Glutathione and Xanthine Metabolic Changes in Tamoxifen Resistant Breast Cancer Cell Lines are Mediated by Down-Regulation of GSS and XDH and Correlated to Poor Prognosis.

Background: Tamoxifen is commonly used in the treatment of hormonal-positive breast cancer. However, 30%-40% of tumors treated with tamoxifen develop resistance; therefore, an important step to overcome this resistance is to understand the underlying molecular and metabolic mechanisms. In the present work, we used metabolic profiling to determine potential biomarkers of tamoxifen resistance, and gene expression levels of enzymes important to these metabolites and then correlated the expression to the survival of patients receiving tamoxifen. Methods: Tamoxifen-resistant cell lines previously developed and characterized in our laboratory were metabolically profiled with nuclear magnetic resonance spectroscopy (NMR) using cryogenic probe, and the findings were correlated with the expression of genes that encode the key enzymes of the significant metabolites. Moreover, the effect of significantly altered genes on the overall survival of patients was assessed using the Kaplan-Meier plotter web tool. Results: We observed a significant increase in the levels of glutamine, taurine, glutathione, and xanthine, and a significant decrease in the branched-chain amino acids, valine, and isoleucine, as well as glutamate and cysteine in the tamoxifen-resistant cells compared to tamoxifen sensitive cells. Moreover, xanthine dehydrogenase and glutathione synthase gene expression were downregulated, whereas glucose-6-phosphate dehydrogenase was upregulated compared to control. Additionally, increased expression of xanthine dehydrogenase was associated with a better outcome for breast cancer patients. Conclusion: Overall, this study sheds light on metabolic pathways that are dysregulated in tamoxifen-resistant cell lines and the potential role of each of these pathways in the development of resistance.

Effect of Cigarette Smoke Exposure and Aspirin Treatment on Neurotransmitters' Tissue Content in Rats' Hippocampus and Amygdala.

Cigarette smoke withdrawal can cause anxiety-like behavior and modulate neurotransmitter-related proteins in the brain. We examined the effects of cigarette smoke with and without aspirin treatment on the concentrations of neurotransmitters, including dopamine, serotonin, glutamate, glutamine, and GABA in the amygdala and hippocampus. Sprague-Dawley rats were randomly assigned to four different groups: (1) control group exposed only to standard room air, (2) cigarette smoke exposed group treated with saline vehicle, (3) cigarette smoke exposed group treated with aspirin (30 mg/kg), and (4) control group treated only with aspirin (30 mg/kg). Cigarette smoke exposure was performed for 2 h/day, 5 days/week, for 31 days. Behavioral testing was carried out weekly, 24 h after cigarette smoke exposure, during acute withdrawal. At the end of week 4, rats were given either distilled water (1 mL) or aspirin 45 min before cigarette exposure for 11 days. Dopamine, serotonin, glutamate, glutamine, and GABA were extracted from both the amygdala and hippocampus and were separated and quantified using a developed and validated HPLC-MS/MS method. Cigarette smoke withdrawal induced anxiety behaviors, and aspirin treatment reduced this effect. Cigarette smoke exposure increased tissue content of dopamine, serotonin, glutamate, glutamine, and GABA, and aspirin treatment reversed this effect. Cigarette smoke caused an increase in tissue content of several neurotransmitters as well as anxiety-like behavior, and these effects were normalized by aspirin treatment.

Rapid and sensitive HPLC-MS/MS method for the quantification of dopamine, GABA, serotonin, glutamine and glutamate in rat brain regions after exposure to tobacco cigarettes.

Tobacco smoking is a preventable main cause of fatal diseases. Accurate measurements of the effects it has on neurotransmitters are essential in developing new strategies for smoking cessation. Moreover, measurements of neurotransmitter levels can aid in developing drugs that counteract the effects of smoking. The aim of this study is to develop and validate a fast, simultaneous and sensitive method for measuring the levels of neurotransmitters in rat brain after the exposure of tobacco cigarettes. The selected neurotransmitters include dopamine, GABA, serotonin, glutamine and glutamate. The method is based on high-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved within 3 min using a Zorbax SB C18 column (3.0 × 100 mm, 1.8 µm particle size). The mobile phase consisted of HPLC-grade water and acetonitrile each containing 0.3% heptafluorobutyric acid and 0.5% formic acid at gradient conditions. The linear range was 0.015-0.07, 825-7,218, 140-520, 63.42-160.75 and 38.25 × 103 to 110.35 × 103  ng/ml for dopamine, GABA, serotonin, glutamine and glutamate, respectively. Inter- and intra-run accuracy were in the range 97.82-103.37% with a precision (CV%) of ≤0.90%. The results revealed that 4 weeks of cigarette exposure significantly increased neurotransmitter levels after exposure to tobacco cigarettes in various brain regions, including the hippocampus and the amygdala. This increase in neurotransmitters levels may in turn activate the nicotine dependence pathway.

