Microsomal Fraction Research Articles

Prohibitin 2 is Involved in Parkin-Mediated Mitophagy in Urothelial Cells of Cattle Infected with Bovine Papillomavirus.

Prohibitin 2 (PHB2), an inner mitochondrial membrane (IMM) protein, has recently been identified as a novel receptor involved in parkin-mediated mitophagy. In the field of veterinary medicine, the role of PHB2 in parkin-mediated mitophagy was described, for the first time, in urothelial cells of cattle, naturally infected with bovine papillomavirus (BPV). The BPV2 and BPV13 E5 oncoprotein, responsible for abortive infections in urothelial cells, was detected by RT-PCR. Severe ultrastructural abnormalities of the inner mitochondrial membrane were detected using transmission electron microscopy. PHB2 formed a functional complex with PHB1. PHB2 was significantly overexpressed in mitochondrial fractions from urothelial mucosa samples taken from cattle harbouring BPV infection. PHB2 overexpression could be attributed to mitochondrial dysfunction, as its expression levels in the cytosolic, microsomal, and nuclear fractions were seen to be unmodified. Immunoprecipitation studies revealed the interaction between PHB2 and phosphorylated forms of both PINK1 and parkin. Furthermore, PHB2 interacted with LC3-II, a marker of autophagosomal membranes and autophagy receptors, such as p62 and optineurin. PHB2 was shown to interact with transcription factor EB (TFEB), which is activated following parkin-mediated mitophagy, and embryonic stem cell-expressed Ras (ERAS), a constitutive protein coded by ERas. Western blot analysis revealed a significant overexpression of unphosphorylated TFEB in mitochondrial and nuclear fractions from urothelial mucosa samples from cattle suffering from BPV infection. Finally, PHB2 interacted with ERAS, believed to be involved in mitophagosome maturation. Taken together, the molecular and ultrastructural findings of this study suggested that BPV infection is responsible for parkin-dependent mitophagy, in the pathway of which PHB2 plays a crucial role.

A comparative study on the in vitro biotransformation of medicagenic acid using human liver microsomes and S9 fractions

Many natural products are prodrugs which are biotransformed and activated after oral administration. The investigation of gastrointestinal and hepatic biotransformation can be facilitated by in vitro screening methods. This study compares two widely used in vitro models for hepatic biotransformation: 1) human S9 fractions and 2) human liver microsomes and cytosolic fractions in a two-step sequence, with the purpose of identifying differences in the biotransformation of medicagenic acid, the putative precursor of active metabolites, responsible for the medicinal effects of the herb Herniaria hirsuta. The combination of liquid chromatography coupled to high-resolution mass spectrometry with subsequent suspect and non-target data analysis allowed the identification of thirteen biotransformation products, four of which are reported here for the first time. Eight biotransformation products resulting from oxidative Phase I reactions were identified. Phase II conjugation reactions resulted in the formation of three glucuronidated and two sulfated biotransformation products.No major differences could be observed between incubations with human liver S9 or when utilizing human microsomal and cytosolic fractions. Apart from two metabolites, both methods rendered the same qualitative metabolic profile, with minor quantitative differences. As a result, both protocols applied in this study can be used to study in vitro human liver biotransformation reactions.

Elucidating pesticide sensitivity of two endogeic earthworm species through the interplay between esterases and glutathione S-transferases

Earthworms are common organisms in soil toxicity-testing framework, and endogeic species are currently recommended due to their ecological role in agroecosystem. However, little is known on their pesticide metabolic capacities. We firstly compared the baseline activity of B-esterases and glutathione-S-transferase in Allolobophora chlorotica and Aporrectodea caliginosa. Secondly, vulnerability of these species to pesticide exposure was assessed by in vitro trials using the organophosphate (OP) chlorpyrifos-ethyl-oxon (CPOx) and ethyl-paraoxon (POx), and by short-term (7 days) in vivo metabolic responses in soil contaminated with pesticides. Among B-esterases, acetylcholinesterase (AChE) activity was abundant in the microsomal fraction (80% and 70% of total activity for A. caliginosa and A. chlorotica, respectively). Carboxylesterase (CbE) activities were measured using three substrates to examine species differences in isoenzyme and sensitivity to both in vitro and in vivo exposure. CbEs were mainly found in the cytosolic fraction (80% and 60% for A. caliginosa and A. chlorotica respectively). GST was exclusively found in the soluble fraction for both species. Both OPs inhibited B-esterases in a concentration-dependent manner. In vitro trials revealed a pesticide-specific response, being A. chlorotica AChE more sensitive to CPOx compared to POx. CbE activity was inhibited at the same extent in both species. The 7-d exposure showed A. chlorotica less sensitive to both OPs, which contrasted with outcomes from in vitro experiments. This non-related functional between both approaches for assessing pesticide toxicity suggests that other mechanisms linked with in vivo OP bioactivation and excretion could have a significant role in the OP toxicity in endogeic earthworms.

