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Drug-induced liver injury (DILI) is a significant public health issue, but standard animal tests and clinical trials sometimes fail to predict DILI due to species differences and the relatively low number of human subjects involved in preapproval studies of a new drug, respectively. In vitro models have long been used to aid DILI prediction, with primary human hepatocytes (PHHs) being generally considered the gold standard. However, despite many efforts and decades of work, traditional culture methods have been unsuccessful in either fully preserving essential liver functions after isolation of PHHs or in emulating interactions between PHHs and hepatic nonparenchymal cells (NPCs), both of which are essential for the development of DILI under in vivo conditions. Recently, various liver-on-a-chip (Liver-Chip) systems have been developed to co-culture hepatocytes and NPCs in a three-dimensional environment on microfluidic channels, enabling better maintenance of primary liver cells and thus improved DILI prediction. The Emulate® Liver-Chip is a commercially available system that can recapitulate some in vivo DILI responses associated with certain compounds whose liver safety profile cannot be accurately evaluated using conventional approaches involving PHHs or animal models due to a lack of innate immune responses or species-dependent toxicity, respectively. Here, we describe detailed procedures for the use of Emulate® Liver-Chips for co-culturing PHHs and NPCs for the purpose of DILI evaluation. First, we describe the procedures for preparing the Liver-Chip. We then outline the steps needed for sequential seeding of PHHs and NPCs in the prepared Liver-Chips. Lastly, we provide a protocol for utilizing cells maintained in perfusion culture in the Liver-Chips to evaluate DILI, using acetaminophen as an example. In all, use of this system and the procedures described here allow better preservation of the functions of human primary liver cells, resulting in an improved in vitro model for DILI assessment. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Liver-Chip preparation Basic Protocol 2: Seeding primary human hepatocytes and nonparenchymal cells on Liver-Chips Basic Protocol 3: Perfusion culture for the study of acetaminophen-induced liver injury.
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Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Animales , Técnicas de Cocultivo , Hepatocitos , HumanosRESUMEN
Neurological disease and disorders remain a large public health threat. Thus, research to improve early detection and/or develop more effective treatment approaches are necessary. Although there are many common techniques and imaging modalities utilized to study these diseases, existing approaches often require a label which can be costly and time consuming. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a label-free, innovative and emerging technique that produces 2D ion density maps representing the distribution of an analyte(s) across a tissue section in relation to tissue histopathology. One main advantage of MALDI IMS over other imaging modalities is its ability to determine the spatial distribution of hundreds of analytes within a single imaging run, without the need for a label or any a priori knowledge. Within the field of neurology and disease there have been several impactful studies in which MALDI IMS has been utilized to better understand the cellular pathology of the disease and or severity. Furthermore, MALDI IMS has made it possible to map specific classes of analytes to regions of the brain that otherwise may have been lost using more traditional methods. This review will highlight key studies that demonstrate the potential of this technology to elucidate previously unknown phenomenon in neurological disease.
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Encéfalo , Neurología , Encéfalo/diagnóstico por imagen , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
There is a lack of experimental reference materials and standards for metabolomics measurements, such as urine, plasma, and other human fluid samples. Reasons include difficulties with supply, distribution, and dissemination of information about the materials. Additionally, there is a long lead time because reference materials need their compositions to be fully characterized with uncertainty, a labor-intensive process for material containing thousands of relevant compounds. Furthermore, data analysis can be hampered by different methods using different software by different vendors. In this work, we propose an alternative implementation of reference materials. Instead of characterizing biological materials based on their composition, we propose using untargeted metabolomic data such as nuclear magnetic resonance (NMR) or gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS) profiles. The profiles are then distributed with the material accompanying the certificate, so that researchers can compare their own metabolomic measurements with the reference profiles. To demonstrate this approach, we conducted an interlaboratory study (ILS) in which seven National Institute of Standards and Technology (NIST) urine Standard Reference Material®s (SRM®s) were distributed to participants, who then returned the metabolomic data to us. We then implemented chemometric methods to analyze the data together to estimate the uncertainties in the current measurement techniques. The participants identified similar patterns in the profiles that distinguished the seven samples. Even when the number of spectral features is substantially different between platforms, a collective analysis still shows significant overlap that allows reliable comparison between participants. Our results show that a urine suite such as that used in this ILS could be employed for testing and harmonization among different platforms. A limited quantity of test materials will be made available for researchers who are willing to repeat the protocols presented here and contribute their data.
