Shotgun Proteomic-Based Approach with a Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometer for Protein Adductomics on a 3D Human Brain Tumor Neurospheroid Culture Model: The Identification of Adduct Formation in Calmodulin-Dependent Protein Kinase-2 and Annexin-A1 Induced by Pesticide Mixture.

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.

Research of Pesticide Metabolites in Human Brain Tumor Tissues by Chemometrics-Based Gas Chromatography-Mass Spectrometry Analysis for a Hypothetical Correlation between Pesticide Exposure and Risk Factor of Central Nervous System Tumors.

Pesticides are widely used, resulting in continuing human exposure with potential health impacts. Some exposures related to agricultural works have been associated with neurological disorders. Since the 2000s, the hypothesis of the role of pesticides in the occurrence of central nervous system (CNS) tumors has been better documented in the literature. However, the etiology of childhood brain cancers still remains largely unknown. The major objective of this work was to assess the potential role of pesticide exposure as a risk factor for CNS tumors based on questionnaires and statistical analysis of information collected from patients hospitalized in the Neurosurgery Department of the Habib Bourguiba Hospital Medium in Sfax, Tunisia, during the period from January 1, 2022, to May 31, 2023. It also aimed to develop a simple and rapid analytical method by the gas chromatography-mass spectrometry technique for the research traces of pesticide metabolites in some collected human brain tumor tissues in order to more emphasize our hypothesis for such a correlation between pesticide exposure and brain tumor development. Patients with a history of high-risk exposure were selected to conduct further analysis. Chemometric methods were adapted to discern intrinsic variation between pathological and control groups and ascertain effective separation with the identification of differentially expressed metabolites accountable for such variations. Three samples revealed traces of pesticide metabolites that were mostly detected at an early age. The histopathological diagnosis was medulloblastoma for a 10-year-old child and high-grade gliomas for 27- and 35-year-old adults. The bivariate analyses (odds ratio >1 and P value <5%) confirmed the great probability of developing cancer by an exposure case. The Cox proportional hazards model revealed the risk of carcinogenicity beyond the age of 50 as a long-term effect of pesticide toxicity. Our study supports the correlation between pesticide exposure and the risk of development of human brain tumors, suggesting that preconception pesticide exposure, and possibly exposure during pregnancy, is associated with an increased childhood brain tumor risk. This hypothesis was enhanced in identifying traces of metabolites from the carbamate insecticide class known for their neurotoxicity and others from pyridazinone, organochlorines (OCs), triazole fungicide, and N-nitroso compounds known for their carcinogenicity. The 2D-OXYBLOT analysis confirmed the neurotoxicity effect of insecticides to induce oxidative damage in CNS cells. Aldicarb was implicated in brain carcinogenicity confirmed by the identification of oxime metabolites in a stress degradation study. Revealing "aziridine" metabolites from the OC class may better emphasize the theory of detecting traces of pesticide metabolites at an early age. Overall, our findings lead to the recommendation of limiting the residential use of pesticides and the support of public health policies serving this objective that we need to be vigilant in the postmarketing surveillance of human health impacts.

Differential Proteome Profiling Analysis under Pesticide Stress by the Use of a Nano-UHPLC-MS/MS Untargeted Proteomic-Based Approach on a 3D-Developed Neurospheroid Model: Identification of Protein Interactions, Prognostic Biomarkers, and Potential Therapeutic Targets in Human IDH Mutant High-Grade Gliomas.

High-grade gliomas represent the most common group of infiltrative primary brain tumors in adults associated with high invasiveness, agressivity, and resistance to therapy, which highlights the need to develop potent drugs with novel mechanisms of action. The aim of this study is to reveal changes in proteome profiles under stressful conditions to identify prognostic biomarkers and altered apoptogenic pathways involved in the anticancer action of human isocitrate dehydrogenase (IDH) mutant high-grade gliomas. Our protocol consists first of a 3D in vitro developing neurospheroid model and then treatment by a pesticide mixture at relevant concentrations. Furthermore, we adopted an untargeted proteomic-based approach with high-resolution mass spectrometry for a comparative analysis of the differentially expressed proteins between treated and nontreated spheroids. Our analysis revealed that the majority of altered proteins were key members in glioma pathogenesis, implicated in the cellular metabolism, biological regulation, binding, and catalytic and structural activity and linked to many cascading regulatory pathways. Our finding revealed that grade-IV astrocytomas promote the downstream of the mitogen-activated-protein-kinases/extracellular-signal-regulated kinase (MAPK1/ERK2) pathway involving massive calcium influx. The gonadotrophin-releasing-hormone signaling enhances MAKP activity and may serve as a negative feedback compensating regulator. Thus, our study can pave the way for effective new therapeutic and diagnostic strategies to improve the overall survival.

