Prem Kumarathasan

Current Status and Future Perspectives of Mass Spectrometry Imaging

Mass spectrometry imaging is employed for mapping proteins, lipids and metabolites in biological tissues in a morphological context. Although initially developed as a tool for biomarker discovery by imaging the distribution of protein/peptide in tissue sections, the high sensitivity and molecular specificity of this technique have enabled its application to biomolecules, other than proteins, even in cells, latent finger prints and whole organisms. Relatively simple, with no requirement for labelling, homogenization, extraction or reconstitution, the technique has found a variety of applications in molecular biology, pathology, pharmacology and toxicology. By discriminating the spatial distribution of biomolecules in serial sections of tissues, biomarkers of lesions and the biological responses to stressors or diseases can be better understood in the context of structure and function. In this review, we have discussed the advances in the different aspects of mass spectrometry imaging processes, application towards different disciplines and relevance to the field of toxicology.

Pulmonary Expression of PreproET-1 and PreproET-3 mRNAs Is Altered Reciprocally in Rats After Inhalation of Air Pollutants

Perturbation of vascular homeostasis is an important mechanism related to the acute health effects of inhaled pollutants. Inhalation of urban particulate matter and ozone by rats has been shown to result in increased synthesis of the potent vasoactive peptide endothelin (ET)-1 in the lungs, with spillover into the circulation. In the present work, we have analyzed the interrelationships between responses of the three major endothelin isoforms, ET-1[1–21], ET-2[1–21], and ET-3[1–21], to inhaled pollutants at the peptide and gene expression levels. Fisher-344 rats were exposed for 4 hrs by nose-only route to clean air, urban particles EHC-93 (0, 50 mg/m3), ozone (0, 0.8 ppm), or ozone and particles together. Circulating levels of both the ET-1 [1–21] and ET-3[1–21] peptides were increased immediately after exposure to particulate matter or ozone. While expression of preproET-1 mRNA in the lungs increased, expression of preproET-3 mRNA decreased immediately after exposure. PreproET-2 mRNA was not detected in the lungs, and exposure to either pollutant did not affect plasma ET-2 levels. Co-exposure to ozone and particles, while altering lung preproET-1 and preproET-3 mRNA levels in a fashion similar to ozone alone, did not cause changes in the circulating levels of the two corresponding peptides. Thus, de novo synthesis of ET-3 in the lungs is not responsible for the increase of circulating plasma ET-3 after inhalation of pollutants, which implies regulation of preproET-3 at a remote site and, hence, systemic impacts of the pollutants. Upregulation of preproET-1 coupled with down-regulation of preproET-3 in the lungs of animals exposed to air pollutants implies a mismatch of local ET-1/ETA receptor–mediated vasoconstriction and ET-3/ETB receptor–mediated vasodilation.

Effects of a Cyclooxygenase-2 Preferential Inhibitor in Young Healthy Dogs Exposed to Air Pollution: A Pilot Study

Residency in cities with high air pollution is associated with neuroinflammation and neurodegeneration in healthy children, young adults, and dogs. Nonsteroidal anti-inflammatory drugs may offer neuroprotection. The authors measured the plasma concentrations of 3-nitrotyrosine and the cerebro-spinal-fluid concentrations of prostaglandin E2 metabolite and the oligomeric form of amyloid derived diffusible ligand; measured the mRNA expression of cyclooxygenase-2, interleukin 1β, CD14, and Aquaporin-4 in target brain areas; and evaluated brain MRI, cognition, and neuropathology in 8 dogs treated with a preferential cyclooxygenase-2 inhibitor (Nimesulide®) versus 7 untreated litter-matched Mexico City dogs. Nimesulide® significantly decreased nitrotyrosine in plasma (p < .0001), frontal gray IL1β (p = .03), and heart IL1β (p = .02). No effect was seen in mRNA COX2, amyloid, and PGE2 in CSF or the MRI white matter lesions. All exposed dogs exhibited olfactory bulb and frontal accumulation of Aβ42 in neurons and blood vessels and frontal vascular subcortical pathology. White matter hyperintense MRI frontal lesions were seen in 4/6 non-treated and 6/8 treated dogs. Nonsteroidal anti-inflammatory drugs may offer limited neuroprotection in the setting of severe air pollution exposures. The search for potentially beneficial drugs useful to ameliorate the brain effects of pollution represents an enormous clinical challenge.