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Variable processing and storage of whole blood and/or plasma are potential confounders in biomarker development and clinical assays. The goal of the study was to investigate how pre-analytical variables impact the human plasma proteome. Whole blood obtained from 16 apparently healthy individuals was collected in six EDTA tubes and processed randomly under six pre-analytical variable conditions including blood storage at 0 °C or RT for 6 h (B6h0C or B6hRT) before processing to plasma, plasma storage at 4 °C or RT for 24 h (P24h4C or P24hRT), low centrifugal force at 1300 × g, (Low×g), and immediate processing to plasma under 2500 × g (control) followed by plasma storage at -80 °C. An aptamer-based proteomic assay was performed to identify significantly changed proteins (fold change ≥1.2, P < 0.05, and false discovery rate < 0.05) relative to the control from a total of 1305 proteins assayed. Pre-analytical conditions Low×g and B6h0C resulted in the most plasma proteome changes with 200 and 148 proteins significantly changed, respectively. Only 36 proteins were changed under B6hRT. Conditions P24h4C and P24hRT yielded changes of 28 and 75 proteins, respectively. The complement system was activated in vitro under the conditions B6hRT, P24h4C, and P24hRT. The results suggest that particular pre-analytical variables should be controlled for clinical measurement of specific biomarkers.
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Plasma/química , Estabilidad Proteica , Proteómica/métodos , Adulto , Aptámeros de Péptidos , Conservación de la Sangre/métodos , Recolección de Muestras de Sangre/métodos , Activación de Complemento , Voluntarios Sanos , Humanos , Proteoma/análisisRESUMEN
Discrepancies in blood sample collection and processing could have a significant impact on levels of metabolites, peptides, and protein biomarkers of inflammation in the blood; thus, sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several preanalytical processing conditions, including different storage times and temperatures for blood or plasma samples and different centrifugation forces on the levels of metabolites, peptides, and inflammation biomarkers in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Temperature was found to be the major factor for metabolite variation, and both time and temperature were identified as major factors for peptide variation. For inflammation biomarkers, temperature played different roles depending on the sample type (blood or plasma). Low temperature affected inflammation biomarkers in blood, while room temperature impacted inflammation biomarkers in plasma.
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Biomarcadores/sangre , Inflamación/sangre , Metabolómica/métodos , Péptidos/sangre , Adolescente , Adulto , Anciano , Recolección de Muestras de Sangre/métodos , Cromatografía Liquida/métodos , Femenino , Humanos , Inflamación/genética , Masculino , Espectrometría de Masas/métodos , Metaboloma/genética , Persona de Mediana Edad , Péptidos/genética , Plasma/química , Adulto JovenRESUMEN
Acetaminophen (APAP), a commonly used over-the-counter analgesic, accounts for approximately fifty percent of the cases of acute liver failure (ALF) in the United States due to overdose, with over half of those unintentional. Current clinical approaches for assessing APAP overdose rely on identifying the precise time of overdose and quantitating acetaminophen alanine aminotransferase (ALT) levels in peripheral blood. Novel specific and sensitive biomarkers may provide additional information regarding patient status post overdose. Previous non-clinical metabolomics studies identified potential urinary biomarkers of APAP-induced hepatotoxicity and metabolites involved pathways of tricarboxylic acid cycle, ketone metabolism, and tryptophan metabolism. In this study, biomarkers identified in the previous non-clinical study were evaluated in urine samples collected from healthy subjects ( N = 6, median age 14.08 years) and overdose patients ( N = 13, median age 13.91 years) as part of an IRB-approved multicenter study of APAP toxicity in children. The clinical results identified metabolites from pathways previously noted, and pathway analysis indicated analogous pathways were significantly altered in both the rats and humans after APAP overdose. The results suggest a metabolomics approach may enable the discovery of specific, translational biomarkers of drug-induced hepatotoxicity that may aid in the assessment of patients.
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Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.
