CYP Activity Research Articles

Chronic exposure to acephate triggers ROS-mediated injuries at organismal and sub-organismal levels of Drosophila melanogaster.

The present study demonstrates ROS-mediated organismal and sub-organismal injuries in Drosophila melanogaster following chronic acephate exposure. Larvae and adults of Drosophila were reared on food supplemented with sub-lethal concentrations (1-6 μg mL-1) of acephate (LC50 8.71 μg mL-1). The longevity of the treated adults was reduced to half at 6 μg mL-1 exposure along with declined neuromuscular coordination and physical activities. Apparent developmental defects in the compound eyes were confirmed through the detection of apoptotic lesions in larval eye imaginal discs. The larval gut manifested tissue damage at various sites. Neural and fat cell viability was reduced by ∼1.89- and ∼3.38-fold at 6 μg mL-1 acephate treatment, respectively. A significant reduction in hemocyte viability confirmed the immunotoxic potential of acephate. Nearly 1-3-fold enhancement in the expression of OS markers (MDA, protein carbonyl contents, SOD, catalase and HSP70) in the treated larvae served as evidence of ROS production. The post-treatment increase in CYP450 and GST activities reflects the 'switch-on' states of the phase-I and phase-II detoxification mechanism. The genotoxic potential of acephate was confirmed through alkaline single cell gel electrophoresis. Thus, the findings of the present study validate the fact that besides traditional cholinesterase inhibition, chronic sub-lethal exposure to acephate potentially induces ROS-mediated toxic responses in Drosophila.

The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes

Tamoxifen, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from (E)- and (Z)-metabolite E by CYP2B6 and CYP2C19, respectively. Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug–drug-interactions.

Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction

Recently, it has been reported that human hepatocyte-like cells can be generated from fibroblasts by direct reprogramming technology. However, the conversion efficiency of human induced hepatocyte-like cells (hiHeps) is not high enough. In addition, comparative analysis with the existing models of hepatocytes, such as human iPS cell-derived hepatocyte-like cells and primary human hepatocytes, has not been sufficiently carried out. In this study, we screened hepatic transcription factors for efficient direct hepatic reprogramming and compared hepatic functions between hiHeps and other existing hepatocyte models. We found that human fibroblasts were efficiently converted into hiHeps by using a combination of ATF5, PROX1, FOXA2, FOXA3, and HNF4A (albumin+/alpha-1 antitrypsin+ cells = 27%, asialoglycoprotein receptor 1+ cells = 22%). The CYP expression levels and CYP activities in hiHeps were higher than those in human iPS cell-derived hepatocyte-like cells, but lower than those in short-term (4 hr) cultured primary human hepatocytes and primary human hepatocytes collected immediately after thawing. These results suggested that functional hiHeps could be efficiently generated by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction. We believe that hiHeps generated by our method will be useful for the drug-discovery activities such as hepatotoxicity screening and drug metabolism tests.

Herba Gelsemii elegantis is detoxified by ramulus et folium Mussaendae pubescentis extract by modulating hepatic cytochrome P450 and glutathione S-transferase enzymes in rats.