The relationship between the content of the potential psychopharmacological agent cyclo-L-prolylglycine in the brain of experimental animals and its antihypoxic effect

In the study of strutural and intracellular distribution of cyclo-t-prolylglycine (CPG) in brain rats in experiments on mongrel white male rats, it was found that CPG determined more in the hippocampus and cerebral cortex with predominant localization in the nuclear fraction of the rat brain, in the mitochondrial and microsomal fractions CPG is distributed approximately equivalently with a small quantitative predominance in the microsomal fraction. In an experimenlt using inbred mice of 2 lines: C57Black/6 and BALB/c, differing in behavior, opposite emotional response to stress, hypoxic influence. It was shown that the content CPG mice of the C57black/6 line are 39 % larger than in the brains of mice of the stress-unstable BALB/c line, which is probably due to differences in the manifestation of its it was shown that the content of CPG in the brain of stress-resistant mice C57Black/6 more than 39 % compared with the content of GPG in brain of mice BALB/c and the relationship between the quantitative content of CPG in the brain of these experimental animals and the manifestation of its antihypoxic effect was established.

Etomidate is devoid of genotoxicty and mutagenicity in human lymphocytes and in the Salmonella typhimurium/microsomal activation test

No carcinogenesis or mutagenesis studies have been carried out with etomidate. The current study showed that etomidate has weak cytotoxic potential after 48 h exposure in human lymphocytes and has no hemolytic activity. The weak cytotoxicity seems to be related with redox imbalance of etomidate (40.9 and 81.9 μM) treated lymphocytes. At both etomidate concentrations, a slight decrease of the levels of GSH intracellular content and a significant increase in the amount of carbonylated proteins were observed after 48 h. The contribution of oxidative stress to genetic toxicity was only perceived when the enzyme Fpg was applied in the comet assay. Etomidate (40.9 and 81.9 μM) is a weak generator of oxidative DNA damage in lymphocytes. These damages to DNA probably were repaired, since no DNA strand breaks were detected in the standard alkaline comet assay (in the presence or absence of hepatic S9 microsomal fraction) without Fpg. Also, no micronucleated lymphocytes or carrying chromosomal aberrations were observed. Finally, etomidate (2046.8 and 4093.5 μM) was not mutagenic in the Salmonella/microsome mutagenicity assay, which used four Salmonella typhimurium strains (TA97a, TA98, TA100, and TA102) to detect frameshift and base-substitution mutations. In summary, etomidate is a weak oxidative DNA damaging anesthetic and is devoid of mutagenic properties in eukaryotic and prokaryotic models.

Human phase I in vitro liver metabolism of two bisphenolic diglycidyl ethers BADGE and BFDGE

Root canal sealers are commonly used to endodontically treat teeth with periapical infections. Some root canal sealers based on epoxy resin contain bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE). The presence of these chemicals is of concern due to the close contact to the blood stream at the apex and the long setting times of up to 24 h. These chemicals, or any of their degradation products or metabolites, can then exert their toxic effects before being excreted. This study aimed to identify the phase I in vitro biotransformation products of BADGE and BFDGE using human liver microsomes. During incubation with microsomal fractions, the epoxides were rapidly hydrolysed in a NADPH independent manner resulting in the formation of BADGE.2H2O and BFDGE.2H2O. Further, oxidative reactions, such as hydroxylation and carboxylation, generated other BADGE metabolites, such as BADGE.2H2O−OH and BADGE.H2O.COOH, respectively. For BFDGE, further oxidation of BFDGE.2H2O led to the newly reported carboxylic acid, BFDGE.H2O.COOH. In total, three specific metabolites have been identified which can serve in future human biomonitoring studies of BADGE and BFDGE.