Hydroxyl radical adduct of 5-aminosalicylic acid: A potential marker of ozone-induced oxidative stress

The use of 5-aminosalicylic acid in assessment of reactive oxygen species formation was investigated by in vitro Fenton and ozonation reactions, and by in vivo ozone-exposure experiments. Enzymatic hydroxylation was evaluated by a microsomal assay. Fischer 344 male rats (250 g) injected with 5-aminosalicylic acid (100 mg x kg(-1) i.p.; 30 min) were exposed to ozone (0, 1, 2 ppm; nose only, 2 h); bronchoalveolar lavage, lung homogenates, and plasma were recovered. Oxidation products of 5-aminosalicylic acid were as follows: salicylic acid, by deamination; 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, from radical or enzymatic hydroxylation; 5-amino-2-hydroxy-N,N-bis(3-carboxy-4-hydroxyphenyl)-1,4-benzoquinonediimine, a condensation product of oxidized 5-aminosalicylic acid; and 5-amino-2,3,4,6-tetrahydroxybenzoic acid, attributed to hydroxyl radical attack without deamination, identified by HPLC electrochemical (HPLC-EC) detector system analysis and by GC-MS analysis of trimethylsilyl derivatives. 5-Aminotetrahydroxybenzoic acid was not formed enzymatically. 5-Aminotetrahydroxybenzoic acid, but not 5-aminosalicylic acid, was significantly elevated in bronchoalveolar lavage (+86%) and lung homogenates (+56%) in response to 2 ppm ozone (p < 0.05); no significant changes were detected in plasma. The data indicate that hydroxylation of 5-aminosalicylic acid is a potential specific probe for in vivo oxidative stress.

Nitrative stress, oxidative stress and plasma endothelin levels after inhalation of particulate matter and ozone

BackgroundWhile exposure to ambient air contaminants is clearly associated with adverse health outcomes, disentangling mechanisms of pollutant interactions remains a challenge.ObjectivesWe aimed at characterizing free radical pathways and the endothelinergic system in rats after inhalation of urban particulate matter, ozone, and a combination of particles plus ozone to gain insight into pollutant-specific toxicity mechanisms and any effect modification due to air pollutant mixtures.MethodsFischer 344 rats were exposed for 4xa0h to a 3 × 3 concentration matrix of ozone (0, 0.4, 0.8xa0ppm) and EHC-93 particles (0, 5, 50xa0mg/m3). Bronchoalveolar lavage fluid (BALF), BAL cells, blood and plasma were analysed for biomarkers of effects immediately and 24xa0h post-exposure.ResultsInhalation of ozone increased (pu2009<u20090.05) lipid oxidation products in BAL cells immediately post-exposure, and increased (pu2009<u20090.05) total protein, neutrophils and mature macrophages in the BALF 24xa0h post-exposure. Ozone increased (pu2009<u20090.05) the formation of reactive oxygen species (ROS), assessed by m-, p-, o-tyrosines in BALF (Ozone main effects, pu2009<u20090.05), while formation of reactive nitrogen species (RNS), indicated by 3-nitrotyrosine, correlated with dose of urban particles (EHC-93 main effects or EHC-93u2009×u2009Ozone interactions, pu2009<u20090.05). Carboxyhemoglobin levels in blood exhibited particle exposure-related increase (pu2009<u20090.05) 24xa0h post recovery. Plasma 3-nitrotyrosine and o-tyrosine were increased (pu2009<u20090.05) after inhalation of particles; the effect on 3-nitrotyrosine was abrogated after exposure to ozone plus particles (EHC-93u2009×u2009Ozone, pu2009<u20090.05). Big endothelin-1 (BET-1) and ET-1 were increased in plasma after inhalation of particles or ozone alone, but the effects appeared to be attenuated by co-exposure to contaminants (EHC-93u2009×u2009Ozone, pu2009<u20090.05). Plasma ET levels were positively correlated (pu2009<u20090.05) with BALF m- and o-tyrosine levels.ConclusionsPollutant-specific changes can be amplified or abrogated following multi-pollutant exposures. Oxidative and nitrative stress in the lung compartment may contribute to secondary extra-pulmonary ROS/RNS formation. Nitrative stress and endothelinergic imbalance emerge as potential key pathways of air pollutant health effects, notably of ambient particulate matter.

