DOI: 10.5061/dryad.bg79cnpbr
title: Targeted lipidomics yields changes in both arachidonic acid and linoleic acid pathways observed in C57BL/6J mice compared to KitW-sh mice after nitrogen mustard exposure

description: Sulfur mustard (SM) has been widely used as a chemical warfare agent
 including most recently in Syria. Mice exposed to SM exhibit an
 increase in pro-inflammatory cytokines followed by
 immune cell infiltration in the lung, however, the mechanisms leading to
 these inflammatory responses has not been completely elucidated. Mast
 cells are one of the first responding innate immune cells found at the
 mucosal surfaces of the lung and have been reported to be activated by SM
 in the skin. Therefore, we hypothesized that nitrogen mustard (NM: a
 surrogate for SM) exposure promotes activation of mast cells causing
 chronic respiratory inflammation. To assess the role of mast cells
 in NM-mediated pulmonary toxicity, we compared the
 effects of NM exposure between C57BL/6
 and B6.Cg-KitW-sh/HNihrJaeBsmJ (KitW-sh; mast cell
 deficient) mice. Lung injury was observed in C57BL/6J mice following NM
 exposure (0.125 mg/kg) at 72 h, which was significantly abrogated in
 KitW-sh mice. Although both strains exhibited damage from NM,
 C57BL/6J mice had higher inflammatory cell infiltration and more elevated
 prostaglandin D2 (PGD2) present in bronchoalveolar lavage fluid
 compared with KitW-sh mice. Additionally, we utilized murine
 bone marrow-derived mast cells to
 assess NM-inducedearly and late activation. Although NM exposure
 did not result in mast cell degranulation, we observed an upregulation in
 PGD2 and IL-6 levels following exposure to NM.
 Results suggest that mast cells play a prominent role in lung injury
 induced by NM and may contribute to the acute and
 potentially long-term lung injury observed caused by SM.
description: Oxylipin Sample
 Preparation All standards
 and internal standards used for LC/MS/MS analysis of arachidonic acid,
 docosahexaenoic acid and linoleic acid derived lipid mediators were
 purchased from Cayman Chemical (Ann Arbor, Michigan). All HPLC solvents
 and extraction solvents were HPLC grade or better. All analysis was done
 at the University of Colorado School of Pharmacy mass spectrometry
 facility. Bronchoalveolar lavage
 fluid (BALF) samples were pretreated for solid phase extraction. Briefly,
 samples were diluted in a 9% methanol, 1% ethanol solution by adding 100
 µl methanol and 10 µl of the internal standard solution (10 pg/µl each of
 5(S)-HETE-d<sub id="461">8</sub>,
 8-iso-PGF2a-d<sub id="462">4</sub>,
 9(S)-HODE-d<sub id="463">4</sub>, LTB4-d<sub id="464">4</sub>, LTD4-d<sub id="465">5</sub>, LTE4-d<sub id="466">5</sub>, PGE2-d<sub id="467">4</sub>, PGF2a-d<sub id="468">9</sub> and RvD2-d<sub id="469">5</sub> in ethanol) and sufficient water to
 create 1 ml of solution. Samples were then homogenized by vortexing and
 purified by solid phase extraction (SPE). Lipid mediators from cells were isolated and
 purified using SPE as follows. The reconstituted extracts were loaded on
 a Strata-X 33 µm 30 mg/1 ml SPE column (Phenomenex, Torrance, California)
 preconditioned with 2 volumes of 1.0 ml methanol. The SPE column was then
 washed with 10% methanol and then eluted directly into a reduced surface
 activity/maximum recovery glass autosampler vial with 1.0 ml of methyl
 formate. The methyl formate was evaporated completely from the vial with a
 stream of nitrogen and then the SPE cartridge was then eluted with 1.0 ml
 of methanol directly into the same autosampler vial. The methanol was
 evaporated to dryness with a stream of nitrogen and then the sample was
 reconstituted with 20 μl of ethanol. The samples are analyzed immediately
 or frozen at −70°C until analysis. Liquid Chromatography-Mass Spectrometry Quantitation of lipid mediators was performed using 2-dimensional reverse phase HPLC tandem mass spectrometry (LC/MS/MS). The HPLC system consisted of an Agilent 1290 autosampler (Agilent Technologies, Santa Clara, California), an Agilent 1200 binary SL loading pump (pump 1), an Agilent 1290 binary analytical pump (pump 2) and a 6 port switching valve. Pump 1 buffers consisted of 0.1% formic acid in water (solvent A) and 9:1 v: v acetonitrile: water with 0.1% formic acid (solvent B). Pump 2 buffers consisted of 0.01% formic acid in water (solvent C) and 1:1 v: v acetonitrile: isopropanol (solvent D). About 5 μl of extracted sample was injected onto an Agilent SB-C18 2.1 × 5 mm 1.8 μm trapping column using pump 1 at 2 ml/min for 0.5 min with a solvent composition of 97% solvent A: 3% solvent B. At 0.51 min the switching valve changed the flow to the trapping column from pump 1 to pump 2. The flow was reversed, and the trapped lipid mediators were eluted onto an Agilent Eclipse Plus C-18 2.1 × 150 mm 1.8 um analytical column using the following gradient at a flow rate of 0.3 ml/min: hold at 75% solvent A : 25% solvent D from 0 to 0.5 min, then a linear gradient from 25% to 75% D over 20 min followed by an increase from 75% to 100% D from 20 to 21 min, then holding at 100% D for 2 min. During the analytical gradient pump 1 washed the injection loop with 100% B for 22.5 min at 0.2 ml/min. Both the trapping column and the analytical column were re-equilibrated at starting conditions for 5 min before the next injection. Mass spectrometric analysis was performed on an Agilent 6490 triple quadrupole mass spectrometer in negative ionization mode. The drying gas was 250°C at a flow rate of 15 ml/min. The sheath gas was 350°C at 12 ml/min. The nebulizer pressure was 35 psi. The capillary voltage was 3500 V. Data for lipid mediators was acquired in dynamic MRM mode using experimentally optimized collision energies obtained by flow injection analysis of authentic standards. Calibration standards for each lipid mediator were analyzed over a range of concentrations from 0.25 to 250 pg on column. Calibration curves for each lipid mediator were constructed using Agilent Masshunter Quantitative Analysis software. Samples were quantitated using the calibration curves to obtain the on-column concentration, followed by multiplication of the results by the appropriate dilution factor to obtain the concentration in pg/ml.
description: High Dose : 0.125 mg/kg Nitrogen Mustard dose
 KIT Wsh mice - Sample numbers ending in K
 C57BL6J mice - samples ending in B BALF-
 Bronchoaleveolar lavage fluid Numbers represent animal
 ID LOD - below the limit of detection
 LOQ- below the limit of quantitation AA-
 arachidonic acid pathway DHA- docosahexaenoic acid
 pathway LA- linoleic acid pathway
 DGLA- Dihomo-y-linolenic acid  EPA-
 eicosapentaenoic acid  LOX - lipoxygenase
 CYP - cytochrome COX- cyclooxygenase
 ROS- reactive oxygen species