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.

Unexpected rapid aerobic transformation of 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical by cytochrome P450 in the presence of NADPH: Evidence against a simple reduction of the nitroxide moiety to the hydroxylamine

Aminoxyl radicals (nitroxides) are a class of compounds with important biomedical applications, serving as antioxidants, spin labels for proteins, spin probes of oximetry, pH, or redox status in electron paramagnetic resonance (EPR), or as contrast agents in magnetic resonance imaging (MRI). However, the fast reduction of the radical moiety in common tetramethyl-substituted cyclic nitroxides within cells, yielding diamagnetic hydroxylamines, limits their use in spectroscopic and imaging studies. In vivo half-lives of commonly used tetramethyl-substituted nitroxides span no more than a few minutes. Therefore, synthetic efforts have focused on enhancing the nitroxide stability towards reduction by varying the electronic and steric environment of the radical. Tetraethyl-substitution at alpha position to the aminoxyl function proved efficient in vitro against reduction by ascorbate or cytosolic extracts. Moreover, 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical (TEEPONE) was used successfully for tridimensional EPR and MRI in vivo imaging of mouse head, with a reported half-life of over 80 min. We decided to investigate the stability of tetraethyl-substituted piperidine nitroxides in the presence of hepatic microsomal fractions, since no detailed study of their “metabolic stability” at the molecular level had been reported despite examples of the use of these nitroxides in vivo. In this context, the rapid aerobic transformation of TEEPONE observed in the presence of rat liver microsomal fractions and NADPH was unexpected. Combining EPR, HPLC-HRMS, and DFT studies on a series of piperidine nitroxides — TEEPONE, 4-oxo-2,2,6,6-tetramethyl(piperidin-1-yloxyl) (TEMPONE), and 2,2,6,6-tetraethyl-4-hydroxy(piperidin-1-yloxyl) (TEEPOL), we propose that the rapid loss in paramagnetic character of TEEPONE is not due to reduction to hydroxylamine but is a consequence of carbon backbone modification initiated by hydrogen radical abstraction in alpha position to the carbonyl by the P450-Fe(V)=O species. Besides, hydrogen radical abstraction by P450 on ethyl substituents, leading to dehydrogenation or hydroxylation products, leaves the aminoxyl function intact but could alter the linewidth of the EPR signal and thus interfere with methods relying on measurement of this parameter (EPR oximetry).

Biomarker considerations in monitoring petrogenic pollution using the mussel Mytilus galloprovincialis.

Mussels are worldwide bioindicators in pollution monitoring since they fulfil the requirements for being good sentinels. However, some methodological concerns arise in the use of particular biomarkers, particularly those displaying low enzymatic rates and/or limited responsiveness to chemicals and biological-related variability. In the present study, the suitability of oxidative stress and detoxification parameters when using mussels as sentinels of polycyclic aromatic hydrocarbon (PAH) pollution is addressed. Present results show that the S9 subcellular fraction of the digestive gland in mussels is an adequate and convenient matrix where to measure most pollution-related biomarkers. Furthermore, this work constitutes the first evidence of the potential suitability of using particular carboxylesterase (CE) activities in determining PAHs exposure in mussels. This fact could imply the replacement of more controversial cytochrome P450 components (phase I oxidation), which are only measurable in microsomal fractions, by CEs (measured in S9 fractions) as good alternatives for phase I reactions in PAH-exposed mussels. Some methodological considerations, such as the need of including commercial purified proteins in biomarker determinations for quality assurance, are evaluated.

