Amal S. Essader

Analysis of the DNA damage produced by a platinum–acridine antitumor agent and its effects in NCI-H460 lung cancer cells

High-performance liquid chromatography in conjunction with electrospray mass spectrometry (LC-ESMS) was used to structurally characterize the adducts formed by the platinum-acridine agent [PtCl(en)(N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide)](NO(3))(2) (compound 1) in cell-free DNA. Compound 1 forms monofunctional adducts exclusively with guanine, based on the fragments identified in enzymatic digests (dG*, dGMP*, dApG*, and dTpG*, where the asterisk denotes bound drug). The time course of accumulation and DNA adduct formation of compound 1 and the clinical drug cisplatin in NCI-H460 lung cancer cells at physiologically relevant drug concentrations (0.1 μM) was studied by inductively-coupled plasma mass spectrometry (ICP-MS). Compound 1 accumulates rapidly in cells and reaches intracellular levels of up to 60-fold higher than those determined for cisplatin. The hybrid agent shows unusually high DNA binding levels: while cisplatin adducts form at a maximum frequency of 5 adducts per 10(6) nucleotides, compound 1 produces 25 adducts per 10(6) nucleotides after only 3 h of continuous incubation with the lung cancer cells. The high overall levels of compound 1 in the cells and in cellular DNA over the entire 12-h treatment period translate into a rapid decrease in cell viability. Possible implications of these findings for the mechanism of action of compound 1 and the agents potential to overcome tumor resistance to cisplatin are discussed.

Calculation of the isoelectric point of tryptic peptides in the pH 3.5–4.5 range based on adjacent amino acid effects

Current algorithms for the calculation of peptide or protein pI, based on the charge associated with individual amino acids, can calculate pI values to within ±0.2 pI units. Here, we present a new pI calculation algorithm that takes into account the effect of adjacent amino acids on the pI value. The algorithm accounts for the effect of adjacent amino acids ± 3 residues away from a charged aspartic or glutamic acid, as well as effects on the free C terminus, and applies a correction term to the corresponding pK values. The correction increments are derived from a 5000‐peptide training set using a genetic optimization approach. The accuracy of the new pI values obtained with this method approaches the error associated with the manufacture of the IPG strip (<±0.03 pI units). The approach is demonstrated for cytosolic cell extracts derived from the breast‐cancer cell line DU4475, and from membrane preparations from human lung‐tissue samples. One potential application of a more highly accurate pI calculation is data filtering of MS/MS outputs that will allow for more complex database searches including gene finding, and validation, and detection of coding single‐nucleotide polymorphisms in their expressed form.

Development, validation, and application of an ultra-performance liquid chromatography–sector field inductively coupled plasma mass spectrometry method for simultaneous determination of six organotin compounds in human serum

Organotin compounds (OTCs) are heavily employed by industry for a wide variety of applications, including the production of plastics and as biocides. Reports of environmental prevalence, differential toxicity between OTCs, and poorly characterized human exposure have fueled the demand for sensitive, selective speciation methods. The objective of this investigation was to develop and validate a rapid, sensitive, and selective analytical method for the simultaneous determination of a suite of organotin compounds, including butyl (mono-, di-, and tri-substituted) and phenyl (mono-, di-, and tri-substituted) species in human serum. The analytical method utilized ultra-performance liquid chromatography (UPLC) coupled with sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). The small (sub-2 µm) particle size of the UPLC column stationary phase and the sensitivity of the SF-ICP-MS enabled separation and sensitive determination of the analyte suite with a runtime of approximately 3 min. Validation activities included demonstration of method linearity over the concentration range of approximately 0.250-13.661 ng mL(-1), depending on the species; intraday precision of less than 21%, interday precision of less than 18%, intraday accuracy of -5.3% to 19%, and interday accuracy of -14% to 15% for all species; specificity, and matrix impact. In addition, sensitivity, and analyte stability under different storage scenarios were evaluated. Analyte stability was found to be limited for most species in freezer, refrigerator, and freeze-thaw conditions. The validated method was then applied for the determination of the OTCs in human serum samples from women participating in the Snart-Foraeldre/MiljØ (Soon-Parents/Environment) Study. The concentration of each OTC ranged from below the experimental limit of quantitation to 10.929 ng tin (Sn) mL(-1) serum. Speciation values were confirmed by a total Sn analysis.

