Etoposide Model-Induced Changes in Antioxidant Studies Histological and Ultrastructural Evaluations of Hepatic, Renal, and Cardiac Tissues of Male Rats / Cambios Inducidos por el Modelo de Etopósido en Estudios de Antioxidantes, Evaluaciones Histológicas y Ultraestructurales de Tejidos Hepáticos, Renales y Cardíacos de Ratas Macho

SUMMARY: Etoposide is an effective antimitotic and antineoplastic agent used to treat various human malignancies. In the present study, Etoposide was injected intraperitoneally into the rats at 1 mg/kg/day for 52 days (52 doses). The control animals received physiological saline (0.5 ml) intraperitoneally daily for 52 doses. The body weight of etoposide-treated rats was significantly reduced compared to control rats. Lipid peroxidation demonstrated an insignificant rise in hepatic tissue, a non-significant decline in renal tissue, and a significant reduction in cardiac tissue. The levels of GSH in hepatic and renal tissue were found to be non-significantly increased but significantly increased in cardiac tissue compared to controls. GR activity was found to be considerably decreased in the treated group. G-S-T levels increased significantly in all treated group. Etoposide injections caused a non-significant change in the GPX level of hepatic tissue, whereas renal and cardiac tissues showed a significant increase. The activity of CAT in hepatic tissue was significantly increased, while CAT activity in renal tissue showed a non-significant decrease, whereas in cardiac tissue, significantly lower levels were observed than in control group. The level of CYTp450 in hepatic and cardiac tissues showed a significant increase; however, renal tissue showed non-significant depletion, whereas CYTb5 in hepatic, renal, and cardiac tissues was significantly lower than controls. The protein content in the hepatic tissue was not significantly increased, whereas the total protein in the renal and cardiac tissues was increased significantly. The research finding is indicative of detoxification activity in the etoposide model.

El etopósido es un agente antimitótico y antineoplásico eficaz que se utiliza para tratar diversas neoplasias malignas humanas. En el presente estudio, se inyectó etopósido por vía intraperitoneal a las ratas a razón de 1 mg/kg/día durante 52 días (52 dosis). Los animales control recibieron solución salina fisiológica (0,5 ml) por vía intraperitoneal diariamente por 52 dosis. El peso corporal de las ratas tratadas con etopósido se redujo significativamente en comparación con las ratas del grupo control. La peroxidación lipídica demostró un aumento insignificante del tejido hepático, una disminución no significativa del tejido renal y una reducción significativa del tejido cardíaco. Se encontró que los niveles de GSH en el tejido hepático y renal no aumentaron significativamente, pero sí aumentaron significativamente en el tejido cardíaco en comparación con los controles. Se encontró que la actividad de GR disminuyó considerablemente en el grupo tratado. Los niveles de G-S-T aumentaron significativamente en todos los grupos tratados. Las inyecciones de etopósido provocaron un cambio no significativo en el nivel de GPX del tejido hepático, mientras que los tejidos renal y cardíaco mostraron un aumento significativo. La actividad de CAT en el tejido hepático aumentó significativamente, mientras que la actividad de CAT en el tejido renal mostró una disminución no significativa, mientras que en el tejido cardíaco se observaron niveles significativamente más bajos que en el grupo de control. El nivel de CYTp450 en los tejidos hepático y cardíaco mostró un aumento significativo; sin embargo, el tejido renal mostró un agotamiento no significativo, mientras que CYTb5 en los tejidos hepático, renal y cardíaco fue significativamente menor que los controles. El contenido de proteínas en el tejido hepático no aumentó significativamente, mientras que la proteína total en los tejidos renal y cardíaco aumentó significativamente. El hallazgo de la investigación es indicativo de la actividad de desintoxicación en el modelo de etopósido.

Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

Stimulatory and Inhibitory Effects of Steroid Hormones and Human Cytochrome P450 (CYP) 3A Inhibitors on Cortisol 6ß-Hydroxylation Catalyzed by CYP3A Subfamilies.

