Gene Expression Profiling in Ecotoxicology

Gene expression profiling is a powerful new end point for ecotoxicology and a means for bringing the genomics revolution to this field. We review the usefulness of gene expression profiling as an end point in ecotoxicology and describe methods for applying this approach to non-model organisms. Since genomes contain thousands of genes representing hundreds of pathways, it is possible to identify toxicant-specific responses from this wide array of possibilities. Stressor-specific signatures in gene expression profiles can be used to diagnose which stressors are impacting populations in the field. Screening for stress-induced genes requires special techniques in organisms without sequenced genomes. These techniques include differential display polymerase chain reaction (DD PCR), suppressive subtractive hybridization PCR (SSH PCR), and representational difference analysis. Gene expression profiling in model organisms like yeast has identified hundreds of genes that are up-regulated in response to various stressors, including several that are well characterized (e.g., hsp78, metallothionein, superoxide dismutase). Using consensus PCR primers from several animal sequences, it is possible to amplify some of these well characterized stress-induced genes from organisms of interest in ecotoxicology. We describe how several stress-induced genes can be grouped into cDNA arrays for rapidly screening samples.     

Profiling of phenolics from Tephrosia cinerea

An HPLC-ESIMS(n) method, based on high-performance liquid chromatography coupled to electrospray positive ionization multistage ion trap mass spectrometry, has been used to rapidly identify and guide the isolation of phenolic constituents from the methanol extract of the aerial parts of T. cinerea. On the basis of the results of the online screening by HPLC-ESIMS (n), 17 phenolic constituents, including two new compounds, namely demethylapollinin 7- O- β-D-glucopyranoside (1) and cineroside A (17), were isolated, and their structures were unambiguously assigned by the extensive use of 1D- and 2D-NMR experiments.     

Olanzapine,but Not Fluoxetine,Treatment Increases Survival in Activity-Based Anorexia in Mice

Anorexia nervosa (AN) is an eating disorder characterized by extreme hypophagia, hyperactivity, and fear of weight gain. No approved pharmacological treatments exist for AN despite high mortality rates. The activity-based anorexia (ABA) phenomenon models aspects of AN in rodents, including progressive weight loss, reduced food intake, and hyperactivity. First, we optimized the ABA paradigm for mice. We compared mouse strains (Balb/cJ, A/J) for susceptibility with ABA, and evaluated the effects of different food access durations (2, 4, 6, 8, and 10 h) on ABA parameters. Balb/cJ mice exhibited significantly shorter survival time (days until 25% bodyweight loss) in the ABA paradigm compared with A/J mice. Furthermore, 6 h of food access reduced survival in mice housed with wheels without reducing survival in mice housed without wheels. We then evaluated the effects of chronic treatment with fluoxetine (4 weeks) or subchronic treatment with olanzapine (OLZ) (1 week) on ABA in BALB/cJ mice. OLZ (12 mg/kg/day) significantly increased survival and reduced food anticipatory activity (FAA). However, OLZ did not alter food intake or running wheel activity during ad-lib feeding (baseline) or restriction conditions, or in mice housed without wheels. Fluoxetine (18 mg/kg/day) increased food intake and reduced FAA, but did not alter survival. Here, we report for the first time that OLZ, but not fluoxetine, reduces ABA in mice. Our findings indicate further need for clinical investigations into the effects of OLZ, but not selective serotonin reuptake inhibitors, on core features of AN.     

The Cannabinoid Receptor Agonist THC Attenuates Weight Loss in a Rodent Model of Activity-Based Anorexia

Anorexia nervosa (AN) is characterized by anhedonia whereby patients experience little pleasure or reward in many aspects of their lives. Reward pathways and the endocannabionid system have been implicated in the mediation of food intake. The potential to exploit these systems to reverse weight loss is investigated in a rodent model of activity-based anorexia (ABA). The effect of subchronic (6 days) Δ⁹-tetrahydrocannabinol (THC) treatment (0.1, 0.5, or 2.0 mg/kg/day) was assessed on chow and high-fat diet (HFD) intake, body weight, running wheel activity (RWA) as well as thermogenesis in brown adipose tissue (BAT) and lipid metabolism in white adipose tissue (WAT). Limited time availability of food and continuous access to running wheels led to anorexia and significantly reduced body weight. THC treatment (0.5 and 2.0 mg/kg/day) transiently stimulated chow intake with a moderate effect on RWA. THC (2.0 mg/kg/day) significantly reduced body weight loss and shifted markers of thermogenesis in BAT and lipid metabolism in WAT in directions consistent with reduced energy expenditure and lipolysis. THC (2.0 mg/kg/day) combined with HFD, produced a transient increase in food intake, reduction in RWA, attenuation of body weight loss, and changes in markers of thermogensis in BAT and lipolysis in the WAT. These changes were significantly greater than those seen in vehicle (HFD), vehicle (chow), and THC (chow)-treated animals. These data show for the first time the effectiveness of the endocannabinoid system in attenuating the weight loss associated with the development of ABA via a mechanism involving reduced energy expenditure.     