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Clofibrato/toxicidad , Sistema Enzimático del Citocromo P-450/genética , Hígado/efectos de los fármacos , Fenobarbital/toxicidad , Animales , Clofibrato/análisis , Sistema Enzimático del Citocromo P-450/metabolismo , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epigenómica , Expresión Génica/efectos de los fármacos , Hígado/enzimología , Masculino , Fenobarbital/análisis , Ratas , Ratas Endogámicas F344RESUMEN
The present study aimed to identify molecular markers of early stages of cardiotoxicity induced by a potent chemotherapeutic agent, doxorubicin (DOX). Male B6C3F1 mice were dosed with 3 mg kg(-1) DOX or saline via tail vein weekly for 2, 3, 4, 6 or 8 weeks (cumulative DOX doses of 6, 9, 12, 18 or 24 mg kg(-1) , respectively) and euthanized a week after the last dose. Mass spectrometry-based and nuclear magnetic resonance spectrometry-based metabolic profiling were employed to identify initial biomarkers of cardiotoxicity before myocardial injury and cardiac pathology, which were not noted until after the 18 and 24 mg kg(-1) cumulative doses, respectively. After a cumulative dose of 6 mg kg(-1) , 18 amino acids and four biogenic amines (acetylornithine, kynurenine, putrescine and serotonin) were significantly increased in cardiac tissue; 16 amino acids and two biogenic amines (acetylornithine and hydroxyproline) were significantly altered in plasma. In addition, 16 acylcarnitines were significantly increased in plasma and five were significantly decreased in cardiac tissue compared to saline-treated controls. Plasma lactate and succinate, involved in the Krebs cycle, were significantly altered after a cumulative dose of 6 mg kg(-1) . A few metabolites remained altered at higher cumulative DOX doses, which could partly indicate a transition from injury processes at 2 weeks to repair processes with additional injury happening concurrently before myocardial injury at 8 weeks. These altered metabolic profiles in mouse heart and plasma during the initial stages of injury progression due to DOX treatment may suggest these metabolites as candidate early biomarkers of cardiotoxicity. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
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Antibióticos Antineoplásicos/toxicidad , Aminas Biogénicas/sangre , Doxorrubicina/toxicidad , Corazón/efectos de los fármacos , Metaboloma/efectos de los fármacos , Miocardio/metabolismo , Animales , Biomarcadores/sangre , Cardiotoxicidad , Relación Dosis-Respuesta a Droga , Inyecciones Intravenosas , Masculino , Ratones EndogámicosRESUMEN
Acetaminophen (APAP) is a commonly used analgesic drug that can cause liver injury, liver necrosis and liver failure. APAP-induced liver injury is associated with glutathione depletion, the formation of APAP protein adducts, the generation of reactive oxygen and nitrogen species and mitochondrial injury. The systems biology omics technologies (transcriptomics, proteomics and metabolomics) have been used to discover potential translational biomarkers of liver injury. The following review provides a summary of the systems biology discovery process, analytical validation of biomarkers and translation of omics biomarkers from the nonclinical to clinical setting in APAP-induced liver injury.
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Acetaminofén/efectos adversos , Analgésicos no Narcóticos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/etnología , Animales , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Glutatión/metabolismo , Humanos , Mitocondrias/patología , Investigación Biomédica Traslacional/métodosRESUMEN
It has been estimated that 10% of acute liver failure is due to "idiosyncratic hepatotoxicity". The inability to identify such compounds with classical preclinical markers of hepatotoxicity has driven the need to discover a mechanism-based biomarker panel for hepatotoxicity. Seven compounds were included in this study: two overt hepatotoxicants (acetaminophen and carbon tetrachloride), two idiosyncratic hepatotoxicants (felbamate and dantrolene), and three non-hepatotoxicants (meloxicam, penicillin and metformin). Male Sprague-Dawley rats were orally gavaged with a single dose of vehicle, low dose or high dose of the compounds. At 6 h and 24 h post-dosing, blood was collected for metabolomics and clinical chemistry analyses, while organs were collected for histopathology analysis. Forty-one metabolites from previous hepatotoxicity studies were semi-quantified and were used to build models to predict hepatotoxicity. The selected metabolites were involved in various pathways, which have been noted to be linked to the underlying mechanisms of hepatotoxicity. PLS models based on all 41 metabolite or smaller subsets of 6 (6 h), 7 (24 h) and 20 (6 h and 24 h) metabolites resulted in models with an accuracy of at least 97.4% for the hold-out test set and 100% for training sets. When applied to the external test sets, the PLS models predicted that 1 of 9 rats at both 6 h and 24 h treated with idiosyncratic liver toxicants was exposed to a hepatotoxic chemical. In conclusion, the biomarker panel might provide information that along with other endpoint data (e.g., transcriptomics and proteomics) may diagnose acute and idiosyncratic hepatotoxicity in a clinical setting.