OBJECTIVE: The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6ß-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6ß-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. METHODS: 6ß-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography. RESULTS: Lower concentrations of DHEA and ANF increased cortisol 6ß-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6ß-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6ß-hydroxylation. Although the Michaelis constant (Km) increased 2-fold in the presence of 20 µM DHEA compared to that of the control, the maximal velocity (Vmax) values gradually increased with increasing DHEA. For ANF, both Km and Vmax values increased, although the Km value decreased at 2.5 µM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6ß-hydroxylation mediated by CYP3A4 with similar inhibition constants. CONCLUSION: The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated.

Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Cannabinoid Metabolites as Inhibitors of Major Hepatic CYP450 Enzymes, with Implications for Cannabis-Drug Interactions.

The legalization of cannabis in many parts of the United States and other countries has led to a need for a more comprehensive understanding of cannabis constituents and their potential for drug-drug interactions. Although (-)-trans-Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are the most abundant cannabinoids present in cannabis, THC metabolites are found in plasma at higher concentrations and for a longer duration than that of the parent cannabinoids. To understand the potential for drug-drug interactions, the inhibition potential of major cannabinoids and their metabolites on major hepatic cytochrome P450 (P450) enzymes was examined. In vitro assays with P450-overexpressing cell microsomes demonstrated that the major THC metabolites 11-hydroxy-Δ9-tetra-hydrocannabinol and 11-nor-9-carboxy-Δ9-THC-glucuronide competitively inhibited several major P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6 (apparent Ki,u values = 0.086 ± 0.066 µM and 0.90 ± 0.54 µM, 0.057 ± 0.044 µM and 2.1 ± 0.81 µM, 0.15 ± 0.067 µM and 2.3 ± 0.54 µM, respectively). 11-Nor-9-carboxy-Δ9- tetrahydrocannabinol exhibited no inhibitory activity against any CYP450 tested. THC competitively inhibited CYP1A2, CYP2B6, CYP2C9, and CYP2D6; CBD competitively inhibited CYP3A4, CYP2B6, CYP2C9, CYP2D6, and CYP2E1; and CBN competitively inhibited CYP2B6, CYP2C9, and CYP2E1. THC and CBD showed mixed-type inhibition for CYP2C19 and CYP1A2, respectively. These data suggest that cannabinoids and major THC metabolites are able to inhibit the activities of multiple P450 enzymes, and basic static modeling of these data suggest the possibility of pharmacokinetic interactions between these cannabinoids and xenobiotics extensively metabolized by CYP2B6, CYP2C9, and CYP2D6. SIGNIFICANCE STATEMENT: Major cannabinoids and their metabolites found in the plasma of cannabis users inhibit several P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6. This study is the first to show the inhibition potential of the most abundant plasma cannabinoid metabolite, THC-COO-Gluc, and suggests that circulating metabolites of cannabinoids play an essential role in CYP450 enzyme inhibition as well as drug-drug interactions.

Mass spectrometry-based targeted proteomics method for the quantification of clinically relevant drug metabolizing enzymes in human specimens.

Biotransformation by phase I and II metabolizing enzymes represents the major determinant for the oral bioavailability of many drugs. To estimate the pharmacokinetics, data on protein abundance of hepatic and extrahepatic tissues, such as the small intestine, are required. Targeted proteomics assays are nowadays state-of-the-art for absolute protein quantification and several methods for quantification of drug metabolizing enzymes have been published. However, some enzymes remain still uncovered by the analytical spectra of those methods. Therefore, we developed and validated a quantification assay for two carboxylesterases (CES-1, CES-2), 17 cytochrome P450 enzymes (CYP) (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F12, CYP4A11) and five UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT2B7, UGT2B15, UGT2B17). Protein quantification was performed by analyzing proteospecific surrogate peptides after tryptic digestion with stable isotope-labelled standards. Chromatographic separation was performed on a Kinetex® 2.6 µm C18 100 Å core-shell column (100 × 2.1 mm) with a gradient elution using 0.1% formic acid and acetonitrile containing 0.1% formic acid with a flow rate of 200 µl/min. Three mass transitions were simultaneously monitored with a scheduled multiple reaction monitoring (sMRM) method for each analyte and standard. The method was partly validated according to current bioanalytical guidelines and met the criteria regarding linearity (0.1-25 nmol/L), within-day and between-day accuracy and precision as well as multiple stability criteria. Finally, the developed method was successfully applied to determine the abundance of the aforementioned enzymes in human intestinal und liver microsomes. Our work offers a new fit for purpose method for the absolute quantification of CES, CYPs and UGTs in various human tissues and can be used for the acquisition of data for physiologically based pharmacokinetic modelling.