Profiling proteasome activity in tissue with fluorescent probes

With proteasome inhibitors in use in the clinic for the treatment of multiple myeloma and with clinical trials in progress investigating the treatment of a variety of hematologic and solid malignancies, accurate methods that allow profiling of proteasome inhibitor specificity and efficacy in patients are in demand. Here, we describe the development, full biochemical validation, and comparison of fluorescent proteasome activity reporters that can be used to profile proteasome activities in living cells with high sensitivity. Seven of the synthesized probes tested label proteasomes in lysates, although the fluorescent dye used affects their specificity. Two differentially labeled probes tested are suitable for studying proteasome activity in living cells by gel-based assays, by confocal laser scanning microscopy, and by flow cytometry. We established methods using these fluorescent reporters to profile proteasome activity in different mouse tissues, carefully avoiding postlysis artifacts, and we show that proteasome subunit activity is regulated in an organ-specific manner. The techniques described here could be used to study in vivo pharmacological properties of proteasome inhibitors.     

Detection of Resistance to Imatinib by Metabolic Profiling

Acquired resistance to imatinib mesylate is an increasing and continued challenge in the treatment of BCR-ABL tyrosine kinase positive leukemias as well as gastrointestinal stromal tumors. Stable isotope-based dynamic metabolic profiling (SIDMAP) studies conducted in parallel with the development and clinical testing of imatinib revealed that this targeted drug is most effective in controlling glucose transport, direct glucose oxidation for RNA ribose synthesis in the pentose cycle, as well as de novo long-chain fatty acid synthesis. Thus imatinib deprives transformed cells of the key substrate of macromolecule synthesis, malignant cell proliferation, and growth. Tracer-based magnetic resonance spectroscopy studies revealed a restitution of mitochondrial glucose metabolism and an increased energy state by reversing the Warburg effect, consistent with a subsequent decrease in anaerobic glycolysis. Recent in vitro SIDMAP studies that involved myeloid cells isolated from patients who developed resistance against imatinib indicated that non-oxidative ribose synthesis from glucose and decreased mitochondrial glucose oxidation are reliable metabolic signatures of drug resistance and disease progression. There is also evidence that imatinib-resistant cells utilize alternate substrates for macromolecule synthesis to overcome limited glucose transport controlled by imatinib. The main clinical implications involve early detection of imatinib resistance and the identification of new metabolic enzyme targets with the potential of overcoming drug resistance downstream of the various genetic and BCR-ABL-expression derived mechanisms. Metabolic profiling is an essential tool used to predict, clinically detect, and treat targeted drug resistance. This need arises from the fact that targeted drugs are narrowly conceived against genes and proteins but the metabolic network is inherently complex and flexible to activate alternative macromolecule synthesis pathways that targeted drugs fail to control.     

Regulatory Aspects of Pharmacokinetic Profiling in Special Populations

The clinical efficacy and safety profile of a new medicinal product is established in phase III studies, which are usually restricted to a well defined patient population. This population may not fully represent the population in which the product will be used once it is on the market. Pharmacokinetic studies in special populations are performed to estimate drug exposure in subpopulations of patients with characteristics that may affect drug exposure. The clinical consequences of altered exposure are then assessed, taking pharmacokinetic/pharmacodynamic relationships into consideration. If needed, specific treatment recommendations should be developed.Recommendations regarding pharmacokinetic characterization in special populations are given in a number of European guidelines. The pharmacokinetic characteristics, therapeutic window and intended use of the medicinal product influence the need for pharmacokinetic studies of a new medicinal product. There are a number of methodological issues to be considered when designing pharmacokinetic studies in special populations: the study design, study population and control group, the dosing regimen to be used, the analytes to be measured, and the distribution and range of the factor to be studied. The data should be presented in sufficient detail to enable assessment by regulatory authorities of the conducted analysis and conclusions drawn. Assessment of the data should include an evaluation of how and to what extent the pharmacokinetics in specific subpopulations deviate from the exposure at the therapeutic dose in the clinical efficacy and safety studies, and if there is a need for specific treatment recommendations. Based on the available information on the pharmacokinetic/pharmacodynamic relationships for efficacy and safety and/or the exposure at the therapeutic dose in the phase III population where efficacy and safety have been demonstrated, target criteria (a target exposure range) should be defined. Within the target exposure range, there should be no clinically relevant difference in efficacy and safety. Should the exposure in a specific group fall outside the defined target criteria, appropriate treatment recommendations need to be developed. The aim should be to develop dosing recommendations that will allow the majority of the patients to obtain exposure within the defined target range. If it is not possible to develop suitable dosing recommendations in a subgroup of patients, there may be a need for specific warnings or wordings regarding monitoring of the patients. It may also be an option to exclude that patient group from the indication. The resulting treatment recommendations should ensure safe and effective use of the drug in the entire population for which it has been approved.     