Lactate and pyruvate levels correlation with lactate dehydrogenase gene expression and glucose consumption in Tamoxifen-resistant MCF-7 cells using capillary electrophoresis with contactless conductivity detection (CE-C4 D).

Breast cancer is the second leading cause of cancer death in women after lung cancer. The first-line treatment of metastatic breast cancer in premenopausal women relies on tamoxifen. The development of tamoxifen resistance is not fully understood. In this study, capillary electrophoresis with capacitively coupled contactless conductivity detector was developed to monitor the changes in lactate and pyruvate levels in supernatant media of three models of developed MCF-7 tamoxifen-resistant cells and correlate these metabolites changes with lactate dehydrogenase genes expression and glucose consumption. The electrophoretic separation was achieved under reversed electroosmotic flow conditions. The linear ranges were 0.15-5 and 0.01-1 mM with a correlation coefficient of 0.9966 and 0.9971 and the limits of detection were 0.01 and 0.02 µM for lactate and pyruvate, respectively. Inter- and intrarun accuracy were in the range of 96.88-105.94% with precision (CV, %) of ≤7.35%. The method was completely validated and the results were in agreement with those obtained using the lactate and glucose assay kits. The results revealed a significant increase in both lactate and pyruvate production in the three tamoxifen-resistant MCF-7 cells models compared to control cells. This increase was correlated with the increase of lactate dehydrogenase genes expression and the increase of glucose consumption.

High Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes.

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography-tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid-ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11-2.25, 0.012-0.227, and 0.02-0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

Changes in Lactate Production, Lactate Dehydrogenase Genes Expression and DNA Methylation in Response to Tamoxifen Resistance Development in MCF-7 Cell Line.

Lactate dehydrogenase (LDH) is a key enzyme in the last step of glycolysis, playing a role in the pyruvate-to-lactate reaction. It is associated with the prognosis and metastasis of many cancers, including breast cancer. In this study, we investigated the changes in LDH gene expression and lactate concentrations in the culture media during tamoxifen resistance development in the MCF-7 cell line, and examined LDHB promoter methylation levels. An upregulation of 2.9 times of LDHB gene expression was observed around the IC50 concentration of tamoxifen in treated cells, while fluctuation in LDHA gene expression levels was found. Furthermore, morphological changes in the cell shape accompanied the changes in gene expression. Bisulfate treatment followed by sequencing of the LDHB promoter was performed to track any change in methylation levels; hypomethylation of CpG areas was found, suggesting that gene expression upregulation could be due to methylation level changes. Changes in LDHA and LDHB gene expression were correlated with the increase in lactate concentration in the culture media of treated MCF-7 cells.

Preferential Accumulation of Phospholipid-PEG and Cholesterol-PEG Decorated Gold Nanorods into Human Skin Layers and Their Photothermal-Based Antibacterial Activity.

Herein, a library of gold nanorods (GNR) decorated with polyethylene glycol-thiol (PEG-SH) containing different functionalities were synthesized and characterized by optical absorption spectroscopy, zeta potential, dynamic light scattering (DLS), transmission electron microscope (TEM) and proton nuclear magnetic resonance (1H-NMR). The colloidal stability of GNR when exposed to skin, and their preferential accumulation into excised human skin layers were investigated. Confocal laser scanning microscopy, transmission electron microscope (TEM) and inductively coupled plasma-optical emission spectroscopy (ICP-OES) were utilized to track the penetration of GNR into different skin layers. The results demonstrated that cholesterol-PEG coated GNR were preferentially loaded up in the upper layers of skin (stratum corneum), while phospholipid-PEG coated counterparts were drastically deposited in skin dermis. Neutral methoxy-PEG-coated GNR were distributed in both SC and dermis skin layers, while charged GNR (anionic-carboxylic acid-PEG-GNR and cationic-amine-PEG-GNR) revealed a minimal accumulation into skin. DSPE-PEG-GNR and Chol-PEG-GNR demonstrated antibacterial activities against Staphylococcus aureus (S aureus) at MIC values of 0.011 nM and 0.75 nM, respectively. Photothermal treatment for S. aureus at sub-MIC concentrations resulted in a significant bactericidal effect when using Chol-PEG-GNR but not DSPE-PEG-GNR. Gold-based nanoscale systems have great value as a promising platform for skin diseases therapy.