Direct Comparison of Total Clearance Prediction: Computational Machine Learning Model versus Bottom-Up Approach Using In Vitro Assay

The in vitro-in vivo extrapolation (IVIVE) approach for predicting total plasma clearance (CLtot) has been widely used to rank order compounds early in discovery. More recently, a computational machine learning approach utilizing physicochemical descriptors and fingerprints calculated from chemical structure information has emerged, enabling virtual predictions even earlier in discovery. Previously, this approach focused more on in vitro intrinsic clearance (CLint) prediction. Herein, we directly compare these two approaches for predicting CLtot in rats. A structurally diverse set of 1114 compounds with known in vivo CLtot, in vitro CLint, and plasma protein binding was used as the basis for this evaluation. The machine learning models were assessed by validation approaches using the time- and cluster-split training and test sets, and five-fold cross validation. Assessed by five-fold validation, the random forest regression (RF) and radial basis function (RBF) models demonstrated better prediction performance in eight attempted machine learning models. The CLtot values predicted by the RF and RBF models were within two-fold of the observed values for 67.7 and 71.9% of cluster-split test set compounds, respectively, while the predictivity was worse in the time-split dataset. The predictivity of both models tended to be improved by incorporating in vitro parameters, unbound fraction in plasma (fu,p), and CLint. CLtot prediction utilizing in vitro CLint and the well-stirred model, correcting for the fraction unbound in blood, was substantially worse compared to machine learning approaches for the same cluster-split test set. The reason that CLtot is underestimated by IVIVE is not fully explained by considering the calculated microsomal unbound fraction (cfu,mic), extended clearance classification system (ECCS), and omitting high clearance compounds in excess of hepatic blood flow. The analysis suggests that in silico machine learning models may have the power to reduce reliance on or replace in vitro and in vivo studies for chemical structure optimization in early drug discovery.

Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004mmol L-1) and high (VM = 153.4 ± 7.7nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5nmolPimin-1mg-1 protein, K0.5 = 0.16 ± 0.01mmol L-1), K+ (VM = 485.3 ± 24.3nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05mmolL-1), Na+ (VM = 425.0 ± 23.4nmol Pi min-1mg-1 protein, K0.5 = 5.1 ± 0.3mmol L-1) and NH4+ (VM = 497.9 ± 24.9nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5mmolL-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8µmolL-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Metabolism, CB1 cannabinoid receptor binding and in vivo activity of synthetic cannabinoid 5F-AKB48: Implications for toxicity

AKB48 and its fluorinated derivative 5F-AKB48 are synthetic cannabinoids (SCs) which have caused hospitalizations and deaths in human users. Abuse of SCs is dangerous because users may mistake them for natural cannabis, which is generally considered to be unlikely to elicit adverse effects. The present studies were designed to investigate the in vitro oxidative metabolism of 5F-AKB48 by human microsomal fractions from different organs and sexes as well as recombinant human cytochrome P450s (P450s). Mass spectrometry data tentatively provides evidence for the existence of mono-, di-, and trihydroxylated metabolites in a successive metabolism. Experiments utilizing P450s revealed that the most active enzymes (CYP2D6, CYP2J2, CYP3A4, and CYP3A5) effectively produced mono- and dihydroxylated metabolites, while CYP3A4/5 also produced significant amounts of the trihydroxylated metabolite. Moreover, although the affinity and potency of Phase I metabolite 4OH-5F-AKB48 is reduced when compared to that of the parent drug, this metabolite nevertheless retains similar high affinity for CB1 receptors, and greater efficacy for G protein activation, when compared to THC. Finally, 5F-AKB48 produced time- and dose-dependent cannabimimetic effects in mice which were more potent, but shorter acting, than those of Δ9-THC, and were attenuated by prior treatment with the CB1 antagonist rimonabant. Based on our data, we hypothesize that while many cases of toxicity result from genetic mutations, which can lead to a decrease or even absence of activity for Phase I drug-metabolizing enzymes, other P450s could potentially increase their role in the metabolism of these SCs. Because many metabolites of SCs remain biologically active, they could contribute to the deleterious effects of these substances.

The Curcumin Derivative, H10, Suppresses Hormone-Dependent Prostate Cancer by Inhibiting 17β-Hydroxysteroid Dehydrogenase Type 3.