Herba Gelsemii elegantis (GE) has been frequently used as a Chinese folk medicine but has high acute toxicity. In Traditional Chinese Medicine, it may be detoxified by Ramulus et Folium Mussaendae pubescentis (MP), but the detoxification mechanism has remained elusive. The present study aimed to evaluate the detoxification mechanisms by which MP modulates the effect of GE in rats, including the inhibition of hepatic cytochrome P (CYP)450 and glutathione S-transferase (GST) enzymes. Male Sprague Dawley rats were orally administered GE at three doses (0.36, 0.43 or 0.54 g/kg) alone and, at the highest dose, in combination with MP (21.6 g/kg) every day for 7 consecutive days. The control group of animals received the same volume of saline. The mRNA and protein expression of hepatic CYPs representative of two subfamilies (CYP2E1 and CYP1A2) were separately assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), western blot and immunohistochemistry assays. The mRNA and protein expression as well as enzyme activity of hepatic GST were assessed by RT-qPCR, western blot and colorimetric assays, respectively. The results indicated that GE significantly inhibited CYP2E1 mRNA and protein expression in a dose-dependent manner. Co-administration of MP increased CYP2E1 mRNA and protein expression as compared with the high GE dose alone. Cells expressing CYP2E1, located around the hepatic vascular plexus under a clear background, were identified by immunohistochemical staining. The results for CYP1A2 were similar to those for CYP2E1. At all concentrations used, GE significantly inhibited GST mu 1 (GSTm1) mRNA and protein expression in a dose-dependent manner, as compared with the control. Combination of GE and MP increased the mRNA and protein expression of GSTm1 as compared with the high dose of GE. However, the differences in GST-pi mRNA and protein expression between the GE and GE + MP groups were not significant. Of note, rats co-treated with MP were significantly protected from the decrease in GST activity produced by GE. The present study indicated that co-administration of GE and MP upregulated the activities of CYP450 and GST enzymes when compared with GE alone. This modulation may explain for the effect of MP in reducing the acute toxicity of GE.

Mechanistic Understanding of the Synergistic Potential of Azole Fungicides in the Aquatic Invertebrate Gammarus pulex.

Azole fungicides are known inhibitors of the important enzyme class cytochrome P450 monooxygenases (CYPs), thereby influencing the detoxification of co-occurring substances via biotransformation. This synergism in mixtures containing an azole has mostly been studied by effect measurements, while the underlying mechanism has been less well investigated. In this study, six azole fungicides (cyproconazole, epoxiconazole, ketoconazole, prochloraz, propiconazole, and tebuconazole) were selected to investigate their synergistic potential and their CYP inhibition strength in the aquatic invertebrate Gammarus pulex. The strobilurin fungicide azoxystrobin was chosen as co-occurring substrate, and the synergistic potential was measured in terms of internal concentrations of azoxystrobin and associated biotransformation products (BTPs). Azoxystrobin is biotransformed by various reactions, and 18 BTPs were identified. By measuring internal concentrations of azoxystrobin and its BTPs with high-resolution tandem mass spectrometry in the presence and absence of azole fungicides followed by toxicokinetic modeling, we showed that the inhibition of CYP-catalyzed biotransformation reactions indeed played a role for the observed synergism. However, synergism was only observed for prochloraz at environmentally realistic concentrations. Increased uptake rate constants, an increase in the total internal concentration of azoxystrobin and its BTPs, in vivo assays for measuring CYP activities, and G. pulex video-tracking suggested that the 2-fold increase in bioaccumulation, and, thereby, the raised toxicity of azoxystrobin in the presence of prochloraz is not only caused by inhibited biotransformation but even more by increased azoxystrobin uptake induced by hyperactivity.

Lipid metabolism and pro-oxidant/antioxidant balance of Halamphora oceanica from the Gulf of Mexico exposed to water accommodated fraction of Maya crude oil

Diatoms play key roles in primary production and carbon fixation at a global scale and in some cases these species live on marine ecosystems impacted by crude oil (CO) spills. Halamphora oceanica, a new diatom species from the Southwest of the Gulf of Mexico was isolated and cultured in the laboratory and was exposed to water accommodated fraction (WAF) of different Maya CO loads at 0.01, 0.1, 1 and 10g/L by 96h. A battery of biomarkers involved in oxidative stress (O2•, H2O2, TBARS, ROOH, RC=O, SOD, CAT, GPx), biotransformation and conjugation (total CYP450 activity and GST) moreover fatty acid (FA) metabolism (FA levels, fatty-acid synthase and acyl-CoA oxidase) were measured. Obtained results suggest that increases of PAHs in the medium (below to EC50) acts as external forces able to turn-on regulatory mechanisms on H. oceanica involved in both, on the PAHs uptake and changing its aerobic metabolism to anaerobic metabolism. However, the growth of this microalgae species evaluated as chlorophyll "a" and pheophytin levels increased as the WAF concentration indicating that PAHs and other hydrosoluble hydrocarbons were used as carbon and energy sources by unidentified enzymes not evaluated in the current study. Our hypothesis was also corroborated by IBRv2. In the current study, we suppose the change from aerobic to anaerobic metabolism as a strategy for Halamphora oceanica survival exposed to petroleum hydrocarbons.