Tailored nanodisc immobilization for one-step purification and reconstitution of cytochrome P450: A tool for membrane proteins' hard cases.

A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion-CYP2B6, diclofenac-CYP2C9, omeprazole-CYP2C19, bufuralol-CYP2D6, and midazolam-CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

A Simple Aggregation-Induced Emission Nanoprobe with Deep Tumor Penetration for Hypoxia Detection and Imaging-Guided Surgery in Vivo.

The pan-cancer detection and precise visualization of tiny tumors in surgery still face great challenges. As tumors grow aggressively, hypoxia is a common feature of solid tumors and has supplied a general way for detecting tumors. Herein, we report a simple aggregation-induced emission nanoprobe-TPE-4NE-O that can specifically switch on their fluorescence in the presence of cytochrome P450 reductase, a reductase which is overexpressed under hypoxia conditions. The probe can selectively light up the hypoxia cells and has shown enhanced deep tumor penetration via charge conversion both in vitro and in vivo. After being modified with FA-DSPE-PEG, higher tumor uptake can be seen and FA-DSPE/TPE-4NE-O showed specific visualization to the hypoxia cancer cells. Excitingly, much brighter fluorescence was accumulated at the tumors in the FA-DSPE/TPE-4NE-O group, even though the tumor was as small as 2.66 mm. The excellent performance of FA-DSPE/TPE-4NE-O in detecting tiny tumors has made it possible for imaging-guided tumor resection. More importantly, the probe exhibited good biocompatibility with negligible organ damage and eliminated a hemolysis risk. The simple but promising probe has supplied a new strategy for pan-cancer detection and tiny tumor visualization, which have shown great potential in clinical translation.

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes.

INTRODUCTION: Ex vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. METHODS: We identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. RESULTS: The optimum incubation time and probe drug concentration was six hours with 5 µM phenacetin (CYP1A2), four hours with 10 µM diclofenac (CYP2C9), 30 min with 1 µM of midazolam (CYP3A4) and 10 min with 1 µM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 µg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 µg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DISCUSSION: This study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata / Determinación de concentraciones letales y niveles de enzimas metabólicas de insecticidas en Triatoma dimidiata

Abstract: Objective: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. Materials and methods: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. Results: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and β-eterase levels. Conclusion: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

Resumen: Objetivo: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. Material y métodos: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. Resultados: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y β-eterasas. Conclusión: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Resistencia a insecticidas en Aedes aegypti y Aedes albopictus (Diptera: Culicidae) de Tapachula, Chiapas, México / Insecticide resistance in Aedes aegypti and Ae. albopictus (Diptera: Culicidae) populations from Tapachula, Chiapas, Mexico

Resumen: Objetivo: Determinar la resistencia a insecticidas en Ae. aegypti y Ae. albopictus de Tapachula, Chiapas, México. Material y métodos: Se utilizaron ovitrampas para obtener huevos de mosquitos Aedes y se realizaron pruebas de susceptibilidad (CDC) y ensayos enzimáticos con la primera generación. Resultados: Aedes aegypti mostró resistencia a deltametrina, permetrina, malatión, clorpirifos, temefos y a bendiocarb (CARB), mientras que Aedes albopictus a malatión y en menor grado a cloripirifos, temefos, permetrina y deltametrina. Ambas especies mostraron altos niveles de enzimas como citocomo P450 y glutatión S-tranferasa, mientras que los niveles de esterasas variaron por especie y sitio muestreado. Se detectó acetilcolinesterasa insensible a insecticidas en ambas especies. Conclusión: En un hábitat urbano de Tapachula, Chiapas, México donde se aplica control con insecticidas Ae. aegypti y Ae. albopictus sólo son susceptibles al propoxur.