Profiling the reactive metabolites of xenobiotics using metabolomic technologies

A predominant pathway of xenobiotic-induced toxicity is initiated by bioactivation. Characterizing reactive intermediates will provide information on the structure of reactive species, thereby defining a potential bioactivation mechanism. Because most reactive metabolites are not stable, it is difficult to detect them directly. Reactive metabolites can form adducts with trapping reagents, such as glutathione, which makes the reactive metabolites detectable. However, it is challenging to "fish" these adducts out from a complex biological matrix, especially for adducts generated via uncommon metabolic pathways. In this regard, we developed a novel approach based upon metabolomic technologies to screen trapped reactive metabolites. The bioactivation of pulegone, acetaminophen, and clozapine were reexamined by using this metabolomic approach. In all these cases, a large number of trapped reactive metabolites were readily identified. These data indicate that this metabolomic approach is an efficient tool to profile xenobiotic bioactivation.     

Profiling drug membrane permeability and activity via biopartitioning chromatography

Drug in vivo pharmacokinetic performances in nature consist of sequential membrane transporting processes and are based on the entry into and exit of drugs from cell, even for metabolism process requiring parent drugs delivered into and metabolites effluxed from the metabolizing cells. Efficient and reliable high throughput screen of membrane permeability properties as early as possible in drug discovery and development program is accordingly desirable. Biopartitioning chromatography (BPC) introduces biomembrane-mimetic structures (such as liposome, phospholipid monolayer, micelle, microemulsion, vesicle and bicelle, etc) into chromatographic system, i.e. liquid system or capillary electrophoresis, and thereby emulates drug-membrane interactions difficult to study in the liquid state by well reproducible, rapid, sensitive and adequately designed chromatographic technique. And recently BPC has been becoming a high-throughput screening platform for drug membrane permeability and biological activity. The theoretical basis, classification and application of BPC were summarized based on the latest advances and our recent works. The development potential and perspectives of this field were also discussed.     

Modeling of Compound Profiling Experiments Using Support Vector Machines

Profiling of compounds against target families has become an important approach in pharmaceutical research for the identification of hits and analysis of selectivity and promiscuity patterns. We report on modeling of profiling experiments involving 429 potential inhibitors and a panel of 24 different kinases using support vector machine (SVM) techniques and naïve Bayesian classification. The experimental matrix contained many different activity profiles. SVM predictions achieved overall high accuracy due to consistently low false‐positive and consistently high true‐negative rates. However, predictions for promiscuous inhibitors were affected by false‐negative rates. Combined target‐based SVM classifiers reached or exceeded the performance of SVM profile prediction methods and were superior to Bayesian classification. The classifiers displayed different prediction characteristics including diverse combinations of false‐positive and true‐negative rates. Predicted and experimentally observed compound activity profiles were compared in detail, revealing activity patterns modeled with different accuracy.     