Pediatric Cytochrome P450 Activity Alterations in Nonalcoholic Steatohepatitis.

Variable drug responses depend on individual variation in the activity of drug-metabolizing enzymes, including cytochrome P450 enzymes (CYP). As the most common chronic liver disease in children and adults, nonalcoholic steatohepatitis (NASH) has been identified as a source of significant interindividual variation in hepatic drug metabolism. Compared with adults, children present age-related differences in pharmacokinetics and pharmacodynamics. The purpose of this study was to determine the impact of fatty liver disease severity on the activity of a variety of CYP enzymes in children and adolescents. Healthy and nonalcoholic fatty liver disease pediatric subjects aged 12-21 years inclusive received an oral cocktail of four probe drugs: caffeine (CYP1A2, 100 mg), omeprazole (CYP2C19, 20 mg), losartan (CYP2C9, 25 mg), and midazolam (CYP3A4, 2 mg). Venous blood and urine were collected before administration and 1, 2, 4, and 6 hours after administration. Concentrations of the parent drugs and CYP-specific metabolites were quantified in plasma and urine using liquid chromatography with tandem mass spectrometry. In plasma, the decreased metabolic area under the curve (AUC) ratio, defined as the metabolite AUC to parent AUC, of omeprazole indicated significant decreases of CYP2C19 (P = 0.002) enzymatic activities in NASH adolescents, while the urine analyses did not show significant differences and were highly variable. A comparison between the present in vivo pediatric studies and a previous ex vivo study in adults indicates distinct differences in the activities of CYP1A2 and CYP2C9. These data demonstrate that pediatric NASH presents an altered pattern of CYP activity and NASH should be considered as a confounder of drug metabolism for certain CYP enzymes. These differences could lead to future investigations that may reveal unexpected variable drug responses that should be considered in pediatric dosage recommendations.

Basal hepatic CYP activities in albino (Sprague–Dawley) and pigmented (Long Evans) rats

Basal hepatic CYP activities in albino (Sprague–Dawley) and pigmented (Long Evans) rats

Tissue Specific Modulation of cyp2c and cyp3a mRNA Levels and Activities by Diet-Induced Obesity in Mice: The Impact of Type 2 Diabetes on Drug Metabolizing Enzymes in Liver and Extra-Hepatic Tissues.

Various diseases such as type 2 diabetes (T2D) may alter drug clearance. The objective of this study was to evaluate the effects of T2D on CYP450 expressions and activities using high-fat diet (HFD) as a model of obesity-dependent diabetes in C57BL6 mice. The cyp450 mRNA expression levels for 15 different isoforms were determined in the liver and extra-hepatic tissues (kidneys, lungs and heart) of HFD-treated animals (n = 45). Modulation of cyp450 metabolic activities by HFD was assessed using eight known substrates for specific human ortholog CYP450 isoforms: in vitro incubations were conducted with liver and extra-hepatic microsomes. Expression levels of cyp3a11 and cyp3a25 mRNA were decreased in the liver (>2–14-fold) and kidneys (>2-fold) of HFD groups which correlated with a significant reduction in midazolam metabolism (by 21- and 5-fold in hepatic and kidney microsomes, respectively, p < 0.001). HFD was associated with decreased activities of cyp2b and cyp2c subfamilies in all organs tested except in the kidneys (for tolbutamide). Other cyp450 hepatic activities were minimally or not affected by HFD. Taken together, our data suggest that substrate-dependent and tissue-dependent modulation of cyp450 metabolic capacities by early phases of T2D are observed, which could modulate drug disposition and pharmacological effects in various tissues.