Liquid chromatography-tandem mass spectrometry for rapid and selective simultaneous determination of fluoroquinolones level in human aqueous humor.

Endophthalmitis, an intraocular infection, may lead to irreversible loss of vision. Antimicrobial chemotherapy is prescribed prior to ocular surgical procedures to avoid endophthalmitis. Fluoroquinolones are the most commonly prescribed and used antibiotics during such procedures. However, the selection of a single entity and proper regimen is not specified in medical guidelines. The objective of this study is to develop a rapid and selective simultaneous high-performance liquid chromatography-tandem mass spectroscopic method to explore the bioavailability of 4 fluoroquinolones, including 0.5% moxifloxacin hydrochloride, 0.3% gatifloxacin, 0.3% ciprofloxacin hydrochloride, and 0.3% ofloxacin, in human aqueous humor after antibiotic topical administration using gemifloxacin as Internal Standard according to the European Medicines Agency (EMA) and US Food and Drug Administration (FDA) guidelines. The newly validated method was capable of accurately and precisely quantifying each antibiotic at the lowest reported lower limit of quantification of 10 ng/mL and operating on a very low pipetting volume of 15 µL, indicating a reliable quantitation of all analytes simultaneously using a very small quantity of aqueous humor with total chromatographic run time of 2.5 min. Sixty-seven patients were divided into 4 groups for each antibiotic. Before the cataract surgery, each group received 4 drops of one of the antibiotics over 1 h, separated by 15 min time interval. After 15-20 min from the last drop, approximately 50-100 µL of aqueous humor was collected during surgery for analysis. The average concentrations of moxifloxacin, gatifloxacin, ofloxacin and ciprofloxacin in aqueous humor samples were 891.8, 271.7, 191.4 and 69.5 ng/mL, respectively. Only moxifloxacin average concentration was higher than the minimum inhibitory concentration for the common endophthalmitis pathogens.

The Safety Assessment of Toxic Metals in Commonly Used Pharmaceutical Herbal Products and Traditional Herbs for Infants in Jordanian Market.

The objective of this study was to assess the levels of contamination by toxic metals (Pb, Al, Ni, Cd and As) that may be present in 25 infant pharmaceutical herbal products and 15 traditional herbs in Jordan. Both products and medicinal herbs are currently prescribed by paediatricians. They are available as over-the-counter medicines and are sold the in herbal market, ensuring easy accessibility for parents. Inductively coupled plasma-optical emission spectroscopy (ICP-OES), with limit of detections (LODs) of 0.10, 1.00, 0.20, 0.15 and 2.00 mg.kg-1 for Pb, Al, Ni, Cd and As respectively, was employed to measure the levels of toxic metals in the samples. Pb, Al and Ni were detected in 88, 76 and 4% of the analysed samples of pharmaceutical herbal products and in 93, 87 and 13% of the analysed samples of traditional herbs, respectively. Neither Cd or As were detected in all analysed samples. The data obtained were subsequently compared by referral to the acceptable limits of toxic heavy metals according to World Health Organisation (WHO) standards. Largely, the results showed acceptable toxic metal levels in the finished pharmaceutical products and the traditional medicinal herbs for infants. One exception to this was Persian Thyme (Satureja thymbra) with Pb content of 41.18 mg.kg-1. Also, the daily intake of detected metals through pharmaceutical herbal products was found to be lower than the daily tolerable intake limit set by the regulatory bodies, except of 8% of products that exceeded the tolerable daily intake of Pb set by US FDA, as compared to traditional medicinal herbs, where the tolerable daily intake for Pb, Al and Ni were exceeded in 40, 60 and 8% of the analysed herbs, respectively. The results obtained revealed that the excessive use of medicinal plants as alternative medicine should be used with caution keeping in mind the safety factor in infants.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016-2018).

