEMENT: Lack of access to pure, catalytically competent human uridine diphosphate glucuronosyl-transferases (UGTs) has long been an impediment to biochemical and biophysical studies of this disease-relevant enzyme family. Here, we demonstrate this barrier can be removed using nanodisc technology-a human UGT2B7•nanodisc is assembled, purified to homogeneity, and shown to have activity comparable to microsomal UGT2B7.

Glucuronidation of Abiraterone and Its Pharmacologically Active Metabolites by UGT1A4, Influence of Polymorphic Variants and Their Potential as Inhibitors of Steroid Glucuronidation.

Abiraterone (Abi) acetate (AA) is a prodrug of Abi, a CYP17A1 inhibitor used to treat patients with advanced prostate cancer. Abi is a selective steroidal inhibitor that blocks the biosynthesis of androgens. It undergoes extensive biotransformation by steroid pathways, leading to the formation of pharmacologically active Δ4-Abi (D4A) and 5α-Abi. This study aimed to characterize the glucuronidation pathway of Abi and its two active metabolites. We show that Abi, its metabolites, and another steroidal inhibitor galeterone (Gal) undergo secondary metabolism to form glucuronides (G) in human liver microsomes with minor formation by intestine and kidney microsomal preparations. The potential clinical relevance of this pathway is supported by the detection by liquid chromatography-tandem mass spectrometry of Abi-G, D4A-G, and 5α-Abi-G in patients under AA therapy. A screening of UGT enzymes reveals that UGT1A4 is the main enzyme involved. This is supported by inhibition experiments using a selective UGT1A4 inhibitor hecogenin. A number of common and rare nonsynonymous variants significantly abrogate the UGT1A4-mediated formation of Abi-G, D4A-G, and 5α-Abi-G in vitro. We also identify Gal, Abi, and its metabolites as highly potent inhibitors of steroid inactivation by the UGT pathway with submicromolar inhibitor constant values. They reduce the glucuronidation of both the adrenal precursors and potent androgens in human liver, prostate cancer cells, and by recombinant UGTs involved in their inactivation. In conclusion, tested CYP17A1 inhibitors are metabolized through UGT1A4, and germline variations affecting this metabolic pathway may also influence drug metabolism. SIGNIFICANCE STATEMENT: The antiandrogen abiraterone (Abi) is a selective steroidal inhibitor of the cytochrome P450 17α-hydroxy/17,20-lyase, an enzyme involved in the biosynthesis of androgens. Abi is metabolized to pharmacologically active metabolites by steroidogenic enzymes. We demonstrate that Abi and its metabolites are glucuronidated in the liver and that their glucuronide derivatives are detected at variable levels in circulation of treated prostate cancer patients. UDP-glucuronosyltransferase (UGT)1A4 is the primary enzyme involved, and nonsynonymous germline variations affect this metabolic pathway in vitro, suggesting a potential influence of drug metabolism and action in patients. Their inhibitory effect on drug and steroid glucuronidation raises the possibility that these pharmacological compounds might affect the UGT-associated drug-metabolizing system and pre-receptor control of androgen metabolism in patients.

Crambe hispanica Subsp. abyssinica Diacylglycerol Acyltransferase Specificities Towards Diacylglycerols and Acyl-CoA Reveal Combinatorial Effects That Greatly Affect Enzymatic Activity and Specificity.

Crambe is an oil crop suitable for industrial purposes due to the high content of erucic acid (22:1) in the seed oil. The final acylation of diacylglycerols (DAG) with acyl-CoA in the production of triacylglycerols (oil) is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. We identified eight forms of DGATs in crambe and characterized them in microsomal preparations of yeast expressing the enzymes using various acyl-CoAs and both di-6:0-DAG and long-chain DAG species as acyl acceptors. All DGATs accepted 22:1-CoA when using di-6:0-DAG as acyl acceptor. When di-22:1-DAG was the acyl acceptor, the DGAT1 type of enzyme utilized 22:1-CoA at a much-reduced rate compared to assays with sn-1-22:1-sn-2-18:1(oleoyl)-DAG, the most frequently available DAG precursor in crambe seeds. None of the DGAT2 enzymes was able to acylate di-22:1-DAG. Our results indicate that formation of trierucin by crambe DGATs is a limiting step for further increasing the levels of 22:1 in the previously developed transgenic crambe lines due to their poor abilities to acylate di-22:1-DAG. We also show that the acyl-CoA specificities and the enzymatic activities are highly influenced by the fatty acid composition of the DAG acyl acceptor. This finding implies that the use of artificial acyl acceptors (e.g. di-6:0-DAG) may not always reflect the actual acyl-CoA specificities of DGATs in planta. The relevance of the here reported pronounced specificities for specific DAG species exerted by DGAT enzymes is discussed in the context of the findings of DAG pools of distinct catalytic origin in triacylglycerol biosynthesis in the seed oil.

Monitoring the Sphingolipid de novo Synthesis by Stable-Isotope Labeling and Liquid Chromatography-Mass Spectrometry.

Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C16 fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d3 and L-serine-d3. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B1). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates – naturally occurring or chemically tailored – as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.

11β-Hydroxysteroid dehydrogenases control access of 7β,27-dihydroxycholesterol to retinoid-related orphan receptor γ

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7β-hydroxycholesterol (7βOHC). We examined the role of 11β-HSDs in the enzymatic control of the intracellular availability of 7β,27-dihydroxycholesterol (7β27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11β-HSD1 and 11β-HSD2 to assess whether 7β27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7β27OHC and 7k27OHC to 11β-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7β27OHC by human 11β-HSD1 and the reverse oxidation reaction of 7β27OHC to 7k27OHC by human 11β-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11β-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7β27OHC are potent inhibitors of the 11β-HSD1-dependent oxoreduction of cortisone and the 11β-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11β-HSD1 and 11β-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11β-HSDs that warrants further investigation.