Abstract: Objective: To determine the insecticide resistance status of Ae. aegypti and Ae. albopictus from Tapachula, México. Materials and methods: Mosquito eggs were collected with the use of ovitraps and CDC susceptibility bioassays and biochemical assays were conducted to determine resistance levels and resistance mechanisms, respectively. Results: Ae. aegypti showed resistance to deltamethrin and permethrin (PYRs), malathion, chlorpyrifos and temephos (OP), and to bendiocarb (CARB), while Ae. albopictus showed resistance to malathion and to a lesser intensity to chlorypirifos, temephos, permethrin and deltamethrin. Both species showed high levels of P450 and GSTs, while levels of esterases varied by species and collection site. Altered acethilcholinesterase was detected in both species. Conclusion: In an urban habitat from Tapachula, Chiapas, Mexico where vector control using insecticides takes place, Ae. aegypti and Ae. albopictus are only susceptible to propoxur.

[Insecticide resistance in Aedes aegypti and Ae. albopictus (Diptera: Culicidae) populations from Tapachula, Chiapas, Mexico]. / Resistencia a insecticidas en Aedes aegypti y Aedes albopictus (Diptera: Culicidae) de Tapachula, Chiapas, México.

OBJECTIVE: To determine the insecticide resistance status of Ae. aegypti and Ae. albopictus from Tapachula, México. MATERIALS AND METHODS: Mosquito eggs were collected with the use of ovitraps and CDC susceptibility bioassays and biochemical assays were conducted to determine resistance levels and resistance mechanisms, respectively. RESULTS: Ae. aegypti showed resistance to deltamethrin and permethrin (PYRs), malathion, chlorpyrifos and temephos (OP), and to bendiocarb (CARB), while Ae. albopictus showed resistance to malathion and to a lesser intensity to chlorypirifos, temephos, permethrin and deltamethrin. Both species showed high levels of P450 and GSTs, while levels of esterases varied by species and collection site. Altered acethilcholinesterase was detected in both species. CONCLUSIONS: In an urban habitat from Tapachula, Chiapas, Mexico where vector control using insecticides takes place, Ae. aegypti and Ae. albopictus are only susceptible to propoxur.

OBJETIVO: Determinar la resistencia a insecticidas en Ae. aegypti y Ae. albopictus de Tapachula, Chiapas, México. MATERIAL Y MÉTODOS: Se utilizaron ovitrampas para obtener huevos de mosquitos Aedes y se realizaron pruebas de susceptibilidad (CDC) y ensayos enzimáticos con la primera generación. RESULTADOS: Aedes aegypti mostró resistencia a deltametrina, permetrina, malatión, clorpirifos, temefos y a bendiocarb (CARB), mientras que Aedes albopictus a malatión y en menor grado a cloripirifos, temefos, permetrina y deltametrina. Ambas especies mostraron altos niveles de enzimas como citocomo P450 y glutatión S-tranferasa, mientras que los niveles de esterasas variaron por especie y sitio muestreado. Se detectó acetilcolinesterasa insensible a insecticidas en ambas especies. CONCLUSIONES: En un hábitat urbano de Tapachula, Chiapas, México donde se aplica control con insecticidas Ae. aegypti y Ae. albopictus sólo son susceptibles al propoxur.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata.