Bush mint (Hyptis suaveolens) and spreading hogweed (Boerhavia diffusa) medicinal plant extracts differentially affect activities of CYP1A2, CYP2D6 and CYP3A4 enzymes

Ethno-pharmacological relevanceHyptis suaveolens (L) Poit and Boerhavia diffusa Linn are medicinal herbal plants commonly found in the tropics and sub-tropics. They are used to treat various conditions among them boils, dyslipidaemia, eczema, malaria, jaundice and gonorrhoea. Thus, the herbal medicinal extracts are now found as part of some commercial herbal formulations. There has not been adequate characterization of these medicinal herbs on their effects on drug metabolising enzymes. Aim of the studyTo investigate the effects of extracts of Hyptis suaveolens (HS) and Boerhavia diffusa (BD) on activity of drug metabolising enzymes, CYP1A2, CYP2D6 and CYP3A4, as well predict their potential for herb-drug interaction. A secondary aim was to identify constituent compounds such as polyphenolics, in the crude extract preparations of Hyptis suaveolens and Boerhavia diffusa and measure them for activity. Materials and methodsCYP450 inhibition assays using recombinant CYP450 (rCYP) and fluorescence screening employing individual isozymes (CYP1A2, CYP2D6 and CYP3A4) were used to determine reversible- and time-dependent inhibition (TDI) profiles of extracts of Hyptis suaveolens and Boerhavia diffusa. Inhibition kinetic parameters, Ki and Kinact were also estimated. UPLC-MS employing a Synapt G2 (ESI negative) coupled to a PDA detector was used to identify polyphenolic compounds in crude extracts of Hyptis suaveolens and Boerhavia diffusa. ResultsThe inhibitory potency of Hyptis suaveolens and Boerhavia diffusa extracts varied among the different enzymes, with CYP1A2 (3.68 ± 0.10µg/mL) being the least inhibited by HS compared to CYP2D6 (1.39 ± 0.01µg/mL) and CYP3A4 (2.36 ± 0.57µg/mL). BD was most potent on CYP3A4 (7.36 ± 0.94µg/mL) compared to both CYP2D6 (17.79 ± 1.02µg/mL) and CYP1A2 (9.48 ± 0.78µg/mL). Extracts of Hyptis suaveolens and Boerhavia diffusa exhibited TDIs on all CYPs. The most prominent phenolic candidates identified in both medicinal herbs using UPLC-MS analysis included caffeic acid, rutin, quercetin, citric acid, ferulic acid and gluconic acid. These phenolic compounds are thought to potentially give HS and BD their therapeutic effects and inhibitory characteristics affecting CYP450 activities. In vivo predictions showed the potential for HS and BD extracts to cause significant interactions if co-administered with other medications. ConclusionsThe study reveals that crude aqueous extracts of HS and BD potentially inhibit drug metabolising isozymes CYP1A2, CYP2D6 and CYP3A4 in a reversible and time-dependent manner. Thus care should be taken when these extracts are co-administered with drugs that are substrates of CYP1A2, CYP2D6 and CYP3A4.

Contributions of cuticle permeability and enzyme detoxification to pyrethroid resistance in the major malaria vector Anopheles gambiae