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Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.
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Acetaminofén/efectos adversos , Camellia sinensis/química , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Extractos Vegetales/farmacología , Animales , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/metabolismo , Glutatión/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Espectroscopía de Resonancia Magnética , Metabolómica/métodos , Ratones , Ratones EndogámicosRESUMEN
Penicillin (PEN) V, a well-known antibiotic widely used in the treatment of Gram-positive bacterial infections, was evaluated in this study. LC/MS- and NMR-based metabolic profiling were employed to examine the effects of PEN on the host's metabolic phenotype. Male Sprague Dawley rats were randomly divided into groups that were orally administered either 0.5% methylcellulose vehicle, 100 or 2400mg PEN/kg body weight once daily for up to 14 consecutive days. Urine, plasma and tissue were collected from groups sacrificed at 6h, 24h or 14d. The body fluids were subjected to clinical chemistry and metabolomics analysis; the tissue samples were processed for histopathology. The only notable clinical chemistry observation was that gamma glutamyltransferase (GGT) significantly decreased at 24h for both dose groups, and significantly decreased at 14d for the high-dose groups. Partial least squares discriminant analysis scores plots of the metabolomics data from urine and plasma samples showed dose- and time-dependent grouping patterns. Time- and dose-dependent decreases in urinary metabolites including indole-containing metabolites (such as 3-methyldioxyindole sulfate generated from bacterial metabolism of tryptophan), organic acids containing phenyl groups (such as hippuric acid, phenyllactic acid and 3-hydroxyanthranilic acid), and metabolites conjugated with sulfate or glucuronide (such as cresol sulfate and aminophenol sulfate) indicated that the gut microflora population was suppressed. Decreases in many host-gut microbiota urinary co-metabolites (indole- and phenyl-containing metabolites, amino acids, vitamins, nucleotides and bile acids) suggested gut microbiota play important roles in the regulation of host metabolism, including dietary nutrient absorption and reprocessing the absorbed nutrients. Decreases in urinary conjugated metabolites (sulfate, glucuronide and glycine conjugates) implied that gut microbiota might have an impact on chemical detoxification mechanisms. In all, these results clearly show that metabolic profiling is a useful tool to better understand the effects of the antibiotic penicillin has on the gut microbiota and the host.
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Antibacterianos/farmacología , Metaboloma/efectos de los fármacos , Penicilinas/farmacología , Aminoácidos/orina , Animales , Ácidos y Sales Biliares/orina , Cromatografía Liquida/métodos , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/microbiología , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas/métodos , Nucleótidos/orina , Plasma/metabolismo , Ratas , Ratas Sprague-Dawley , Orina/químicaRESUMEN
Numerous factors in conjunction with an individual's genetic make up will determine predisposition to disease, adverse or beneficial effects of drug treatment or therapy, and disease progression. A major limitation of current clinical measures is that the disease phenotype, which is comprised of the genotype and other environmental factors, is underestimated. Rather, each disease is treated similarly even though the disease process is highly complex. Methods that evaluate the interaction of genotype and environmental factors would likely be a better indicator of patients' response to medical treatments. The omics technologies, specifically metabolomics, will play a major role in the movement towards personalized medicine. Metabolomics is phenotype driven and should provide better clinical biomarkers. Furthermore, recent studies have shown that associations between genetic variants and downstream metabolite changes can provide a unique description of an individual's genotype and phenotype, which will further enhance the movement towards personalized medicine.