Profiling Cytosine Oxidation in DNA by LC-MS/MS

Spontaneous and oxidant-induced damage to cytosine is probably the main cause of CG to TA transition mutations in mammalian genomes. The reaction of hydroxyl radical (·OH) and one-electron oxidants with cytosine derivatives produces numerous oxidation products, which have been identified in large part by model studies with monomers and short oligonucleotides. Here, we developed an analytical method based on LC-MS/MS to detect 10 oxidized bases in DNA, including 5 oxidation products of cytosine. The utility of this method is demonstrated by the measurement of base damage in isolated calf thymus DNA exposed to ionizing radiation in aerated aqueous solutions (0-200 Gy) and to well-known Fenton-like reactions (Fe(2+) or Cu(+) with H(2)O(2) and ascorbate). The following cytosine modifications were quantified as modified 2'-deoxyribonucleosides upon exposure of DNA to ionizing radiation in aqueous aerated solution: 5-hydroxyhydantoin (Hyd-Ura) > 5-hydroxyuracil (5-OHUra) > 5-hydroxycytosine (5-OHCyt) > 5,6-dihydroxy-5,6-dihydrouracil (Ura-Gly) > 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (Imid-Cyt). The total yield of cytosine oxidation products was comparable to that of thymine oxidation products (5,6-dihydroxy-5,6-dihydrothymine (Thy-Gly), 5-hydroxy-5-methylhydantotin (Hyd-Thy), 5-(hydroxymethyl)uracil (5-HmUra), and 5-formyluracil (5-ForUra)) as well as the yield of 8-oxo-7,8-dihydroguanine (8-oxoGua). The major oxidation product of cytosine in DNA was Hyd-Ura. In contrast, the formation of Imid-Cyt was a minor pathway of DNA damage, although it is the major product arising from irradiation of the monomers, cytosine, and 2'-deoxycytidine. The reaction of Fenton-like reagents with DNA gave a different distribution of cytosine derived products compared to ionizing radiation, which likely reflects the reaction of metal ions with intermediate peroxyl radicals or hydroperoxides. The analysis of the main cytosine oxidation products will help elucidate the complex mechanism of oxidative degradation of cytosine in DNA and probe the consequences of these reactions in biology and medicine.     

Profiling of metal ions leached from pharmaceutical packaging materials

Metal leachables from packaging components can affect the safety and efficacy of a pharmaceutical formulation. As liquid formulations continue to contain surfactants, salts, and chelating agents coupled with lower drug levels, the interaction between the formulation and the packaging material becomes more important. This study examines the interaction of commonly used packaging materials with extraction solvents representative of liquid formulations found in the pharmaceutical industry stressed under conditions encountered during accelerated stability studies.     

非轴对称端壁叶栅内部流场特性的实验

摘要：通过风洞实验对三角函数非轴对称端壁造型法和压差非轴对称端壁造型法设计的叶栅流道在设计工况和变工况下的流场参数进行了测量。实验结果表明：不同来流条件时，非轴对称端壁造型对于流场参数影响也呈现出一些新特点。由于压力面与吸力面上压力改变的幅度大小会因为来流条件的变化而不同，当地总压损失系数会随着马赫数的降低而减小，二次流速度矢量分布规律不会随着马赫数的变化而不同，但二次流速度大小及通道涡系强度会随着马赫数的降低而减小。     

Targeted Kinase Selectivity from Kinase Profiling Data

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Profiling urinary metabolites of naproxen by liquid chromatography-electrospray mass spectrometry

Glucuronidation, an important metabolic process for the biotransformation of drugs into easily eliminable water-soluble detoxification products, can also lead to biologically active or toxic glucuronide conjugates. The present work describes a liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) approach for the characterization of naproxen and O-6-desmethylnaproxen glucuronides. The method is fast and efficient and permitted to individuate alpha and beta isomers of both naproxen and O-6-desmethylnaproxen glucuronides. The procedure could be potentially extended to the characterization of other drug metabolites.     

Solubility Profiling of HIV Protease Inhibitors in Human Intestinal Fluids

The present study pursued to profile the intestinal solubility of nine HIV protease inhibitors (PIs) in fasted- and fed-state human intestinal fluids (FaHIF, FeHIF) aspirated from four volunteers. In addition, the ability of fasted- and fed-state simulated intestinal fluids (FaSSIF, FeSSIF) to predict the intestinal solubility was evaluated. All PIs were poorly soluble in FaHIF (from 7-μM for ritonavir to 327-μM for darunavir) and FeHIF (from 15-μM for atazanavir to 409μM for darunavir). For four of nine PIs, food intake significantly enhanced the solubilizing capacity of intestinal fluids (up to 18.4-fold increase for ritonavir). The intersubject variability (average coefficient of variance CV_fed = 60.6%, CV_fasted = 40.4%) was higher as compared with the intrasubject variability (CV_fed = 41.3%, CV_fasted = 20.5%). PI solubilities correlated reasonably well between FaSSIF and FaHIF (R = 0.817), but not between FeSSIF and FeHIF (R = 0.617). To conclude, postprandial conditions increased the inter- and intrasubject variability of the PIs. The inability of FeSSIF to accurately predict the FeHIF solubility emphasizes the need for a multivariate approach to determine solubility profiles, taking into account solid-state characteristics, pH, mixed bile acid/phospholipid micelles, and digestive products. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3800–3807, 2013