Development and validation of a high-throughput method for the determination of titanium dioxide in rodent lung and lung-associated lymph node tissues

Abstract There is a need to determine the tissue burdens of inhaled titanium dioxide due to the widespread industrial use of this compound. A high-throughput method for the determination of TiO2 as titanium in rodent lung and lung-associated lymph node tissues by inductively coupled plasma optical emission spectrometry (ICP-OES) was developed and validated. Samples were digested in open-vessel, disposable centrifuge tubes in the presence of nitric and hydrofluoric acids. The determined method quantitation limit (MQL) was 4.2 µg TiO2/g tissue. Replicate control samples prepared at this level were determined with a high level of accuracy (100%) and precision (2% RSD). After validation, the described method was applied to determine the concentration of TiO2 (as titanium) in 600 rodent tissue samples spanning a 16-month time period. During this time period, laboratory control samples (n = 48) were analyzed to assess the long-term precision and accuracy of the method. The average recovery of titanium in these samples was 102±4.8%.

Validation of a Method for the Determination of Total Chromium in Rat Feces by Inductively Coupled Plasma Optical Emission Spectrometry

Abstract The validation of a method for the determination of total chromium in Fischer-344 rat feces by inductively coupled plasma optical emission spectrometry following a rapid, atmospheric-pressure microwave digestion is described. The performance of the method was evaluated over the concentration range of 5.00 to 200 µg Cr/g feces. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limit of detection and limit of quantitation data. Data from a cross-validation method for B6C3F1 mouse feces are also presented. Following validation, the method was applied to analyze samples collected in support of two chronic toxicological investigations.

Development and Optimization of a Procedure for the Determination of Indium-Tin Oxide Particle Size and Concentration in Cellular Media

Indium-tin oxide (ITO) is a solid mixture often comprised of approximately 90% indium oxide and 10% tin oxide by weight. ITO is employed as a transparent conductive coating for flat panel, liquid crystal, and plasma displays and is typically deposited after sintering as a thin film on a substrate through a variety of processes. Unfortunately, there have been published reports of workers who have been adversely impacted through exposure to this compound. When these reports are coupled with the industrial utility of ITO, future toxicological investigations are likely. Comprehensive characterization of ITO test materials would be an essential component of these studies. Ideally, characterization would include assessment of ITO particle size as administered during dosing because this physicochemical parameter can significantly impact solubility. The objective of this task was to develop, optimize, and apply a dynamic light scattering (DLS) sample preparation and measurement protocol for determination of particle size for a suite of sintered/non-sintered ITO samples prepared in cellular growth media at nominal 0.3 mg/mL doses. Sonication time was evaluated from 15 to 90 minutes over a 24-hour, continuous DLS measurement period. Resulting suspensions from samples subjected to 30 minute sonication times were stable with respect to hydrodynamic particle size between 10 and 24 hours after sonication. Throughout the 24-hour DLS measurement period, suspension aliquots were analyzed by inductively coupled plasma mass spectrometry to determine indium concentration. The developed protocol enabled investigators to use ITO suspensions of known hydrodynamic particle size and concentration in their in vitro cellular study.

Characterization of Zinc Carbonate Basic as a Source of Zinc in a Rodent Study Investigating the Effects of Dietary Deficiency or Excess

ABSTRACT Zinc deficiency and excess can result in adverse health outcomes. There is conflicting evidence regarding whether excess or deficient zinc in the diet can contribute to carcinogenicity. The objective of this study was to characterize zinc carbonate basic for use as a source of dietary zinc in a rodent toxicity and carcinogenicity study investigating the effects of zinc deficiency and excess. Because of the complex chemistries of zinc carbonate basic compounds, inconsistent nomenclature, and literature and reference spectra gaps, it was necessary to use multiple analytical techniques, including Karl Fischer titration, combustion analysis, inductively coupled plasma–optical emission spectrometry, X-ray diffraction, infrared spectroscopy, X-ray fluorescence spectrometry, and thermogravimetric analysis to characterize the test article. Based on the collective evidence and through the process of elimination, the test article was found to be composed mainly of zinc carbonate basic with zinc oxide as a minor component. The zinc content was determined to be 56.6% (w/w) with heavy metals such as arsenic, cadmium, mercury, and lead below the limit of quantitation of less than or equal to 0.01%. The test material was stable at ambient temperature. Based on the work described in this manuscript, the test article was suitable for use as a source of zinc in studies of deficiency and excess in the diet.