One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.

Time-Resolved Pharmacological Studies using Automated, On-line Monitoring of Five Parallel Suspension Cultures.

Early stage pharmacological studies rely on in vitro methodologies for screening and testing compounds. Conventional assays based on endpoint measurements provide limited information because the lack in temporal resolution may not determine the pharmacological effect at its maximum. We developed an on-line, automated system for near real-time monitoring of extracellular content from five parallel suspension cultures, combining cell density measurements with a high-resolution separations every 12 minutes for 4 days. Selector and switching valves provide the fluidic control required to sample from one culture during the analysis of the previous sample from another culture, a time-saving measure that is fundamental to the throughput of the presented system. The system was applied to study the metabolic effects of the drugs rotenone, ß-lapachone and clioquinol using lactate as metabolic indicator. For each drug, 96 assays were executed on the extracellular matrix at three concentrations with two controls in parallel, consuming only 5.78 mL of media from each culture over four days, less than 60 µL per analysis. The automated system provides high sample throughput, good temporal resolution and low sample consumption combined with a rugged analytical method with adequate sensitivity, providing a promising new platform for pharmacological and biotechnological studies.

Capillary electrophoresis for automated on-line monitoring of suspension cultures: Correlating cell density, nutrients and metabolites in near real-time.

Increasingly stringent demands on the production of biopharmaceuticals demand monitoring of process parameters that impact on their quality. We developed an automated platform for on-line, near real-time monitoring of suspension cultures by integrating microfluidic components for cell counting and filtration with a high-resolution separation technique. This enabled the correlation of the growth of a human lymphocyte cell line with changes in the essential metabolic markers, glucose, glutamine, leucine/isoleucine and lactate, determined by Sequential Injection-Capillary Electrophoresis (SI-CE). Using 8.1 mL of media (41 µL per run), the metabolic status and cell density were recorded every 30 min over 4 days. The presented platform is flexible, simple and automated and allows for fast, robust and sensitive analysis with low sample consumption and high sample throughput. It is compatible with up- and out-scaling, and as such provides a promising new solution to meet the future demands in process monitoring in the biopharmaceutical industry.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2012-2014).

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods, covering the period July 2012-July 2014. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to ITP, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.

On-line sequential injection-capillary electrophoresis for near-real-time monitoring of extracellular lactate in cell culture flasks.

Cell culture has replaced many in vivo studies because of ethical and regulatory measures as well as the possibility of increased throughput. Analytical assays to determine (bio)chemical changes are often based on end-point measurements rather than on a series of sequential determinations. The purpose of this work is to develop an analytical system for monitoring cell culture based on sequential injection-capillary electrophoresis (SI-CE) with capacitively coupled contactless conductivity detection (C(4)D). The system was applied for monitoring lactate production, an important metabolic indicator, during mammalian cell culture. Using a background electrolyte consisting of 25mM tris(hydroxymethyl)aminomethane, 35mM cyclohexyl-2-aminoethanesulfonic acid with 0.02% poly(ethyleneimine) (PEI) at pH 8.65 and a multilayer polymer coated capillary, lactate could be resolved from other compounds present in media with relative standard deviations 0.07% for intraday electrophoretic mobility and an analysis time of less than 10min. Using the human embryonic kidney cell line HEK293, lactate concentrations in the cell culture medium were measured every 20min over 3 days, requiring only 8.73µL of sample per run. Combining simplicity, portability, automation, high sample throughput, low limits of detection, low sample consumption and the ability to up- and outscale, this new methodology represents a promising technique for near real-time monitoring of chemical changes in diverse cell culture applications.

Capillary electrophoresis for monitoring bioprocesses.

Chemical characterization and monitoring of fermentation broths and cell culture media provide significant information on the changes occurring within these complex and dynamic systems. Analytical methods based on CE in capillaries and microchips are attractive for integration in instrumental tools to obtain this critical data, improving the understanding and control of bioprocesses. In this review, the use of CE for chemical characterization and monitoring fermentations is discussed, organized by analyte class, including organic acids, pharmaceuticals, proteins, sugars, amino acids, and metabolites published between 1992 and October 2012. A section is dedicated to the roles CE plays throughout the wine making process, where applications range from characterization and increase in fundamental understanding of the fermentation to forensic applications, verifying the authenticity of the wine.