OBJECTIVE: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. MATERIALS AND METHODS: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. RESULTS: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and ß-eterase levels. CONCLUSIONS: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

OBJETIVO: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. MATERIAL Y MÉTODOS: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. RESULTADOS: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y ß-eterasas. CONCLUSIONES: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Enrichment-free High-throughput Liquid Chromatography-Multiple-Reaction Monitoring Quantification of Cytochrome P450 Proteins in Plated Human Hepatocytes Direct from 96-Well Plates Enables Routine Protein Induction Measurements.

Despite the availability of liquid chromatography (LC)-mass spectrometry (MS) methods for quantifying cytochrome P450 (P450) proteins, incorporation of P450 protein quantification into induction study workflows has not been widely adopted. To more readily enable P450 protein quantification in induction study workflows, DMPK research groups need a simple, robust, cost-effective, high-throughput method compatible with 96-well-plated human hepatocyte formats. Here, we provide such a methodology. Our method bypasses both microsomal enrichment and antibody-based enrichment to go directly from the plate to LC-MS/MS analysis. We use this "plate-to-peaks" approach for quantifying CYP3A4, CYP2B6, and CYP1A2, the major inducible hepatic P450s representative of pregnane X receptor-, constitutive androstane receptor-, and aryl hydrocarbon receptor-mediated induction, respectively. We leveraged our induction study-aligned assay format to assess induction across mRNA, protein, and enzyme activity using known induction control compounds. As expected, results from the three methods using model inducers were broadly concordant, but the magnitude of the induction response differed. Induction of CYP3A4 using 10 µM rifampicin was 12-fold for RNA, eightfold for protein, and threefold for activity; for CYP1A2 with 50 µM omeprazole, induction was 30-fold for RNA, 13-fold for protein, and 17-fold for activity; for CYP2B6 with 50 µM phenytoin, induction was 23-fold for RNA, twofold for protein, and fivefold for activity. Most importantly, we anticipate the relative ease of this method will enable researchers to routinely adopt P450 protein quantification as part of nonclinical evaluation of P450 induction. SIGNIFICANCE STATEMENT: Current methodologies for quantifying P450 proteins by liquid chromatography (LC)-tandem mass spectrometry are either cumbersome, too costly, or both to be widely adopted into induction study workflows by the ADME research community. We present a simplified LC-MS/MS methodology for quantifying P450 proteins directly from human hepatocytes, without any form of enrichment, in 96-well induction assay plate format that should be readily adoptable by any ADME laboratory with LC-multiple-reaction monitoring capabilities.

PPARα plays an important role in the migration activity, and the expression of CYP2S1 and CYP1B1 in chrysin-treated HCT116 cells.

OBJECTIVE: This study aimed to examine the metabolising effect of chrysin by investigating the mRNA expression levels of PPARα and its related cellular mechanisms in HCT116 cells. RESULTS: The mRNA expression of PPARα was significantly induced in HCT116 cells following treatment with chrysin for 36 h, but the mRNA expression of PPARα was inhibited, when the cells were treated with a combination of chrysin and MK886 (PPARα inhibitor). This phenomenon proved that the incorporation of MK886 lowers the expression levels of PPARα, thus enabling us to study the function of PPARα. The cell population of the G0/G1 phase significantly increased in chrysin-treated cells, which was accompanied by a decrease in the percentage of S phase cell population after 12 h of treatment. However, treatments of HCT116 cells with chrysin only or a combination of chrysin and MK886 did not show the opposite situation in the G0/G1 and S phase cell populations, indicating that the expression of PPARα may not be associated with the cell cycle in the treated cells. The migration rate in chrysin-treated HCT116 cells was reduced significantly after 24 and 36 h of treatments. However, the activity was revived, when the expression of PPARα was inhibited, indicating that the migration activity of chrysin-treated cells is likely correlated with the expression of PPARα. Comparison of the CYP2S1 and CYP1B1 mRNA expression in chrysin only treated, and a combination of chrysin and MK886-treated HCT116 cells for 24 and 36 h showed a significant difference in the expression levels, indicating that PPARα inhibitor could also modify the expression of CYP2S1 and CYP1B1. CONCLUSION: The study indicates that PPARα may play an essential role in regulating the migration activity, and the expression of CYP2S1 and CYP1B1 in chrysin-treated colorectal cancer cells.