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The rapid growth in the development of nanoparticles for uses in a variety of applications including targeted drug delivery, cancer therapy, imaging, and as biological sensors has led to questions about potential toxicity of such particles to humans. High-throughput methods are necessary to evaluate the potential toxicity of nanoparticles. The omics technologies are particularly well suited to evaluate toxicity in both in vitro and in vivo systems. Metabolomics, specifically, can rapidly screen for biomarkers related to predefined pathways or processes in biofluids and tissues. Specifically, oxidative stress has been implicated as a potential mechanism of toxicity in nanoparticles and is generally difficult to measure by conventional methods. Furthermore, metabolomics can provide mechanistic insight into nanotoxicity. This chapter focuses on the application of both LC/MS and NMR-based metabolomics approaches to study the potential toxicity of nanoparticles.
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Metabolómica/métodos , Nanopartículas/toxicidad , Pruebas de Toxicidad/métodos , Animales , Cromatografía Liquida , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de MasasRESUMEN
Following kidney failure in domesticated pets in the US and kidney issues requiring hospitalization with some deaths in children in China, investigators determined the cause was adulteration of pet foods and baby formula with melamine. It has since been noted that exposure of rats to melamine and cyanuric acid forms melamine cyanurate crystals in the kidney leading to acute nephrotoxicity. This metabolomics study aimed to identify biomarkers of melamine and cyanuric acid-induced renal injury. Male and female Fischer 344 rats were fed a diet fortified with varying doses of melamine and cyanuric acid for 28 days. Analysis of urinary amino acids showed hydroxyproline was increased in both sexes in a manner consistent with the clinical chemistry and histopathology data; most prominent when total urine output was taken into account. Furthermore, rats with the highest levels of urinary hydroxyproline were the only rats that exhibited fibrosis within the kidney. Clinical chemistry and histopathology indicated male rats were slightly more affected than female rats following dosing with the 120 and 180 ppm formulations; hydroxyproline excretion also supports this finding. Hydroxyproline may be a noninvasive urinary biomarker for detection of acute kidney injury potentially associated with kidney fibrosis.
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Biomarcadores/análisis , Hidroxiprolina/orina , Enfermedades Renales/inducido químicamente , Enfermedades Renales/diagnóstico , Triazinas/toxicidad , Animales , Femenino , Fibrosis , Enfermedades Renales/patología , Masculino , Metabolómica/métodos , Ratas , Ratas Endogámicas F344RESUMEN
An improved three-dimensional quantitative spectral data-activity relationship (3D-QSDAR) methodology was used to build and validate models relating the activity of 130 estrogen receptor binders to specific structural features. In 3D-QSDAR, each compound is represented by a unique fingerprint constructed from (13)C chemical shift pairs and associated interatomic distances. Grids of different granularity can be used to partition the abstract fingerprint space into congruent "bins" for which the optimal size was previously unexplored. For this purpose, the endocrine disruptor knowledge base data were used to generate 50 3D-QSDAR models with bins ranging in size from 2 ppm × 2 ppm × 0.5 Å to 20 ppm × 20 ppm × 2.5 Å, each of which was validated using 100 training/test set partitions. Best average predictivity in terms of R(2)test was achieved at 10 ppm ×10 ppm × Z Å (Z = 0.5, ..., 2.5 Å). It was hypothesized that this optimum depends on the chemical shifts' estimation error (±4.13 ppm) and the precision of the calculated interatomic distances. The highest ranked bins from partial least-squares weights were found to be associated with structural features known to be essential for binding to the estrogen receptor.
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Estrógenos/química , Receptores de Estrógenos/química , Sitios de Unión , Estrógenos/metabolismo , Predicción , Espectroscopía de Resonancia Magnética , Relación Estructura-Actividad Cuantitativa , Receptores de Estrógenos/metabolismoRESUMEN
The Liver Toxicity Biomarker Study is a systems toxicology approach to discover biomarkers that are indicative of a drug's potential to cause human idiosyncratic drug-induced liver injury. In phase I, the molecular effects in rat liver and blood plasma induced by tolcapone (a "toxic" drug) were compared with the molecular effects in the same tissues by dosing with entacapone (a "clean" drug, similar to tolcapone in chemical structure and primary pharmacological mechanism). Two durations of drug exposure, 3 and 28 days, were employed. Comprehensive molecular analysis of rat liver and plasma samples yielded marker analytes for various drug-vehicle or drug-drug comparisons. An important finding was that the marker analytes associated with tolcapone only partially overlapped with marker analytes associated with entacapone, despite the fact that both drugs have similar chemical structures and the same primary pharmacological mechanism of action. This result indicates that the molecular analyses employed in the study are detecting substantial "off-target" markers for the two drugs. An additional interesting finding was the modest overlap of the marker data sets for 3-day exposure and 28-day exposure, indicating that the molecular changes in liver and plasma caused by short- and long-term drug treatments do not share common characteristics.