Physical and chemical stability of cytarabine in polypropylene syringes.

BACKGROUND: Cytarabine is widely used to treat leukemia and lymphoma. Currently, Cyrabol®, powder for injection, is one of the specialties marketed in Tunisia. However, no stability data when diluted with 0.9% NaCl are available. The aim of this study is to evaluate the physical and chemical stability of cytarabine (Cyrabol®) solution after dilution in 0.9% NaCl (1 mg/mL, 5 mg/mL and 10 mg/mL) in polypropylene syringes under different storage conditions. METHODS: Cytarabine solutions (1 mg/mL, 5 mg/mL and 10 mg/mL) in 0.9% NaCl were prepared in polypropylene syringes and stored for 28 days under different conditions. Cytarabine preparations in glass containers were prepared as a control to detect any adsorption. Chemical stability was assessed by a stability-indicating high-performance liquid chromatography method. The stability-indicating capacity of the method was proved by forced degradation tests. Linearity, precision and limit of detection and quantification were performed according to the International Conference on Harmonisation recommendations. Physical stability was checked by visual inspection. RESULTS: The method was proven to be a validated stability-indicating assay. At 2-8°C, all tested solutions were chemically stable for 28 days. However, at 25°C, the main degradation product gradually increased during the study and the chemical stability of 1 mg/mL, 5 mg/mL and 10 mg/mL solutions was 14 days, 8 days and 5 days, respectively. Similar results were observed in the glass containers. CONCLUSION: The highest physical and chemical stability of cytarabine diluted in 0.9% NaCl in polypropylene syringes was observed at 2-8°C. At 25°C, better stability was found in the 1 mg/mL solution compared with those at higher concentrations (5 mg/mL and 10 mg/mL).

Development of an extraction method for anabolic androgenic steroids in dietary supplements and analysis by gas chromatography-mass spectrometry: Application for doping-control.

Several studies have highlighted that nutritional supplements may contain undeclared anabolic steroids that are banned by the International Olympic Committee/World Anti-Doping Agency. Any kind of abuse with these drugs is extremely dangerous because of their side effects. Thus, the control of food additives in order to protect the best consumer health and to limit fraudulent practices in the field of sports is essential. This paper describes a simple and effective qualitative gas chromatography-mass spectrometry (GC-MS) method to detect anabolic androgenic steroids (AAS): androsterone, nandrolene, dehydroepiandrosterone, 5ɑ-androstane-3ß, 17ß-diol, dihydrotestosterone, testosterone, methenolone acetate, methandienone, boldenone and fluoxymesterone, in food supplements. Methyltestosterone was used as internal standard. Target compounds were extracted with a mixture of N-pentane and di-ethylether (7.5:2.5, v/v). A good extraction recovery was obtained by our method for all the AAS (R > 88%). Crude extract was derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Separation was performed on a GC connected to quadrupole MS detector using a 5% phenylmethylsiloxane fused silica capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 µm). Helium was used as carrier gas with a flow rate of 0.3 µl min-1 (measured at 6.1 psi 190 °C). The MS was operated in electron ionization mode (70 eV) and in selected ion monitoring (SIM). The mass spectra of the standard compounds were acquired in full SCAN mode (50-700 m/z) by infusion of a reference solution at 50 µg/ml. Three higher diagnostic ions were monitored for each compound of interest. All AAS get separated with good peak shapes and resolution factor. The total analysis time by our optimised method was only 20 min. The developed method was validated according to Laboratories International Standard regulations for specificity, precision in both liquid and solid matrixes, and memory effect. The Tolerance Interval was judged true. The limit of detection was about 10 ng/g for solid samples and 10 ng/ml for liquid samples. The developed method was then applied to the research of steroids in nine Tunisian commercially dietary supplements using for each compound of interest SIM mode for screening then SCAN mode for confirmation. One of the monitoring samples was positive to methandienone not declared on the label. Our analytical method can be beneficial for AAS screening in dietary supplements.

Photodegradation of aqueous argatroban investigated by LC/MSn: Photoproducts, transformation processes and potential implications.

Argatroban (ARGA), used as intravenous anticoagulant drug, has been reported to photodegrade under light exposure, requiring specific precautions at handling, storage and administration. Thus, for the first time, aqueous ARGA photodegradation under aerobic conditions has been described in terms of photoproducts, phototransformation processes and potential implications. ARGA significant photoproducts were successfully separated and characterized by gradient reversed-phase liquid chromatography coupled with high-resolution multistage mass spectrometry (LC/HR-MSn). Hitherto still not available in literature, ARGA in-depth fragmentation study was conducted so as to thoroughly sort out the main mechanisms specific to the molecule and therefore, to propose a fragmentation pattern relevant to the identification of ARGA related substances. Thereafter, in view of the structural characteristics of the photoproducts formed, ARGA photodegradation pathways could be worked out, showing that whether by direct photolysis or through photosensitization, the methyltetrahydroquinoline nitrogen and that of guanidine group would be mainly involved in photolysis initiation reactions, through one-electron oxidation along with proton loss. Desulfonation, cyclisation affording compounds of diazinane type, and/or rearrangements with transfer of the methyltetrahydroquinoline group toward the guanidine function were observed accordingly. Having a good insight into ARGA photodegradation pathways allows for consistent measures in view of mitigating or avoiding the drug decay and the related potential effects.

An Integrated View of the Influence of Temperature, Pressure, and Humidity on the Stability of Trimorphic Cysteamine Hydrochloride.

Understanding the phase behavior of pharmaceuticals is important for dosage form development and regulatory requirements, in particular after the incident with ritonavir. In the present paper, a comprehensive study of the solid-state phase behavior of cysteamine hydrochloride used in the treatment of nephropathic cystinosis and recently granted orphan designation by the European Commission is presented employing (high-pressure) calorimetry, water vapor sorption, and X-ray diffraction as a function of temperature. A new crystal form (I2/a, form III) has been discovered, and its structure has been solved by X-ray powder diffraction, while two other crystalline forms are already known. The relative thermodynamic stabilities of the commercial form I and of the newly discovered form III have been established; they possess an overall enantiotropic phase relationship, with form I stable at room temperature and form III stable above 37 °C. Its melting temperature was found at 67.3 ± 0.5 °C. Cysteamine hydrochloride is hygroscopic and immediately forms a concentrated saturated solution in water with a surprisingly high concentration of 47.5 mol % above a relative humidity of 35%. No hydrate has been observed. A temperature-composition phase diagram is presented that has been obtained with the unary pressure-temperature phase diagram, measurements, and calculations. For development, form I would be the best form to use in any solid dosage form, which should be thoroughly protected against humidity.

Degradation pathways study of the natriuretic and ß-adrenoceptor antagonist tienoxolol using liquid chromatography-electrospray ionization multistage mass spectrometry.

Tienoxolol is a pharmacologically active molecule designed with the functional groups ketothiophene, alkyl benzoate and arylpropanolamine so as to combine a diuretic and a ß-adrenoreceptor antagonist into a single molecule. Its degradation products generated in several stress media have been determined by high-pressure liquid chromatography (HPLC) coupled to a hybrid mass spectrometer with a triple quadrupole-linear trap. A Polaris(®) column with a C18-A stationary phase and a linear gradient mobile phase composed of a mixture of trifluoroacetic acid 1% (v/v) and acetonitrile allowed for optimal separation. Structural elucidation of the degradation products has been based on MS/MS techniques, by comparing their fragmentation patterns to the precursor's data. Up to seven degradation products of the active ingredient, resulting from hydrolysis, oxidation, dehydration and transamidation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics have been studied to assess the molecule's shelf life and to identify the most important degradation factor.