To tackle the problem of insecticide resistance, all resistance mechanisms need to be studied. This study investigated the involvement of the cuticle in pyrethroid resistance in a strain of Anopheles gambiae, MRS, free of kdr mutations. Bioassays revealed MRS to be resistant to pyrethroids and DDT, indicated by increasing knockdown times and resistance ratios. Moreover, biochemical analysis indicated that metabolic resistance based on enhanced CYP450 activity may also play a role. Insecticide penetration assays showed that there were significantly lower amounts of insecticide in the MRS strain than in the susceptible control. Analysis of the levels of the selected transcripts by qPCR showed that CYP6M2, a major pyrethroid metaboliser, CYP4G16, a gene implicated in resistance via its contribution to the biosynthesis of elevated epicuticular hydrocarbons that delay insecticide uptake, and the cuticle genes CPAP3-E and CPLCX1 were upregulated after insecticide exposure. Other metabolic (CYP6P3, GSTe2) and cuticle (CPLCG3, CPRs) genes were also constitutively upregulated. Microscopic analysis showed that the cuticle layers of the MRS strain were significantly thicker than those of the susceptible strain. This study allowed us to assess the contribution made by the cuticle and metabolic mechanisms to pyrethroid resistance in Anopheles gambiae without target-site mutations.

Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450.

PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells’ activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug–drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.

De Novo Biosynthesis of Apigenin, Luteolin, and Eriodictyol in the Actinomycete Streptomyces albus and Production Improvement by Feeding and Spore Conditioning.

Nutraceutical compounds as plant flavonoids play an important role in prevention and modulation of diverse heath conditions, as they exert interesting antifungal, antibacterial, antioxidant, and antitumor effects. They also possess anti-inflammatory activities in arthritis, cardiovascular disease or neurological diseases, as well as modulatory effects on the CYP450 activity on diverse drugs. Most flavonoids are bioactive molecules of plant origin, but their industrial production is sometimes hindered due to reasons as low concentration in the plant tissues, presence in only some species or as a complex mixture or inactive glycosides in plant vacuolae. In this work, we describe the de novo biosynthesis of two important flavones, apigenin and luteolin, and one known flavanone, eriodictyol. Their plant biosynthetic pathways have been reconstructed for heterologous expression in Streptomyces albus, an actinomycete bacterium manageable at industrial production level. Also, production levels for apigenin have been improved by feeding with naringenin precursor, and timing for settlement of secondary metabolism has been advanced by spore conditioning. In the cases of eriodictyol and luteolin, their production in this important type of biotechnology-prone bacteria, the actinomycetes, had not been described in the literature yet.

In vivo Phenotyping Methods: Cytochrome P450 Probes with Emphasis on the Cocktail Approach.

Differences in drug response among patients are common. Most major drugs are effective in only 25 to 60 percent of the patients, in part due to the CYP enzymes, whose activity vary up to 50-fold between individuals for some index metabolic reactions. Several factors affect CYP activity, among which genetic polymorphisms have been studied as the major cause for long time. Age, gender, disease states, and environmental influences such as smoking, concomitant drug treatment or exposure to environmental chemicals are also important. This article reviews the available literature on multiple phenotypes assessment as an important tool to predict possible therapeutic failures or toxic reactions to conventional drug doses during patient evaluation. Probe drugs can be used in various combinations allowing for the in vivo assessment of multiple pathways of drug metabolism in a single experiment, configuring a new tool known as phenotyping "cocktails". There are several drug cocktails with different advantages and disadvantages. Most of them have sufficient clinical evidence and data validation to support their use in clinical setting as a surrogate for the risk of adverse reaction in the course of therapy, leading to a better balance between efficacy and safety. Probes characteristics and metabolic ratio measurements are important in the evaluation of phenotyping cocktails as near-future applications.

Modulating nasal mucosal permeation using metabolic saturation and enzyme inhibition techniques.

Presystemic elimination resulting from local enzymatic degradation can play a key role in limiting the bioavailability of intranasally administered drugs. The aim of this study was to evaluate the transfer of a metabolically susceptible drug across the nasal mucosa to illustrate the relative contributions of drug diffusivity and metabolic susceptibility on overall nasal mucosal permeation and to understand the effects of changes in enzymatic activity on the transfer across nasal epithelial and submucosal tissues. The concentration-dependent permeation of melatonin, a CYP450 substrate, across excised bovine nasal olfactory and respiratory explants was studied along with quantifying the extent of melatonin 6-hydroxylation. Microsomal preparations were also used to determine the kinetic parameters for melatonin to 6-hydroxymelatonin biotransformation. Enzyme saturation at higher melatonin concentrations and inclusion of a CYP450 inhibitor both resulted in the significant increase in melatonin permeation across the nasal mucosa. Metabolic loss of melatonin during nasal permeation demonstrates CYP450 activity in the nasal epithelium and submucosal tissues. The extent of biotransformation of melatonin during its transport across the nasal mucosal explants suggests that, although the nasal route bypasses hepatic first-pass metabolism, nasal bioavailability can be significantly influenced by mucosal enzymatic activity.

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MSn ), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). For metabolism studies, urine was collected over a 24h period after administration of the compounds to male Wistar rats at 20mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC-HR-MS/MS. 5-MeO-2-Me-DALT (24 phase I and 12 phase II metabolites), 5-MeO-2-Me-ALCHT (24 phase I and 14 phase II metabolites), and 5-MeO-2-Me-DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O-demethylation, hydroxylation, N-dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O-demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N-dealkylation. For SUSAs, GC-MS, LC-MSn , and LC-HR-MS/MS were applied to rat urine samples after 1 or 0.1mg/kg BW doses, respectively. In contrast to the GC-MS SUSA, both LC-MS SUSAs were able to detect an intake of 5-MeO-2-Me-ALCHT and 5-MeO-2-Me-DIPT via their metabolites following 1mg/kg BW administrations and 5-MeO-2-Me-DALT following 0.1mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.

Exploring the Role of CYP3A4 Mediated Drug Metabolism in the Pharmacological Modulation of Nitric Oxide Production.

Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation, and cytochrome P450, the major enzymes involved in drug metabolism, share striking similarities. Therefore, it makes sense that cytochrome P450 drug mediated biotransformations might play an important role in the pharmacological modulation of nitric oxide synthase. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and nitric oxide modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes in order assess the implication of CYP450 activities over production of nitric oxide. In vitro systems based in human liver microsomes and activated mouse macrophages were developed for these purposes. Additionally in vitro production the hepatic metabolites of dual inhibitor, roxithromycin, was investigated achieving the identification and isolation of main hepatic biotransformation products. Our results suggested that for some macrolide compounds, the cytochrome P450 3A4 derived drug metabolites have an important effect on nitric oxide production and might critically contribute to the pharmacological immunomodulatory activity observed.

Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture

The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

The Hsp90 machinery facilitates the transport of diphtheria toxin into human cells

Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.

Optimization of intestinal microsomal preparation in the rat: A systematic approach to assess the influence of various methodologies on metabolic activity and scaling factors.

The metabolic capacity of the intestine and its importance as the initial barrier to systemic exposure can lead to underestimation of first‐pass, and thus overestimation of oral bioavailability. However, the in vitro tools informing estimates of in vivo intestinal metabolism are limited by the complexity of the in vitro matrix preparation and uncertainty with the scaling factors for in vitro to in vivo extrapolation. A number of methods currently exist in the literature for the preparation of intestinal microsomes; however, the impact of key steps in the preparation procedure has not been critically assessed. In the current study, changes in enterocyte isolation, the impact of buffer constituents heparin and glycerol, as well as sonication as a direct method of homogenization were assessed systematically. Furthermore, fresh vs. frozen tissue samples and the impact of microsome freeze thawing was assessed. The rat intestinal microsomes were characterized for CYP content as well as metabolic activity using testosterone and 4‐nitropheonol as probes for CYP and UGT activity, respectively. Comparisons in metabolic activity and scaled unbound intestinal intrinsic clearance (CL intu,gut) were made to commercially available microsomes using 25 drugs with a diverse range of metabolic pathways and intestinal metabolic stabilities. An optimal, robust and reproducible microsomal preparation method for investigation of intestinal metabolism is proposed. The importance of characterization of the in vitro matrix and the potential impact of intestinal scaling factors on the in vitro–in vivo extrapolation of F G needs to be investigated further. © 2017 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.