Mass spectrometry imaging in zebrafish larvae for assessing drug safety and metabolism.

Drug safety assessment in the early phases of drug discovery is critical to facilitate the rapid development of novel therapeutics. Recently, teleost zebrafish (Danio rerio) has emerged as a promising vertebrate model for the assessment of drug safety. Zebrafish is a convenient model because of its small size, high fecundity, embryo transparency, and ex utero development. In this study, we developed a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) method applied to zebrafish larvae to investigate safety and metabolism of sahaquine (Sq), an anticancer agent inhibiting histone deacetylase 6. This technique improves on prior studies using liquid chromatography-mass spectrometry (LC-MS) by adding analysis of the drug spatial distribution. Using this method, it was determined that Sq dissolved in fish water (1-2000 µM) did not reach the larval body and was mainly distributed throughout the yolk. High Sq concentration (800 µM) administered intravenously allowed the compound to reach the larval body but did not induce phenotypic abnormalities. Sq was metabolized into its glucuronidated form within 24 h and was excreted within 72 h. MALDI MSI was instrumental in showing that Sq-glucuronide was mainly formed in the gut and slightly in yolk syncytial layer, and provided valuable insights into xenobiotics elimination in zebrafish larvae. This study indicates that Sq has a good safety profile and merits further investigations in other disease models. In addition, the optimized MALDI MSI protocol provided here can be widely applied to study distribution and metabolic fate of other structurally related molecules.

A matrix-assisted laser desorption/ionization imaging mass spectrometric approach to study weight-related changes within thyroid tissue.

Obesity is associated with numerous comorbidities along with abnormalities of the endocrine system, more commonly manifesting as dysfunctions of the thyroid gland such as goiter. Changes in weight, especially an increase, could lead to an increase in the incidence of thyroid dysfunction; however, its pathophysiology remains to be elucidated. In the present study, we aimed to interrogate the changes in the protein distribution and abundance between the lean patients and patients with obesity thyroid tissue sections through utilizing this technique. The FFPE-fixed thyroid tissue blocks from the selected cases and controls were identified and targeted for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) analysis. Patients in the 30 to 75 years age group and undergoing total thyroidectomy for benign thyroid disease were recruited. Patients with thyroid cancers, autoimmune disorders, and other inflammatory conditions were excluded from the study. The selected patients were divided into two groups according to their BMIs: lean (BMI < 25) and obese (BMI > 35). An initial trial set was used as a pilot study for the optimization of the MALDI IMS protocol that was next applied to the selected thyroid tissues. MALDI IMS data from all the samples were aligned and statistical analysis carried out by k-means and linear discriminant analysis (LDA) classification model using principle component analysis (PCA) results were evaluated between the two groups: controls (lean) and cases (obese). Receiver operator characteristic (ROC) curves were alternatively used to calculate the variability of the identified peptides. The discriminating peptides were also independently identified and related to their corresponding proteins by using liquid chromatography and tandem mass spectrometry (LC-MS/MS) analyses. Eight peptides mainly from thyroglobulin were found to be upregulated whereas 10 others were found to be downregulated in the lean compared to the obese group. Through this technique, we will be able to better understand the relationship between the disease entity and obesity.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism.

Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.

Mechanisms and Antitumor Activity of a Binary EGFR/DNA-Targeting Strategy Overcomes Resistance of Glioblastoma Stem Cells to Temozolomide.

PURPOSE: Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy, temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN: ZR2002 is a "combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs, mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines, and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS: ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR, downstream Erk1/2 phosphorylation, increased DNA strand breaks, and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line, decreased EGFR, Erk1/2, and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS: These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability, blood-brain barrier permeability, and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM.

Detection of exogenous substances in latent fingermarks by silver-assisted LDI imaging MS: perspectives in forensic sciences.

For over one hundred years, the fingerprint has reigned as one of the most trusted pieces of forensic evidence for suspect identification. In the last few decades, the modernization of chemical analysis technologies led scientists to explore new possibilities to further analyse fingermarks sampled from a crime scene. Indeed, the detection of chemicals a suspect has been in contact with before or during the crime can provide valuable insights into criminal investigations. In this regard, imaging mass spectrometry (IMS) has shown to be a powerful tool for the analysis of fingermarks by combining suspect identification and the detection of numerous endogenous and exogenous compounds. A novel approach developed in our laboratory, silver-assisted laser desorption ionization (AgLDI), was adopted to allow for the chemical analysis of latent fingermarks left on nonconductive surfaces (such as paper, cardboard, plastic and forensic lifting tape) with a time-of-flight mass spectrometer. In this study, we continue to evaluate the potential of AgLDI IMS to provide circumstantial evidence by detecting exogenous substances. We first demonstrate that owner-specific chemical signatures can be recovered from fingermarks based on the presence of several cosmetics and personal care products. We then show the possibility of detecting and imaging fingermarks containing three common illicit drugs, namely tetrahydrocannabinol, cocaine and heroin. Finally, we demonstrate that the methodology also allows us to successfully image bloody fingermarks after appropriate forensic enhancement treatments. Overall, we believe that AgLDI IMS has significant potential that could positively contribute to forensic investigations.

Development of laser desorption imaging mass spectrometry methods to investigate the molecular composition of latent fingermarks.

For a century, fingermark analysis has been one of the most important and common methods in forensic investigations. Modern chemical analysis technologies have added the potential to determine the molecular composition of fingermarks and possibly identify chemicals a suspect may have come into contact with. Improvements in analytical detection of the molecular composition of fingermarks is therefore of great importance. In this regard, matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) imaging mass spectrometry (IMS) have proven to be useful technologies for fingermark analysis. In these analyses, the choice of ionizing agent and its mode of deposition are critical steps for the identification of molecular markers. Here we propose two novel and complementary IMS approaches for endogenous and exogenous substance detection in fingermarks: sublimation of 2-mercaptobenzothiazol (2-MBT) matrix and silver sputtering.