Alteration in aromatic hydroxylation and lipid oxidation status in the lungs of rats exposed to ozone.

Fischer 344 rats were exposed to ozone by inhalation to identify sensitive indices of acute exposure. 5-Aminosalicylic acid (5-ASA) hydroxylation in bronchoalveolar lavage (BAL), an indicator of hydroxyl radical (*OH) formation, and lipid oxidation in various regions of airways, representing oxidative stress, were measured to verify whether they can function as markers of exposure. BAL cells and supernatants taken from rats that received saline or 5-ASA (ip, 50 mg/kg) prior to ozone exposure (0, 0.4, or 0.8 ppm for 4 h) were analyzed for products of lipid oxidation. *OH formation was assessed by analysis of the BAL supernatant for 5-aminotetrahydroxybenzoic acid (5-ATHBA), a hydroxylation product of 5-ASA. The tetrahydroxy derivative of 5-ASA was higher in the BAL of ozone-treated rats than in air controls, reaching significance (p <. 05) at 0.8 ppm of ozone, The products of lipid oxidation propanal and hexanal were higher in BAL cells taken from rats exposed to ozone, reaching significance (p <. 05) at a 0.8 ppm ozone level, compared to air control animals, irrespective of whether they received saline or 5-ASA prior to ozone exposure. Increases in cholesterol levels were also seen in BAL cells after rats were exposed to ozone. However, there were no significant dose-related changes in the lipid oxidation products in BAL supernatants after exposure to ozone. Lipid oxidation products in BAL cells and 5-ATHBA in lavage exhibited the potential to serve as markers of ozone exposure. This work was supported by Health Canada (#4320105) and Toxic Substances Research Initiatives (TSRI #60).

Experimental Atmosphere Monitoring in Gasoline Vapor Inhalation Studies

Conditions affecting the composition and stability of test atmospheres of gasoline were investigated. Vapor was generated at 60°C from a methanol-gasoline blend (85:15, v:v) in a carrier air flow (25 L/min) and mixed with dilution air (580 L/min) for distribution to 2.5-m3 inhalation chambers. Three nominal concentrations (equivalent to 5150, 515, 51.5 ppm methanol) were produced under dynamic flow conditions. Analyses of vapor were carried out by gas chromatography with mass spectrometer and flame ionization detectors. Analyte concentrations determined for the high, medium, and low exposure levels were within ±1%, ±4%, and ±7% of the nominal values, respectively. Within-day variation and day-to-day variation in the analyte concentrations were <5%. Virtually constant levels of low boiling (<115°C) components (e.g., 2,2,4-trimethyl pentane) were observed over a daily 6-h exposure period. However, increase in high boilers such as 1,3,5-trimethyl benzene in the vapor with time was associated with the accumul...

A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies: IN VITRO PARTICLE SCREENING USING TOXICOPROTEOMICS

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre‐fractionated by molecular weight cut‐off filtration, digested enzymatically and were analyzed by matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre‐fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose‐dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency‐based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.