Evaluation of Zhenwu Decoction Effects on CYP450 Enzymes in Rats Using a Cocktail Method by UPLC-MS/MS.

This thesis is aimed at shedding light on the effects of the Zhenwu decoction (ZWD) on the activities and mRNA expressions of seven CYP450 isoenzymes. In the first step, we determined the main chemical compounds of ZWD by high-performance liquid chromatography (HPLC). Next, 48 male (SD) rats were randomly divided into the normal saline (NS) group and the ZWD low- (2.1875 g/kg), medium- (4.375 g/kg), and high- (8.75 g/kg) dose groups (12 per group). All rats were gavaged once daily for 28 consecutive days. A mixed solution of seven probe drugs was injected into 24 rats through the caudal vein after the last intragastric administration. Lastly, a validated cocktail method and real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to detect pharmacokinetic parameters and mRNA expressions, respectively. Compared with the NS group, ZWD at medium- and high-dose groups could significantly induce CYP2C6 (P < 0.05) activity, while the mRNA expression (P < 0.05) increased only in the high-dose group. Additionally, CYP2C11 activity was induced and consistent with mRNA expression (P < 0.05). Moreover, ZWD could induce the activity of CYP3A1 (P < 0.05), but the mRNA expression showed no significant differences except in high-dose groups. Additionally, ZWD has no effects on CYP1A2, CYP2B1, CYP2C7, and CYP2D2. In conclusion, the significant inductive effects of ZWD on three CYP450 isoenzymes indicated that when ZWD was coadministrated with drugs mediated by these enzymes, not only should the potential herb-drug interactions (HDIs) be observed, but the dosage adjustment and tissue drug concentration should also be considered. Furthermore, the approach described in this article can be applied to study the importance of gender, age, and disease factors to HDI prediction.

CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms.

BACKGROUND: Cytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner. RESULTS: CYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences. CONCLUSIONS: CYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

Pharmacokinetics of omeprazole in rats with dextran sulfate sodium-induced ulcerative colitis.

Omeprazole is a commonly used drug in patients with ulcerative colitis (UC). This study investigated the pharmacokinetics of omeprazole in rats with UC induced by dextran sulfate sodium (DSS). The pharmacokinetics of intravenously administered omeprazole (20 mg/kg) was investigated in normal and UC rats using LC-MS/MS. The formation of 5-OH omeprazole, a main metabolite of omeprazole, in rat liver microsomes (RLMs) from normal and UC rats was compared. The protein levels of CYP1A2, CYP2D1, and CYP3A1 in the liver were measured by Western blot. Compared with normal rats, UC rats had increased plasma concentrations of omeprazole, resulting in an increased AUC0-240 min and decreased CL. DSS treatment decreased the formation rate of 5-OH omeprazole in RLMs but did not change the affinity of the enzymes. The Vmax and CLint of RLMs from UC rats were 62% and 48% those of RLMs from normal rats, respectively. The hepatic CYP1A2 and CYP3A1 protein levels in UC rats were 42.6 and 45.2% lower than those in normal rats, respectively; however, the protein levels of CYP2D1 in the two groups were similar. The activity and expression of some hepatic CYP450 isoforms were decreased by UC, leading to changes in the pharmacokinetics of omeprazole.

Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine.

The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n = 4) and ileum (n = 12) using quantification concatemer-based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-α), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P = 0.002, R 2 = 0.63), 2C9-2J2 (P = 0.004, R 2 = 0.40), 2D6-2J2 (P = 0.002, R 2 = 0.50)] and UGT [1A1-2B7 (P = 0.02, R 2 = 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-α (P < 0.0001, R 2 = 0.77), UGT1A6 (P = 0.009, R 2 = 0.38), and CYP3A4 (P = 0.007, R 2 = 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals. SIGNIFICANCE STATEMENT: A number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.