The 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) enzyme is a potential therapeutic target for hormone-dependent prostate cancer, as it is the key enzyme in the last step of testosterone (T) biosynthesis. A curcumin analog, H10, was optimized for inhibiting T production in LC540 cells that stably overexpressed 17β-HSD3 enzyme (LC540 [17β-HSD3]) (P < 0.01), without affecting progesterone (P) synthesis. H10 downregulated the production of T in the microsomal fraction of rat testes containing the 17β-HSD3 enzyme from 100 to 78.41 ± 7.41%, 51.86 ± 10.03%, and 45.14 ± 8.49% at doses of 10, 20, and 40 μM, respectively. There were no significant differences among the groups with respect to the protein expression levels of 17β-HSD3, 3βHSD1, CYP17a1, CYP11a1, and STAR, which participate in 17β-HSD3-mediated conversion of androgens to T (P > 0.05). This indicated that H10 only inhibited the enzymatic activity of 17β-HSD3 in vitro. Furthermore, H10 inhibited the adione-stimulated growth of xenografts established from LNCaP cells in nude mice in vivo. We conclude that H10 could serve as an effective inhibitor of 17β-HSD3, which in turn would inhibit the biosynthesis of androgens and progression of prostate cancer.

Comparative liver metabolic enzyme activity of cytochrome P450 and glutathione-S-transferase in crocodile (Crocodylus siamensis) and livestock

This study aimed to compare the metabolism of detoxification liver enzymes activity of CYP1A2, CYP2E1, the GST enzymes activity for class pi, mu, alfa, and the universal GST between crocodile and livestock, plus the relationship of these two enzyme activities in the crocodile. Interestingly, the CYP1A2 and CYP2E1 were existent in the freshwater crocodile livers microsomal enzyme activities, and these two enzymes could be determined in both pig and chicken. Also, the universal GST, alpha, mu, and pi class of phase II were presented in the crocodile liver cytosolic fractions, and these three enzymes could also be found in pig and chicken. The kinetic activity (Vmax/Km ratio) of GST activity towards CDNB was higher in the crocodile than chicken, and pig; 80.02, 57.80 and 45.25 ml/min/mg protein, respectively. The Vmax/Km ratio GST activity towards t-PBO was highest in crocodile. In contrast, the GST activity towards EA was highest in pig, chicken and crocodile in this order. However, the Vmax/Km ratio GST activity towards CHP was very low in all species. The crocodile liver microsome fraction could metabolites AFB1 to AFM1, suggesting that the CYP1A2 activity was actively presented. The kinetic enzyme activity of crocodile liver towards CDNB revealed the highest velocity compared with other livestock species; this indicates that crocodile liver enzyme activities were very active for the detoxification function towards all xenobiotic, especially for GST activity, towards carcinogenic agent when compared with other livestock.

Seasonal variability of chemical composition and mutagenic effect of organic PM2.5 pollutants collected in the urban area of Wrocław (Poland)

The objective of the study was the assessment of the mutagenicity of chemical pollutants adsorbed on suspended particulate matter with aerodynamic diameter < 2.5 μm (PM2.5) in the four seasons. Samples were collected from the urban agglomeration of Wroclaw, Poland and evaluated for mutagenicity using two Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation with microsomal fraction S9.The work covered sampling of suspended dusts in four seasons: summer, spring, autumn and winter. The dust samples were collected on glass filters using air aspirator and the organic matter of PM2.5 was extracted using Soxhlet extractor. The levels of polycyclic aromatic hydrocarbon compounds (PAH), nitro-PAH and dinitro-PAH were determined in the extract.Variable degree of air pollution with mutagenic substances was determined at the selected study site. A greater, negative effect of chemical compounds on DNA was determined in dust samples collected in the autumn-winter season in comparison to samples collected in the spring-summer season. In the majority of tests, higher mutagenicity was obtained in analyses conducted on total extracts in comparison to tests conducted in the presence of PAH pollutant fractions. The obtained mutagenic ratio values pointed to the presence of chemical compounds with a character of both promutagens and direct mutagens.Samples collected in the autumn-winter season were observed to have a higher diversity of organic substances absorbed on PM2.5 dusts. Particular samples differed in the total content and percent contribution of particular PAHs, nitro-PAHs, and other organic compounds. In addition, the identified substances included compounds belonging to different chemical classes: aliphatic compounds, cycloalkanes, mono- and bicycling arenes, polycyclic arenes, compounds containing oxygen, nitrogen, and sulphur.

18F]Atorvastatin: synthesis of a potential molecular imaging tool for the assessment of statin-related mechanisms of action

BackgroundStatins are lipid-lowering agents that inhibit cholesterol synthesis and are clinically used in the primary and secondary prevention of cardiovascular diseases. However, a considerable group of patients does not respond to statin treatment, and the reason for this is still not completely understood. [18F]Atorvastatin, the 18F-labeled version of one of the most widely prescribed statins, may be a useful tool for statin-related research.Results[18F]Atorvastatin was synthesized via an optimized ruthenium-mediated late-stage 18F-deoxyfluorination. The defluoro-hydroxy precursor was produced via Paal-Knorr pyrrole synthesis and was followed by coordination of the phenol to a ruthenium complex, affording the labeling precursor in approximately 10% overall yield. Optimization and automation of the labeling procedure reliably yielded an injectable solution of [18F]atorvastatin in 19% ± 6% (d.c.) with a molar activity of 65 ± 32 GBq·μmol−1. Incubation of [18F]atorvastatin in human serum did not lead to decomposition. Furthermore, we have shown the ability of [18F]atorvastatin to cross the hepatic cell membrane to the cytosolic and microsomal fractions where HMG-CoA reductase is known to be highly expressed. Blocking assays using rat liver sections confirmed the specific binding to HMG-CoA reductase. Autoradiography on rat aorta stimulated to develop atherosclerotic plaques revealed that [18F]atorvastatin significantly accumulates in this tissue when compared to the healthy model.ConclusionsThe improved ruthenium-mediated 18F-deoxyfluorination procedure overcomes previous hurdles such as the addition of salt additives, the drying steps, or the use of different solvent mixtures at different phases of the process, which increases its practical use, and may allow faster translation to clinical settings. Based on tissue uptake evaluations, [18F]atorvastatin showed the potential to be used as a tool for the understanding of the mechanism of action of statins. Further knowledge of the in vivo biodistribution of [18F]atorvastatin may help to better understand the origin of off-target effects and potentially allow to distinguish between statin-resistant and non-resistant patients.

Predicting liver cytosol stability of small molecules

Over the last few decades, chemists have become skilled at designing compounds that avoid cytochrome P (CYP) 450 mediated metabolism. Typical screening assays are performed in liver microsomal fractions and it is possible to overlook the contribution of cytosolic enzymes until much later in the drug discovery process. Few data exist on cytosolic enzyme-mediated metabolism and no reliable tools are available to chemists to help design away from such liabilities. In this study, we screened 1450 compounds for liver cytosol-mediated metabolic stability and extracted transformation rules that might help medicinal chemists in optimizing compounds with these liabilities. In vitro half-life data were collected by performing in-house experiments in mouse (CD-1 male) and human (mixed gender) cytosol fractions. Matched molecular pairs analysis was performed in conjunction with qualitative-structure activity relationship modeling to identify chemical structure transformations affecting cytosolic stability. The transformation rules were prospectively validated on the test set. In addition, selected rules were validated on a diverse chemical library and the resulting pairs were experimentally tested to confirm whether the identified transformations could be generalized. The validation results, comprising nearly 250 library compounds and corresponding half-life data, are made publicly available. The datasets were also used to generate in silico classification models, based on different molecular descriptors and machine learning methods, to predict cytosol-mediated liabilities. To the best of our knowledge, this is the first systematic in silico effort to address cytosolic enzyme-mediated liabilities.

Assessment of CYP2A6 and CYP2C19 Genetic Variation on Cotinine‐N‐Oxide Formation and their Potential Effect on the Nicotine Metabolic Ratio (NMR)