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Benzofenonas/toxicidad , Catecoles/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Nitrilos/toxicidad , Nitrofenoles/toxicidad , Animales , Biomarcadores/análisis , Proteínas Sanguíneas/análisis , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Femenino , Perfilación de la Expresión Génica , Hígado/química , Hígado/metabolismo , Masculino , Metaboloma/efectos de los fármacos , Metabolómica , Proteoma/análisis , Proteoma/efectos de los fármacos , Proteómica , Ratas , Proyectos de Investigación , Tolcapona , Pruebas de Toxicidad Aguda/métodos , Pruebas de Toxicidad Crónica/métodosRESUMEN
An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals--drugs, pesticides, and environmental pollutants--interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP) enzymes. In the present work, spectral data-activity relationship (SDAR) and structure-activity relationship (SAR) approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV) test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR) spectral descriptors. In the present work, both 1D ¹³C and 1D ¹5N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D ¹³C-NMR and ¹5N-NMR spectra caused an increase in the tenfold cross-validation (CV) performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models.
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Inhibidores del Citocromo P-450 CYP2D6 , Citocromo P-450 CYP2D6/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Polypharmacy increasingly has become a topic of public health concern, particularly as the U.S. population ages. Drug labels often contain insufficient information to enable the clinician to safely use multiple drugs. Because many of the drugs are bio-transformed by cytochrome P450 (CYP) enzymes, inhibition of CYP activity has long been associated with potentially adverse health effects. In an attempt to reduce the uncertainty pertaining to CYP-mediated drug-drug/chemical interactions, an interagency collaborative group developed a consensus approach to prioritizing information concerning CYP inhibition. The consensus involved computational molecular docking, spectral data-activity relationship (SDAR), and structure-activity relationship (SAR) models that addressed the clinical potency of CYP inhibition. The models were built upon chemicals that were categorized as either potent or weak inhibitors of the CYP3A4 isozyme. The categorization was carried out using information from clinical trials because currently available in vitro high-throughput screening data were not fully representative of the in vivo potency of inhibition. During categorization it was found that compounds, which break the Lipinski rule of five by molecular weight, were about twice more likely to be inhibitors of CYP3A4 compared to those, which obey the rule. Similarly, among inhibitors that break the rule, potent inhibitors were 2-3 times more frequent. The molecular docking classification relied on logistic regression, by which the docking scores from different docking algorithms, CYP3A4 three-dimensional structures, and binding sites on them were combined in a unified probabilistic model. The SDAR models employed a multiple linear regression approach applied to binned 1D ¹³C-NMR and 1D ¹5N-NMR spectral descriptors. Structure-based and physical-chemical descriptors were used as the basis for developing SAR models by the decision forest method. Thirty-three potent inhibitors and 88 weak inhibitors of CYP3A4 were used to train the models. Using these models, a synthetic majority rules consensus classifier was implemented, while the confidence of estimation was assigned following the percent agreement strategy. The classifier was applied to a testing set of 120 inhibitors not included in the development of the models. Five compounds of the test set, including known strong inhibitors dalfopristin and tioconazole, were classified as probable potent inhibitors of CYP3A4. Other known strong inhibitors, such as lopinavir, oltipraz, quercetin, raloxifene, and troglitazone, were among 18 compounds classified as plausible potent inhibitors of CYP3A4. The consensus estimation of inhibition potency is expected to aid in the nomination of pharmaceuticals, dietary supplements, environmental pollutants, and occupational and other chemicals for in-depth evaluation of the CYP3A4 inhibitory activity. It may serve also as an estimate of chemical interactions via CYP3A4 metabolic pharmacokinetic pathways occurring through polypharmacy and nutritional and environmental exposures to chemical mixtures.