Liquid chromatography-tandem mass spectrometry metabolic profiling of nazartinib reveals the formation of unexpected reactive metabolites.

Nazartinib (EGF816, NZB) is a promising third-generation human epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. This novel irreversible mutant-selective EGFR inhibitor targets EGFR containing both the resistance mutation (T790M) and the activating mutations (L858R and Del19), while it does not affect wild-type EGFR. However, the metabolic pathway and bioactivation mechanisms of NZB are still unexplored. Thus, using liquid chromatography–tandem mass spectrometry, we screened for products of NZB metabolism formed in vitro by human liver microsomal preparations and investigated the formation of reactive intermediates using potassium cyanide as a nucleophile trap. Unexpectedly, the azepane ring was not bioactivated. Instead, the carbon atom between the aliphatic linear tertiary amine and electron-withdrawing system (butenoyl amide group) was bioactivated, generating iminium intermediates as reactive species. Six NZB phase I metabolites, formed by hydroxylation, oxidation and N-demethylation, were characterized. Moreover, two reactive iminium ions were characterized and their corresponding bioactivation mechanisms were proposed. Based on our results, we speculate that bioactivation of NZB can be blocked by small sterically hindering groups, isosteric replacement or a spacer. This approach might reduce the toxicity of NZB by avoiding the generation of reactive species.

New approaches to old problems: Removal of phospholipase A2 results in highly active microsomal membranes from the honey bee, Apis mellifera

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.

Highly Variable Expression of CYP1A1 in Human Liver and Impact on Pharmacokinetics of Riociguat and Granisetron in Humans.

Cytochrome P450s (CYPs), a superfamily of enzymes, are involved in the biotransformation of endogenous and xenobiotic chemicals and mainly responsible for the metabolic clearance of widely prescribed drugs. Out of the 57 human isoforms, only a few, most notably CYP3A4, are considered to be important in this process. CYP1A1, one of the three isoforms of the CYP1 family, is widely believed to play an important role in the metabolism and activation of numerous procarcinogens, e.g., polyaromatic hydrocarbons (PAHs) or aromatic amines. It is also known that CYP1A1 is highly inducible by endogenous and exogenous factors, e.g., PAHs. However, CYP1A1 has not been considered to play a significant role in the metabolic clearance of drugs, since this isoform has been detected only in extrahepatic tissues in small amounts. In contrast to conventional wisdom, we herein demonstrate the expression of CYP1A1 protein in human liver microsomal preparations. The expression levels of CYP1A1 were quantified by Western blot and LC/MS analyses and corresponded well with enzymatic activities of highly selective CYP1A1 reactions. In a panel of 29 individual liver microsomal preparations, highly variable and substantial expression levels (up to ∼10 pmol/mg) were measured. Together with the high selectivity and especially the high metabolic efficiency of CYP1A1 shown for granisetron and riociguat, it is demonstrated that CYP1A1 plays an important role in the metabolic clearance of these drugs and is responsible for the clinically observed interindividual variability in their pharmacokinetics. Therefore, the importance of CYP1A1 in drug discovery and development needs to be reconsidered.

Non‐Additive Effects of Changing the Composition of the Cytochrome P450 Ensemble in Human Liver Microsomes: the Focus on CYP2E1 and Alcohol‐Drug Interactions

Recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a straight correlation between the composition of human cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of physical interactions between multiple P450 species co‐localized in the microsomal membrane. Due to complex effects of these interactions any change in the content of a particular P450 enzyme may affect drug metabolism in a complex, hard‐to‐predict manner. Of particular practical importance are the functional effects of the changes in the composition of P450 ensemble induced by alcohol consumption. Although the multi‐fold increase in the content of cytochrome P450 2E1 (CYP2E1) in liver observed in both alcoholics and moderate alcohol consumers is well documented, the role of CYP2E1 in the instances of alcohol‐drug interactions is commonly considered insignificant due to a minor role of this enzyme in drug metabolism. However, the involvement of CYP2E1 in alcohol‐drug interactions may stretch far beyond its immediate effects on the metabolism of its substrates.The aim of this study is to probe the effect of increasing concentrations of CYP2E1 in HLM on the metabolism of drug substrates of other P450 species. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into HLM that allows creating the microsomal preparations with altered composition of the P450 ensemble. Incorporation of CYP2E1 into HLM results in an increase in the rate of CYP2E1‐dependent metabolism of chlorzoxazone, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Studies with a series of substrates of drug‐metabolizing P450 species showed that CYP2E1 incorporation also results in pronounced alterations in the metabolism of substrates of several other P450 enzymes. The most important effect is observed on CYP2C19. The changes in the effective KM and Vmax values with such probe substrates as 7‐etoxy‐4‐cyanocoumarin (CEC, the substrate of CYP2C19 and CYP1A2), 7‐dimethylamino‐4‐trifluoromethylcoumarin (coumarin‐152, the substrate of CYP2B6, CYP2C19 and CYP2C9) and N‐butyl 7‐methoxy‐4‐(aminomethyl)‐coumarin (BMAMC, the substrate of CYP1A2, CYP2C19 and CYP2D6) reveal a substantial decrease in the involvement of CYP2C19 in their metabolism. Furthermore, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5. These observations suggest that ethanol‐dependent induction CYP2E1 may be involved in pronounced pharmacokinetic interactions of alcohol with such drug substrates of CYP2C19 and CYP3A enzymes as benzodiazepines (diazepam), tricyclic antidepressants (amitriptylline, imipramine) and anticonvulsants (phenytoin, carbamazepine).Support or Funding InformationThis research was supported by grant R21‐AA024548 from NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.