SignificanceThe nicotine metabolic ratio (NMR) of 3‐hydroxycotinine (3HC)/cotinine in smokers is a determinant of CYP2A6 activity and is widely used in clinical practice to help select smoking cessation pharmacotherapy. The major mode of metabolism of nicotine is via the formation of cotinine and subsequently to 3HC by the enzyme CYP2A6. Cotinine is also metabolized to cotinine‐N‐glucuronide and to cotinine‐N‐oxide (COX), accounting for ~7 and 5% of urinary nicotine metabolites, respectively, in smokers. The goal of the present study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway.MethodsWe used specific inhibitors of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in cotinine‐N‐oxidation reactions using pooled human liver microsomes (HLM). COX formation was monitored by UPLC‐MS/MS, and enzyme kinetic analysis was performed using microsomal fractions from CYP‐overexpressing HEK293 cell lines. Additionally, genotype‐phenotype analysis was performed for a panel of 113 HLM specimens.ResultsInhibition of COX formation was only observed when using inhibitors of CYPs 2A6, 2B6, 2C19, 2E1, and 3A4. CYPs 2A6 and 2C19 exhibited similar oxidation activity against cotinine with Vmax/KM values of 4.4, and 4.2 nL•min−1•mg−1, respectively; the Vmax/KM value for the 2B6 isoform was of 2.2 nL•min−1•mg−1. The activity observed for both CYPs 2E1 and 3A4 were low, with KM’s greater than 10 mM. We measured the levels of COX formation in 113 HLM specimens and genotyped variants in CYPs 2A6 (*2, *9, and *14), 2B6 (*2, *5, *9, rs4803419 and rs8109525) and 2C19 (*2 and *17) using qPCR. An association between decreased levels of COX formation and CYP variants in the 2C19 (up to 35% reduction) and 2A6 (up to 38% reduction) isoforms was observed. No statistically significant correlation was observed with variants in the 2B6 isoform.ConclusionsThese results demonstrate that genetic variants in CYPs 2A6 and 2C19 are associated with decreased COX formation, potentially affecting the levels of free cotinine and consequently the calculated NMR in smokers and clinical practices surrounding smoking cessation therapies.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program and CONACyT I2T2 (to Y. Perez‐Paramo), Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)

Role of Sucrose Synthase in the Metabolism of Indican

Indigo,a blue dye, is obtained from the cultivated plant species Polygonum tinctorium. It is derived from indican (indoxyl β‐D‐glucoside), one of the secondary metabolites stored in the vacuole of the leaf cells. In the cells, indican is biosynthesized by the catalysis of indoxyl b‐D‐glucoside synthase (IGS) that uses indoxyl and UDP‐glucose as its substrates. Indoxyl is possibly synthesized from indole by the catalysis of a monooxygenase, such as cytochrome P450 (CYP), whereas, sucrose synthase (SUS) or UDP‐glucose pyrophosphatase catalyzes the synthesis of UDP‐glucose. In the metabolism of indican, except IGS, the related proteins have not been identified to date; therefore, this study aimed to identify the specific proteins involved in the metabolism of the compound indican. Indoxyl is usually a very unstable substrate and is easily oxidized to indigo; due to its instability, it must be allowed to complete its indican synthesis reaction as fast as possible. In the plant secondary metabolism, the ER membrane‐associated CYPs that hydroxylate metabolites are well known. Furthermore, these CYPs may function as a scaffold for a temporary complex, known as metabolon, on the ER membrane. We assumed that the IGS also associates with some ER membrane proteins, because a part of IGS localizes on the microsome. We then performed protein–protein interaction analysis (coimmunoprecipitation and pull‐down assay) and detected some candidate proteins. Some proteins including CYPs and SUS were identified by the MS/MS analysis. We then examined the involvement of SUS in the metabolism of indican by performing protein–protein interaction analysis and cell fractionation using recombinant SUS.The full‐length cDNA of SUS gene obtained using the RACE‐PCR encodes a soluble cytosol protein composed of 806 amino acids. The recombinant SUS expressed in Escherichia coli was used to perform the experiments, as shown below. Firstly, we examined the intracellular localization of SUS using the cytosol and microsomal fractions prepared from the crude extract of leaf cells. Similar to IGS, SUS was also detected in both the fractions. The interaction between IGS and SUS was examined by coimmunoprecipitation using specific antibodies. We found that both proteins interacted with one another, suggesting that SUS might be a supply source for UDP‐glucose in the reaction involving the synthesis of indican. We are going to further discuss the data required to elucidate the entire indican metabolic pathway and to confirm the presence of its metabolon.

THE EFFECT OF VITAMIN E ON THE BIOCHEMICAL PARAMETERS IN THE EXPERIMENT

The effect of vitamin E on rat liver cytochrome P-450 in experimental acute pancreatitis (AP) was studied. The animals were divided into 4 groups. The obtained data were compared with the indicators of the 1–st group (intact). During the experiment, the development of AP showed a decrease in the content of cytochrome P450 in the microsomal fraction. The administration of vitamin E to animals of the 4th group led to an increase in the content of cytochrome P-450, strengthening the protection of the liver, the abolition of inhibition of the monooxygenase system of the liver.