Evaluating a targeted multiple reaction monitoring approach to global untargeted lipidomic analyses of human plasma.

RATIONALE: The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. METHODS: Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. RESULTS: Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. CONCLUSIONS: The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

Screening ToxCast™ for Chemicals That Affect Cholesterol Biosynthesis: Studies in Cell Culture and Human Induced Pluripotent Stem Cell-Derived Neuroprogenitors.

BACKGROUND: Changes in cholesterol metabolism are common hallmarks of neurodevelopmental pathologies. A diverse array of genetic disorders of cholesterol metabolism support this claim as do multiple lines of research that demonstrate chemical inhibition of cholesterol biosynthesis compromises neurodevelopment. Recent work has revealed that a number of commonly used pharmaceuticals induce changes in cholesterol metabolism that are similar to changes induced by genetic disorders with devastating neurodevelopmental deficiencies. OBJECTIVES: We tested the hypothesis that common environmental toxicants may also impair cholesterol metabolism and thereby possibly contribute to neurodevelopmental toxicity. METHODS: Using high-throughput screening with a targeted lipidomic analysis and the mouse neuroblastoma cell line, Neuro-2a, the ToxCast™ chemical library was screened for compounds that impact sterol metabolism. Validation of chemical effects was conducted by assessing cholesterol biosynthesis in human induced pluripotent stem cell (hiPSC)-derived neuroprogenitors using an isotopically labeled cholesterol precursor and by monitoring product formation with UPLC-MS/MS. RESULTS: Twenty-nine compounds were identified as validated lead-hits, and four were prioritized for further study (endosulfan sulfate, tributyltin chloride, fenpropimorph, and spiroxamine). All four compounds were validated to cause hypocholesterolemia in Neuro-2a cells. The morpholine-like fungicides, fenpropimorph and spiroxamine, mirrored their Neuro-2a activity in four immortalized human cell lines and in a human neuroprogenitor model derived from hiPSCs, but endosulfan sulfate and tributyltin chloride did not. CONCLUSIONS: These data reveal the existence of environmental compounds that interrupt cholesterol biosynthesis and that methodologically hiPSC neuroprogenitor cells provide a particularly sensitive system to monitor the effect of small molecules on de novo cholesterol formation. https://doi.org/10.1289/EHP5053.

Striatal Cholesterol Precursors Are Altered with Age in Female Huntington's Disease Model Mice.

BACKGROUND: Cholesterol is necessary for proper neurodevelopment and neuronal health. The brain relies on neural and astrocytic de novo cholesterol synthesis. Huntington's disease presents with altered levels of cholesterol precursors however it is unknown when the disruption in this molecular pathway occurs and whether Manganese (Mn) may alter these metabolic alterations. OBJECTIVE: To examine the effect of Mn exposure on cholesterol biosynthesis in pre-manifest and manifest Huntington's disease mice. METHODS: 12-week (pre-manifest) male and female and 42-week old (manifest) female YAC128 and littermate control (WT) mice received 3 subcutaneous Mn or vehicle injections. Animals were sacrificed 24 hours after the final injection and striatum, cerebral cortex and cerebellum were collected to measure cholesterol and cholesterol precursors using LC/MS-MS. RESULTS: Striatal desmosterol and cholesterol are increased in pre-manifest HD females compared to age-matched WT female mice. Striatal lanosterol, 8-DHC and desmosterol and cholesterol are reduced in manifest HD females compared to age-and sex-matched WT mice with minimal effects in the cortex and cerebellum. Mn treatment had no effect in the pre-manifest or manifest female brain except reduced lanosterol levels in the cortex of pre-manifest female mice. Neither Mn or HD altered brain cholesterol precursor levels in the pre-manifest HD or WT male mouse. CONCLUSIONS: Cholesterol biosynthesis is impaired in early disease stage in female HD mice only and continues throughout disease. These alterations appear largely striatal-specific. Acute systemic exposure to Mn did not significantly alter cholesterol biosynthesis in the striatum at any disease stage.

Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

Oxidative Stress from Environmental Exposures.

Oxidative stress is arguably the most common mechanism in the toxicology of environmental agents, unifying the action of broad classes of physichochemically disparate environmental pollutants, including oxidant gases, organic compounds, particulate surfaces, and metal ions. As advances in redox biology identify previously unrecognized targets for disruption by exposure to xenobiotics, redox toxicology has emerged as a new field of investigation. Environmental contaminants can induce oxidative stress on cells through mechanisms that are direct, indirect or involve the disruption of metabolic or bioenergetic processes that are regulated by thiol redox switches. Live-cell imaging has proven to be a powerful approach to the study of environmental oxidative stress. Cells are equipped with multiple complementary energy-dependent systems for maintaining redox homeostasis in the face of environmental oxidative stress.

Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components.

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H2O2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H2O2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.

Probes for protein adduction in cholesterol biosynthesis disorders: Alkynyl lanosterol as a viable sterol precursor.

The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.

Detecting Protein Sulfenylation in Cells Exposed to a Toxicant.

Protein sulfenylation is a post-translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide multiple levels of mechanistic insight towards understanding the impact of a toxicological exposure. For instance, sulfenylation is caused by only a handful of reactive chemical species. Any altered sulfenylation suggests a change in cellular health, and the elucidation of the specific protein target that undergoes sulfenylation can help ascertain downstream targets and associated adverse outcomes. This document describes straightforward approaches to detect protein sulfenylation of total protein as well as individual proteins of interest with a focus on immunoblotting approaches. © 2017 by John Wiley & Sons, Inc.

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress.

BACKGROUND: Oxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes. SCOPE OF REVIEW: The study of events involved in redox toxicology requires methodology capable of detecting transient modifications at relatively low signal strength. This article reviews the advantages of live-cell imaging for redox toxicology studies. MAJOR CONCLUSIONS: Toxicological studies with xenobiotics of supra-physiological reactivity require careful consideration when using fluorogenic sensors in order to avoid potential artifacts and false negatives. Fortunately, experiments conducted for the purpose of validating the use of these sensors in toxicological applications often yield unexpected insights into the mechanisms through which xenobiotic exposure induces oxidant stress. GENERAL SIGNIFICANCE: Live-cell imaging using a new generation of small molecule and genetically encoded fluorophores with excellent sensitivity and specificity affords unprecedented spatiotemporal resolution that is optimal for redox toxicology studies. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress.

Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ) induce oxidative stress by redox cycling, which generates hydrogen peroxide (H2O2). Cysteinyl thiolate residues on regulatory proteins are subjected to oxidative modification by H2O2 in physiological contexts and are also toxicological targets of oxidant stress induced by environmental contaminants. We investigated whether exposure to environmentally relevant concentrations of 1,2-NQ can induce H2O2-dependent oxidation of cysteinyl thiols in regulatory proteins as a readout of oxidant stress in human airway epithelial cells. BEAS-2B cells were exposed to 0-1000 µM 1,2-NQ for 0-30 min, and levels of H2O2 were measured by ratiometric spectrofluorometry of HyPer. H2O2-dependent protein sulfenylation was measured using immunohistochemistry, immunoblotting, and isotopic mass spectrometry. Catalase overexpression was used to investigate the relationship between H2O2 generation and protein sulfenylation in cells exposed to 1,2-NQ. Multiple experimental approaches showed that exposure to 1,2-NQ at concentrations as low as 3 µM induces H2O2-dependent protein sulfenylation in BEAS-2B cells. Moreover, the time of onset and duration of 1,2-NQ-induced sulfenylation of the regulatory proteins GAPDH and PTP1B showed significant differences. Oxidative modification of regulatory cysteinyl thiols in human lung cells exposed to relevant concentrations of an ambient air contaminant represents a novel marker of oxidative environmental stress.

SRC-mediated EGF receptor activation regulates ozone-induced interleukin 8 expression in human bronchial epithelial cells.

BACKGROUND: Human exposure to ozone (O3) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. OBJECTIVE: We examined the role of EGFR activation in O3-induced expression of the chemokine interleukin 8 (IL-8) in human bronchial epithelial cells (HBEC). METHODS: We detected phosphorylated EGFR using immunoblotting. EGFR dimerization was examined through cross-linking reaction and immunoblotting, and levels of IL-8 protein were measured using ELISA. RESULTS: Exposure to O3 (0.25-1.0 ppm) induced rapid and marked increase in EGFR phosphorylation at the autophosphorylation site Y1068 and the transphosphorylation site Y845, implicating the involvement of Src kinase. Further investigation showed that O3 stimulation induced phosphorylation of Src at Y416, indicative of Src activation. Pharmacological inhibition of Src kinase activity abrogated O3-induced EGFR phosphorylation at tyrosines 1068 and 845. Moreover, pretreatment of BEAS-2B cells with inhibitor of either EGFR or Src kinase activities significantly blocked O3-induced IL-8 expression. CONCLUSION: O3 exposure increased IL-8 expression through Src-mediated EGFR transactivation in HBEC.

Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells.

Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn(2+)) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn(2+) toxicity is not fully understood. H2O2 and Zn(2+) have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn(2+) to cause cellular H2O2 production. To determine the role of Zn(2+)-induced H2O2 production in the human airway epithelial cell response to Zn(2+) exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50µM Zn(2+) for 5min in the presence of 1µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn(2+) exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn(2+)-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn(2+)-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn(2+) leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms.