Soluble Epoxide Hydrolase Inhibitor Attenuates …Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation ... ... The

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Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation and Airway

Hyperresponsiveness in Mice

Jun Yang1, Jennifer Bratt

2, Lisa Franzi

2, Junyan Liu

1, Guodong Zhang

1, Amir A. Zeki

2,

Christoph F. A. Vogel3,4

, Keisha Williams2, Hua Dong

1, Yanping Lin

1, Sung Hee

Hwang1, Nicholas J. Kenyon

2, Bruce D. Hammock

1 *

1 Department of Entomology and Comprehensive Cancer Center,

2 Department of Internal

Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, 3 Department of

Environmental Toxicology, 4

Center for Health and the Environment. University of

California, Davis, CA 95616.

Address correspondence to: Bruce D. Hammock

Department of Entomology

University of California at Davis

One Shields Avenue, Davis, CA 95616.

Tel: (530) 752-7519

Fax: (530) 751-1537

E-mail: [email protected]

Page 1 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC

Copyright © 2014 by the American Thoracic Society

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Abstract

Rationale: Control of airway inflammation is critical in asthma treatment. The soluble

epoxide hydrolase (sEH) has recently been demonstrated as a novel therapeutic target for

treating inflammation, including lung inflammation. Here, we hypothesize that

pharmacological inhibition of sEH can modulate the inflammatory response in a murine

ovalbumin (OVA) model of asthma.

Methods: BALB/c mice were sensitized and exposed to OVA over 6 weeks. The sEH

inhibitor (sEHI) was administrated for two weeks. Respiratory system compliance,

resistance and forced exhale nitric oxide were measured. Lung lavage cell counts were

performed and a selected panel of cytokines and chemokines in the lung lavage fluid

were measured by a Multiplex immunoassay kit. A liquid chromatography tandem mass

spectrometry method was used to measure 87 regulatory lipids mediators in plasma, lung

tissue homogenates, and lung lavage fluid.

Results: The pharmacological inhibition of sEH increased the concentrations of the anti-

inflammatory epoxy eicosatrienoic acids (EETs) and simultaneously decreased the

concentrations of the pro-inflammatory dihydroxyeicosatrienoic acids (DHETs) and

dihydroxyoctadecenoic acids (DHOMEs). All monitored inflammatory markers, which

including FeNO levels, and total cell and eosinophil numbers in the lung lavage of OVA-

exposed mice were reduced by sEHI. The Th2 cytokines (IL-4, IL-5) and chemokines

(Eotaxin and RANTES) were dramatically reduced after administration of sEHI.

Moreover, the resistance and dynamic lung compliance were also improved by sEHI.



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Conclusions: We demonstrated that the administration of an sEHI attenuates allergic

airway inflammation and airway responsiveness in a murine model. sEHI may have

potential as a novel therapeutic strategy for allergic asthma.

Keywords

Soluble epoxide hydrolase; asthma; inflammation; lipid mediators; Th2 cytokines

Running Title

sEHI attenuates lung inflammation in asthma

Listing of contributions of authors:

Conception and design: BDH, JY, NJK;

Analysis and Interpretation: JY, JB, LF, JYL, GDZ, AAZ, CFAV, KW, HD,

YPL;

Drafting the manuscript: JY, BDH, NJK

Word Count:

Abstract: 250 words

Methods: 459 words

Total: 2959 words

Figures: 6



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INTRODUCTION

Three hundred million people worldwide suffer from asthma either episodically or

persistently (1). The cornerstones of treatment for persistent asthma are inhaled

corticosteroids (ICS) and beta-agonist bronchodilators; however, a significant minority of

asthma patients does not respond well to these therapies (2). Thus, there is ongoing effort

to develop novel treatment strategies (3) such as specific antagonists of Th2 cytokines

and mediators.

Epoxyeicosatrienoic acids (EETs, or EpETrEs according to LIPIDMAPS nomenclature)

are a class of important lipid mediators with critical physiological functions that include

vasodilation, anti-inflammation, anti-hypertension, organ protection, and analgesic

effects (4). Specifically in lung health and disease, EETs are reported to affect lung

epithelial ion transport (5-7), relax the pre-contracted bronchi (8), reduce inflammation

(9, 10), regulate endothelial permeability in the lung (11), and regulate pulmonary

vascular pressures (12, 13). Thus, modulation of endogenous EETs is an attractive

approach to potentially control the symptoms of asthma, which include chronic airway

inflammation and airway hyperresponsiveness (AHR).

The soluble epoxide hydrolase (sEH) hydrolyzes these bioactive EETs to corresponding

diols, which are less beneficial, if not toxic. Using potent inhibitors of sEH to stabilize

endogenous EETs (14-17), sEH has recently been demonstrated in animal models as a

novel therapeutic target (4) for treating cardiovascular diseases (18-20), inflammation (21,

22), pain (23-25), and pulmonary diseases such as pulmonary hypertension (26, 27) and

tobacco smoke-induced chronic obstructive pulmonary disease (COPD) (9, 10).



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In the present study, we hypothesized that pharmacological inhibition of sEH can

modulate the inflammatory response in a well-established murine ovalbumin (OVA)-

exposure asthma model. We found that administration of a sEH inhibitor (t-TUCB)

reduced the total inflammatory cell infiltration into the airway and lung, and inhibited

OVA-induced influx of eosinophil. The profiling of regulatory lipid mediators shows that

administration of t-TUCB not only increased the anti-inflammatory lipid mediators-EETs,

but also increased other anti-inflammatory mediators such as 17-hydroxy

docosahexaenoic acid (17-HDoHE) and decreased the pro-inflammatory lipid mediators

including DHOMEs (DiHOMEs) and LTB4 in the plasma, lung tissue, and lavage.

Stabilization of the anti-inflammatory epoxide lipid mediators through pharmacological

inhibition of metabolism by the enzyme-sEH decreased production of Th2 cytokines at

both protein and mRNA levels after OVA induction. Furthermore, compliance and

resistance of the respiratory system were also improved after the administration of sEHI.

These findings support the hypothesis that sEH is a potential target to treat asthma.

METHODS

Animals

Pathogen free male BALB/c mice, aged 8-10 weeks, were purchased from Charles River

Laboratory (Wilmington, MA). All mice were maintained in a HEPA-filtered laminar

flow cage rack with a 12-hour light/dark cycle and allowed free access to food and water.

Figure 1A shows the animal protocol. All procedures with mice were performed in

accordance with an IACUC approved protocol.



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Drug Solutions and Exposure of Mice to Ovalbumin Aerosol

The sEHI, trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]-cyclohexyloxy}-benzoic

acid (t-TUCB), was synthesized as previously described (17). The sEHI was dissolved in

0.05% Tween-80 water solution. The sEHI solution (1 or 3 mg/Kg) was administered

subcutaneously to the mice every day for 14 days. On the last day, the drugs were

administrated half an hour before the exposure of ovalbumin aerosol. The exposure

procedures of ovalbumin used have been described in detail previously (28).

Lung Compliance and Resistance Measurements

The dynamic lung compliance (Cdyn) and resistance of the respiratory system (Rrs) were

simultaneously measured with a whole body plethysmograph for restrained animals

(Buxco Inc., Troy, NY) 1–3 hours after termination of the final OVA exposure. The

whole procedure for this is described in detail in the supplementary Information (SI).

Measurement of Exhaled NO

A 5-minute sample of exhaled gases was collected from the cannulated mice through the

ventilator exhalation port immediately after insertion of the mouse into a plethysmograph

as previously described (29).

Cytokine and Chemokine Assays

The concentrations of selected cytokines and chemokines (Eotaxin, IFN-γ, IL-1β,IL2,

IL4, IL5, IL6, IL10, IL13, RANTES, TNF-α) from BALF supernatant were measured

with commercially available multiplex immunoassays according to the manufacturer’s

instructions (Millipore, St. Charles, MO).



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Measurement of t-TUCB Concentration

Blood (10 µL) was diluted by with 0.1% aqueous EDTA (50 µL) and mixed vigorously.

Samples were then extracted using 200 µL of ethyl acetate twice and dried by Speedvac.

In the end, the residue was reconstituted to 50 µL of internal standard solution and

measured by LC/MS/MS.

Regulatory Lipid Mediator Profiling

Profiles of regulatory mediators were measured using the LC/MS/MS method as

published previously (30). Aliquots of plasma (250 µL), BAL supernatant (2mL), or lung

tissue (~100 mg) were used for the measurements, respectively. Extraction protocols are

fully described in the SI.

Quantitative Real-time Reverse Transcription–PCR (RT-PCR)

Total RNA was isolated from lung tissue using Trizol and a Quick-RNA Mini prep

isolation kit (Zymo Research, Irvine, CA), cDNA synthesis and the RT-PCR processes

are fully described in the SI.

Statistical Analysis

Data are presented as mean values ± SEM. Data were analyzed using unpaired values

compared by two tailed Student's t-test or one-way or two-way ANOVA with Tukey's

post test where appropriate using the Prism 5.0 software package (GraphPad, Inc., San

Diego, CA), with statistical significance defined as a p value of 0.05 or less.

RESULTS



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sEHI was Successfully Delivered and Well Engaged.

After 14 days of subcutaneous injection of sEHI (t-TUCB), concentrations of t-TUCB in

blood (Figure 2A) reached 55.6 ± 13.2 nM (1mg/Kg) and 213 ± 38 nM (3 mg/Kg), which

are 42.8 times and 164 times higher, respectively, than the IC50 of t-TUCB in mouse on

the murine recombinant sEH (1.3 nM) using trans-diphenylpropene oxide (t-DPPO) as

substrate (17). As a result of enzyme inhibition, the concentrations of 14,15-EpETrE, one

endogenous substrate of sEH, increased by 3-3.6 fold from 0.78 nM to 2.56 or 2.86 nM

with administration of 1 or 3 mg/Kg t-TUCB compared to the vehicle control (Figure

2B). Increased concentrations of 14,15-EpETrE after exposure to t-TUCB confirmed the

efficacy of t-TUCB as an sEHI in this study.

Administration of sEHI Increased Anti-inflammatory Mediator Concentrations and

Decreased Pro-inflammatory Mediator Concentrations.

We analyzed the regulatory lipid mediators from BALF, plasma, and lung tissue

homogenate using LC/MS/MS. Figure 3 shows the results presented as heat maps. Figure

3A shows a simplified arachidonic acid cascade listing the major lipid mediators. Figures

3B-D show the significantly changed regulatory lipid mediators after administration of t-

TUCB. In general, for the P450 and sEH pathways, administration of sEHI increased

epoxides in plasma and BALF, and decreased diols in BALF and lung homogenates. In

plasma, administration of sEHI increased some COX and LOX metabolites (11-HETE, 9-

HETE, 5-HETE, 5-HEPE). The low dose of sEHI significantly reduced the pro-

inflammatory mediators: 6-keto-PGF1α (the metabolite and surrogate of prostacyclin-

PGI2) and LTB4. In BALF, administration of sEHI increased LOX metabolites including



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11-HETE, 9-HETE, 5-HETE, 15-HEPE, 17HDoHE, 8-HETE. In lung homogenates,

administration of sEHI increased the COX metabolites 6-keto-PGF1α, TXB2, PGF2α,

PGE3 and PGJ2, 11-HETE; increased the LOX metabolites including 15-HETE, 15(s)

HETrE, 15-HEPE, 17-HDoHE, and 8-HETE.

Administration of sEHI Reduced Th2 Cytokines and Chemokines

Several inflammatory cytokines were induced after OVA exposure (Figure 4 and

supplementary Figure E1). After administration of sEHI, IL-4 and IL-5 in lung lavage

fluid decreased to almost the base line of the control animals (Figures 4A-B). By contrast,

there were no clear trends for those Th1 and innate immune cytokines assayed

(Supplementary Figure E1). Because of the methodological issues involved in IL-13

detection, we could not make firm conclusions regarding involvement of IL-13 in this

model system. The chemokine eotaxin was induced after OVA exposure, and its levels

were blunted by inhibition of sEH (Figure 4C). These data suggest that inhibition of sEH

could reduce the Th2-specific cytokines and chemokines, which are important in

eosinophil trafficking, recruitment, and maturation in airways. Lung expression of IL-4

and IL-5 also showed that RNA levels of these Th2 cytokines were down-regulated by

the administration of sEHI (Figure 4D-E).

Administration of sEHI Reduced Inflammatory Cell Infiltration in Lung Tissues

and Lavage Fluid.

As Figure 5A shows, sensitization and exposure of mice to OVA induced a significant

inflammatory cell infiltration into the airway. The total cell count in BAL reached

approximately 2.6 × 106 cells / mL. Administration of the inhibitor of sEH, t-TUCB at 1



10

mg/Kg dose, decreased the number of inflammatory cells infiltrated into the lavage fluid.

Furthermore, 3 mg/Kg t-TUCB significantly reduced the total cell number in BAL to

approximately 46.2%. Analysis of these results by ANOVA showed that there was

significant reduction of total live cell number after administration of sEHI (p=0.04).

Figure 5B shows the differential cell counts determined by the Hema-3 stain set. After

OVA exposure, eosinophil is the dominant inflammatory cell type in BAL comprising up

to 75% of the total inflammatory cell infiltrate. After administration of sEHI, the

percentage of eosinophil was reduced to 65%. t-TUCB at a dose of 3 mg/Kg significantly

reduced the eosinophil infiltration into lung lavage from 1.63× 106 to 7.05 × 10

5. The

ANOVA analysis shows that there is significant reduction of total live cell number after

administration of sEHI (p=0.049). It indicates that sEHI not only reduced the total

inflammatory cell infiltration into the airway but also altered the ratio of inflammatory

cells present in the BAL (eosinophil/macrophage ratio from 4.39 to 2.30). This result also

corresponds to the reduction of Th2 cytokines in the lavage and lung tissue. Exhaled

nitric oxide (NO) is a biomarker of airway eosinophil inflammation, and was increased

from 5.43 ppb to 15.1 ppb after ovalbumin exposure (Figure 5C). Treatment with

1mg/Kg and 3 mg/Kg of t-TUCB decreased the induction of this inflammatory biomarker

to 3.68 ppb and 6.57 ppb respectively. The ANOVA analysis with Bonferroni post test

shows that there is significant reduction of FeNO after administration of sEHI

(p=0.0006). In lung tissues, there was marked inflammatory cell influx in the peri-

bronchiolar space post-OVA exposure as shown in the H&E stain result (Figures 5, D-E)

consistent with the lung lavage data (Figure 5A-B). After inhibition of sEH,

inflammatory cells in the lung tissue were reduced in a dose dependent manner (Figures



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5, F-G). These figures show that administration of sEHI reduced the lung inflammatory

cell infiltration, which is consistent with the inflammatory cell results in the BAL cell

counts.

Taken together, there is severe eosinophil dominant inflammation in both airway and

alveolar of the mice after exposure of ovalbumin. Administration of sEHI reduced this

inflammation as shown in total live cell number, inflammatory cell differentiation, H&E

stained lung tissue, and FeNO.

sEHI Reduced Methacholine-induced Changes in Resistance and Dynamic

Compliance of the Respiratory System.

Exposure to OVA reduced the baseline compliance from 0.036 ml/cm H2O (FA) to 0.023

mL/cm H2O (OVA + vehicle). Administration of 3 mg/Kg sEHI rescued the baseline

compliance to 0.03 ml/cm H2O (Figure 6A). Although 1 mg/Kg sEHI did not rescue the

baseline compliance, both sEHI treatment groups did help to prevent the change in

compliance with methacholine challenge (p<0.0001 for overall effect by treatment group,

p=0.0005 for the doses, assessed by two-way ANOVA).

OVA exposure increased both baseline resistance and the slope of resistance change in

response to the methacholine challenge, as presented in Figure 6B. Administration of

sEHI rescued the baseline resistance in a dose dependent manner (p=0.0035 for overall

effect by treatment group, p=0.0018 for the doses of methacholine, assessed by two-way

ANOVA). At 1 mg/Kg, sEHI reduced the slope of resistance change along with

methacholine, whereas sEHI at a dose of 3 mg/Kg shows a more complex pattern: a

shallow initial slope with low methacholine dose, then rapid increase in slope with the

higher dose of methacholine.



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DISCUSSION

To summarize briefly, we demonstrated that administration of a soluble epoxide

hydrolase inhibitor markedly attenuates the allergic airway inflammation and AHR

caused by exposing mice to ovalbumin. Specifically, the sEHI reduced the total

inflammatory cell number by 50%. Compared with two clinically available compounds,

montelukast and dexamethasone tested in the similar animal model, sEHI had a greater

effect at reducing the inflammatory cells infiltration more than montelukast and

dexamethasone did (40% and 28%) (31). In addition, sEHI reduced IL-5 levels to 12.5%

of those mice treated with ovalbumin and vehicle. Taken together, sEHI is a promising

potential candidate drug to treat allergic asthma.

We and others have previously shown the anti-inflammatory effects of sEHIs on different

disease models (9, 21, 22, 32). The mechanism is believed to be through stabilizing of the

anti-inflammatory EET, which regulates NFκ-B translocation (33) or reducing production

of pro-inflammatory diols including DHOMEs (34) and dihydroxy eicosatrienoic acid

(DHET) (35). Here, our data show that the concentration of sEHI in the plasma

significantly altered the circulating EETs and DHETs levels present in plasma, BALF

and lung homogenates (Figure 3C, 3D). Node (33) reported that EETs (EpETrEs) can

reduce endothelial cell VCAM-1 expression in response to TNF-α, IL-1α, and LPS. At

the same time, administration of sEHI significantly reduced pro-inflammatory DHETs in

BALF and lung homogenate (Figure 3C, 3D). The DHETs were reported as essential for

monocyte chemotaxis to MCP-1 (35). In particular, sEHI reduced in the lung

homogenates the DHOMEs, metabolites of leukotoxin and iso-leukotoxin. It was found

that these DHOMEs are more toxic than EpOMEs and are associated with multiple organ



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failure and adult respiratory distress syndrome (34). Taken together, the effect of the

sEHI, t-TUCB, on the concentrations of both EETs and DHETs may contribute to the

anti-inflammatory effects of sEHI in this murine asthmatic model.

In this study, we also observed that additional lipid mediators were affected upon

administration of t-TUCB. The pro-inflammatory lipid mediator-LTB4 was decreased in

the plasma after the low dose of administration of sEHI. It was reported that LTB4

participates in the allergic sensitization process in animal models (36). Therefore, the

effect of inhibition of sEH might also benefit from reduction of pro-inflammatory LTB4.

Another lipid mediator, 17HDoHE, which is a precursor to resolvins and possesses

biological activity that inhibits TNFα-induced IL-1β expression (37), was increased in

both lavage and lung homogenates after administration of sEHI. Resolvins have been

reported to promote the resolution of allergic airways response (38).

Our findings add to those reported elsewhere by describing the effects of sEH inhibition

on allergic airway inflammation. Several studies (10, 21) have demonstrated that sEHI

can reduce the inflammatory cytokines IL-1β, IL-6, INF-γ. In the present study, we found

that sEHI reduced Th2 cytokines and chemokines, which are known to play major roles

in the asthmatic immune response (39). Specifically, the pronounced effect of sEHI on

IL-5 and eotaxin-1, a key cytokine and chemokine responsible for the release of

eosinophil from the bone marrow and homing of eosinophil to the lung, is rather

intriguing. Indeed mepolizumab, a monoclonal antibody against IL-5, used for the

treatment of severe asthma, is garnering significant attention in the clinical realm (40).

The inhibition of sEH may be an alternative strategy for decreasing IL-5 levels in concert

with other key mediators of lung inflammation.



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There are reports indicating that some of these regulatory lipid mediators, such as EETs

have direct function on the bronchi (6, 41). We observed that administration of sEHI

increased EETs in the BALF as shown in Figure 5C, which may directly rescue the

airway hyperreactivity. However, inflammation may also play a role in regulating lung

compliance and resistance. The anti-inflammatory effects of sEHI might have contributed

to the improvement of lung function after sEHI administration in this acute model of

asthma. In addition, it is worth keeping in mind that lipid mediators such as HETEs are

reported (42) to have effects on the airways. Alterations in the levels of various lipid

mediators may explain why treatment with 3 mg/kg of sEHI did not show improved

rescue (in comparison to treatment with 1 mg/kg) of the resistance induced by 2.0 mg/mL

of methacholine (Figure 6B). Direct pulmonary administration of an sEH inhibitor would

provide additional evidence on how sEHI regulates lung compliance and resistance. To

date we have not developed an effective system for administering sEHI directly to the

lung.

In our current study, neither COX2 nor 5-LOX in lung homogenates were significantly

suppressed by sEHI administration in OVA-exposed mice (supplementary Figure E2).

These findings suggest, at least in lung homogenates, that the major effects of sEHI are

unlikely due to the NFκ-B pathway.

Taken together, the administration of sEHI increased the anti-inflammatory mediators

both systemically and in the airways, as indicated by the lipid mediator levels from the

lavage, while simultaneously decreased pro-inflammatory mediators in the lung tissues

and airways. These lipid mediators changes influenced the reduction and down-regulation

of Th2 cytokines and chemokines expression in the airways. The reduction of these Th2



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cytokines and chemokines further decreased the recruitment of inflammatory cells

infiltration into the lungs and airways. The reduction in overall lung inflammation and the

increase of EETs in airway contributes to the alleviation of AHR.

The data are consistent with the sEH (EPHX2) being primarily responsible for the

conversion of fatty acid epoxides to the corresponding diols in the lung and with t-TUCB

inhibiting this catalytic activity. The activity of mEH (EPHX1) on this substrate is low

and pulmonary levels of the mEH are low. Also we have found no evidence for catalytic

activity of EH3 (EPHX3) or 4 in the lung. Although there is no evidence of inhibition of

EPHX1, 3, 4 or an unknown enzyme by t-TUCB, we cannot exclude the possibility of off

target effects with absolute certainty.

Among the limitations of this study was that we used only prophylactic and not

therapeutic treatment. Long term pulmonary inflammation including asthma leads to

chronic changes in the lung. However, our short-term model did not assess the effects of

sEHI on fibrotic biomarkers since this would require a longer term exposure to

inflammation, particularly that is associated with chronic diseases involving the use of

multiple drugs with different mechanisms of action. Future studies need to address

possible beneficial and detrimental effects of long term sEHI use.

Acknowledgements

This work was supported by American Asthma Association AAF 09-0269,

NIEHS grant R01 ES002710 and NIEHS Superfund Research Program grant P42

ES004699. Analytical work was partially supported by the NIH and NIDDK grant U24

DK097154. NIH HL105573 provided the NO work equipment. JY was supported by a



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Fellowship from Cystic Fibrosis Research, Inc. BDH is a George and Judy Marcus senior

fellow of the American Asthma Foundation. AAZ was funded by #UL1TR000002 and

CTSC NIH KL2 (K12) Award TR000134. JB was supported by T32HL007013.



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Figure Legends

Figure 1. The protocol for exposure and treatments. The BALB/c mice were sensitized by i.p.

(intraperitoneal) injection of ovalbumin (OVA) and alum adjuvant solution for 4 weeks and then

were exposed to OVA aerosol six times. Depending on the treatment groups, mice were injected

subcutaneous (s.c.) with vehicle solution, 1 mg/Kg t-TUCB or 3mg/Kg t-TUCB by s.c. every

other day from day 26 to day 38. Previous pharmacokinetic studies with t-TUCB in mice

indicated that these doses will give good exposure.

Figure 2. The concentration of t-TUCB in blood (A) and 14,15-EpETrE (EET) concentration in

plasma (B) suggest that the inhibitor was delivered successfully in vivo and well engaged. Blood

was drawn 2-6 hours after administration of the last dose. * means significant difference from

vehicle group. # means significantly difference between 1 mg/Kg and 3 mg/Kg t-TUCB groups.

All of three groups were exposed to OVA. (n=5 in all groups except n=4 in Air+Vehicle group)

Figure 3. Heatmaps generated from regulatory lipid mediators show that administration of t-

TUCB increased anti-inflammatory lipid mediators and decreased pro-inflammatory lipid

mediators in vivo. (A) A simplified depiction of the arachidonic acid cascade. (B-D) Heat maps

based on regulatory lipid mediators result of plasma (B), Bronchoalveolar lavage fluid (BALF)

(C), and lung homogenates (D). The color is corresponding to the fold change as shown in the

legend. Bright red means increase more than 2 times significantly (p<0.05); dark red means

increase less than 2 times significantly; black means no significant change; dark green means

decrease less than 2 times significantly; bright green means decrease by more than 2 times

significantly. (For plasma and BALF, n=5 in all groups except n=4 in Air+Vehicle group; for

lung homogenates result, n=4 in all groups except n=3 in Air+Vehicle group)

Figure 4. The administration of t-TUCB dramatically decreased the Th2 cytokines (IL-4 and IL-5)

(A and B) and chemokine (Eotaxin) (C) production in BALF and down-regulated the gene

expression of these Th2 cytokines (D and E) in lung homogenates. * means significant difference

from OVA+vehicle group. (n=5 in all groups except n=4 in non-immunized+Vehicle group).

Figure 5. (A) Administration of t-TUCB reduced the infiltrated inflammatory cells (#cell/mL) in

BAL. (B) The result of differentiation cells by Hema-3 stain shows that sEHI reduced the

number of eosinophil (#cell/mL) in BAL. (C) Administration of t-TUCB reduced the

inflammatory marker-FeNO. (D-G). The H&E stain results of lung tissues shows that sEHI

reduces the infiltration of inflammatory cells into lung tissues. (D) is from the air control group;

(E) is from the vehicle control after OVA exposure; (F) is from 1 mg/kg t-TUCB treated group;

(G) is from 3 mg/kg t-TUCB treated group. * means significant difference from OVA+vehicle

group.

Figure 6. Dynamic lung compliance (A) and airway hyperreactivity (B) results show that

administration of t-TUCB rescued OVA-induced asthmatic AHR. (n=4 for OVA+ Vehicle group



and OVA+ 3 mg/Kg t-TUCB group; n=3 for non-ummunized+Vehicle group and OVA+ 1

mg/Kg t-TUCB group.)



Figure 1



Figure 2



Figure 3

(A)

(B) Plasma

(C) BALF

(D) Lung Homogenate





Figure 4.





Figure 5



(D) (E)

(F) (G)



Figure 6.



1

Supplementary Files

Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation and Airway

Hyperresponsiveness in Mice

Jun Yang1, Jennifer Bratt

2, Lisa Franzi

2, Junyan Liu

1, Guodong Zhang

1, Amir A. Zeki

2,

Christoph F. A. Vogel3,4

, Keisha Williams2, Hua Dong

1, Yanping Lin

1, Sung Hee

Hwang1, Nicholas J. Kenyon

2, Bruce D. Hammock

1 *

1 Department of Entomology and Comprehensive Cancer Center,

2 Department of Internal

Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, 3 Department of

Environmental Toxicology, 4

Center for Health and the Environment. University of

California, Davis, CA 95616.

Address correspondence to: Bruce D. Hammock

Department of Entomology

University of California at Davis

One Shields Avenue, Davis, CA 95616.

Tel: (530) 752-7519

Fax: (530) 751-1537

E-mail: [email protected]



2

SUPPLEMENTARY MATERIALS AND METHODS

Lung compliance and resistance measurements

The dynamic lung compliance (Cdyn) and resistance of the respiratory system (Rst) was

measured with a plethysmograph for restrained animals (Buxco Inc., Troy, NY) 1–3

hours after termination of the final (6th) OVA exposure. Mice were deeply anesthetized

and sedated with medetomidine, 0.5 mg/kg (Domitor, Orion Pharma, Finland), and

tiletamine/zolpidem, 50 mg/kg (Telazol, Fort Dodge Laboratories, Fort Dodge, IA) and

surgically cannulated and ventilated at 7–8 cc/kg using a mouse ventilator (MiniVent,

Harvard Apparatus, Cambridge, MA) for the duration of the procedure. Cdyn (mL/cm of

H2O) and Rst (cm of H2O*sec/mL) measurements were made at baseline and

immediately following serial 3 minute nebulizations of saline and methacholine (0.5, 1.0

and 2.0 mg/ml), with 2-minute recovery periods allowed after each exposure.

Lung Tissue Processing

Immediately after lung physiology measurements, mice were killed with an overdose of

Beuthanasia-D. After blood collection, lungs were lavaged twice with 1 ml of PBS (pH

7.4), and then centrifuged at 2,500 rpm on a tabletop centrifuge for 10 minutes. The total

lavage live cell number and differential cell counts were determined.

Mice had their lungs fixed or immediately frozen and stored at -80 °C. Lungs were fixed

for histological evaluation with 1% paraformaldehyde. After fixation and paraffin

embedding, the lungs were stained with hematoxylin and eosin to qualitatively assess

peribronchiolar inflammation. Half of right lungs were immersed in RNA Later solution

(Life Technology, Carlsbad, CA) for the followed RT-PCR measurements.



3

Airway Inflammatory Cell Populations

Total cell counts in the lung lavage fluid were determined using a hemocytometer and

trypan blue exclusion; 100 µL of the remaining cell suspension was processed onto slides

using a cytocentrifuge at 1650 rpm for 7 minutes for determination of the cell differential

counts. Slides were air-dried, stained with a Hema3 stain set as described in the

manufacturer's instructions (Fisher Scientific, Kalamazoo, MI).

LC/MS/MS analysis for t-TUCB

The liquid chromatography system used for analysis was an Agilent 1200 SL liquid

chromatography series (Agilent Corporation, Palo Alto, CA). The autosampler was kept

at 4 °C. Liquid chromatography was performed on an ACQUITY UPLC BEH C18, 1.7

µm, 2.1× 50 mm column (Waters, Milford, MA). Mobile phase A was water with 0.1%

glacial acetic acid. Mobile phase B consisted of acetonitrile with 0.1% glacial acetic acid.

Gradient elution was performed at a flow rate of 400 µL/min. The gradient began with

50% B and reached 95% B at 3 min. After holding at 95% for 0.5 min, the composition

of mobile phase went back to 50% at 3.5 min for another 0.5 min. The column was

connected to a 4000 QTrap tandem mass spectrometer (Applied Biosystems Instrument

Corporation, Foster City, CA) equipped with an electrospray source (Turbo V). The

instrument was operated in negative MRM mode. Individual analyte standards were

infused into the mass spectrometer and MRM transitions and source parameters

optimized for t-TUCB. The MRM transition for t-TUCB is 437.2/137.1; the transition for

CUDA is 339.2/214.3.

Extraction Protocol for Plasma Oxylipin Profile



4

The Oasis cartridges (Waters,) were washed with ethyl acetate and methanol, and

equilibrated to the initial condition with the solution containing H2O and methanol.

Diluted supernatant was loaded on the cartridge and washed with SPE solution (H2O with

5% methanol with 0.1% acetic acid) twice. The cartridges were dried by vacuum and

analytes were eluted with 0.5 mL of methanol and 1.5 mL of ethyl acetate to tubes with 6

µL of trapping solution (30% glycerol in methanol). The elutes were dried by vacuum

(SpeedVac) and reconstitute with internal solution (200 nM CUDA methanol solution).

Extraction Protocol for BALF Oxylipin Profile

Ten microLiters of antioxidant solution (0.2 mg/mL of BHT and EDTA) were added to 2

mL BAL fluid supernatant followed by the addition of 10 uL of surrogate solutions, in

which 9 deuterated internal standards were included. BAL fluids were then loaded on the

Oasis cartridges as mentioned above. The followed SPE protocol is same with the plasma

extraction protocol described above.

Extraction Protocol for Lung Homogenates Oxylipin Profile

Ten microLiters of anti-oxidant solution were added to 100mg of lung tissue followed by

the addition of 10 µL of surrogate solution. After addition of 400 µL of ice-cold

methanol with 0.1 % of acetic acid and 0.1% of BHT, the lung tissues were stored at -

80°C freezer for 30 min. Then, the lung tissue samples were homogenized using Mixer

Mill MM301 (Retsch, Haan, Germany) at 30 Hz for 10 min. The homogenates were

stored at -80°C freezer overnight. After centrifuged at 10,000 rpm for 10 min, the

supernatant were collected and the remaining pellets were washed with 100 µL of ice-

cold methanol with 0.1 % of acetic acid and 0.1% of BHT and centrifuged again. The



5

supernatants of each sample were combined and diluted with 2 mL of H2O and load onto

SPE cartridges. Then the SPE protocol is the same as the one for plasma extraction

protocol.

Quantitative Real-time Reverse Transcription–PCR (RT-PCR)

Total RNA was isolated from tissue using Trizol and a Quick-RNA Mini prep isolation

kit (Zymo Research, Irvine, CA), and cDNA synthesis and RT-PCR process are fully

described in the supplementary files. was done as previously described (1). Quantitative

detection of β-actin and differentially expressed genes was performed with LightCycler

LC480 (Roche, Indianapolis, IN) using the Fast SYBR Green Master Mix (Life

Technologies, Grand Island, NY) according to the manufacturer’s instructions. DNA-free

total RNA (1.0 µg) was reverse-transcribed using 4 U Omniscript reverse transcriptase

(RT; Qiagen) and 1 µg oligo(dT)15 in a final volume of 40 µl. The primers for each gene

were designed on the basis of the respective cDNA or mRNA sequences using OLIGO

primer analysis software provided by Steve Rozen and the Whitehead Institute/MIT

Center for Genome Research (2). So, that the targets were 100–200 bp in length. The

following primer sequences were used: mouse β-actin (forward primer, 5′-

AGCCATGTACGTAGCCATCC-3′; reverse primer, 5′-

CTCTCAGCTGTGGTGGTGAA-3′), mouse IL-4 (forward primer, 5’-

TCAACCCCCAGCTAGTTGTC-3’; reverse primer, 5’-

TGTTCTTCGTTGCTGTGAGG-5’), mouse IL-5 (forward primer, 5’-

GAAGTGTGGCGAGGAGAGAC-3’; reverse primer, 5’-

GCACAGTTTTGTGGGGTTTT-3’) and mouse IL-13 (forward primer:5’-

CAGCTCCCTGGTTCTCTCAC-3’); reverse primer: 5’-



6

CCACACTCCATACCATGCTG-3’). PCR amplification was carried out in a total

volume of 20 µl containing 2 µl cDNA, 10 µl 2 × Fast SYBR Green Master Mix, and 0.2

µM of each primer. The PCR cycling conditions were 95°C for 30 sec followed by 40

cycles of 94°C for 3 sec, and 60°C for 30 sec. Detection of the fluorescent product was

performed at the end of the 72°C extension period. Negative controls were concomitantly

run to confirm that the samples were not cross-contaminated. A sample with DNase- and

RNase-free water instead of RNA was concomitantly examined for each of the reaction

units described above. To confirm the amplification specificity, the PCR products were

subjected to melting curve analysis.

Western blot method

The lung tissue were solicited in a homogenization buffer containing 0.1% SDS, protease

inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 574 µM phenylmethanesulfonyl

fluoride (PMSF) in PBS. Homogenates were centrifuged at 10,000 rpm for 20 minutes

and the resulting supernatant was stored at -80 °C until further use.

Antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) unless

otherwise stated. Total protein concentration of homogenate samples was determined

using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Samples

containing 20 µg of protein were incubated at 65 °C for 15 minutes, electroporated under

reducing conditions, and transferred to a polyvinylidene difluoride (PVDF) membrane.

Membranes were blocked in 5% dry milk in PBS for 1 hour at 25 °C, then incubated in

0.4 µg/ml of goat, anti-mouse Arg1, 0.4 µg/ml of goat, anti-mouse Arg2, or 0.4 µg/ml of



7

rabbit anti-human α-actinin overnight at 4 °C, then incubated for 1 hour at 25 °C in 40

ng/ml of horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (Pierce

Biotechnology, Rockford, IL) or 40 ng/ml of horseradish peroxidase (HRP) conjugated

goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL). Bands were visualized using

the Immobilon western chemiluminescent HRP substrate kit (Millipore, Billerica, MA)

and images captured using Image Reader LAS-3000 version 2.1 (FUJIFILM, Cypress,

CA).

References

1. Vogel, C. F., Sciullo, E., Park, S., Liedtke, C., Trautwein, C., Matsumura, F.,

2004. Dioxin increases C/EBPbeta transcription by activating cAMP/protein

kinase A. J Biol Chem 279, 8886-94.

2. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for

biologist programmers. Methods Mol Biol 132, 365-86.

SUPPLEMENTARY FIGURE LEGENDS

Figure E1. The inflammatory cytokine panel as well as the IL-4, IL-5, eotaxin shown in

Figure 4 responded differently to the OVA exposure and administration of sEHI.

Figure E2. COX2 (A) and 5-LOX (B) induced by OVA challenge but were not reduced

by administration of sEHI.



8

Supplementary Table E1. The concentrations of lipid mediators in Plasma, BALF and

lung homogenates. The units in plasma and BALF are in nmol/L. The unit in lung

homogenates is in pmol/g tissue.

Supplementary Table E2. The epoxides to diols ratios supported the engagement of sEHI

in plasma, BALF and lung homegenates.

This table is in Excel format. It can be accessed from this issue’s table of contents online

at www.atsjournal.org.

Figure E1.



9



10

Figure E2.

(A)

(B)



Origin # Group 6-keto-PGF1aPGE2 PGD2 PGF2a PGD1 15-deoxy-PGJ211-HETE

115 Vehicle 0.54 0.14 0.41 0.15 0.09 0.72 0.18

116 Vehicle 0.68 0.11 0.43 0.26 0.07 0.72 0.63

117 Vehicle 0.69 0.10 0.46 0.00 0.05 0.67 0.31

118 Vehicle 0.73 0.08 0.51 0.00 0.03 0.42 0.73

119 Vehicle 0.67 0.08 0.43 0.00 0.10 1.02 0.24

120 Vehicle 0.85 0.09 0.44 0.15 0.42 0.99 0.22

133 Low 1728 0.58 0.15 0.51 0.00 0.08 1.14 0.81

134 Low 1728 0.36 0.44 0.45 0.81 0.06 1.83 0.70

135 Low 1728 0.51 0.16 0.55 0.00 0.08 0.53 1.01

136 Low 1728 0.77 0.11 0.55 0.00 0.09 0.69 0.92

137 Low 1728 0.70 0.09 0.41 0.00 0.05 1.11 0.35

138 Low 1728 0.77 0.06 0.43 0.63 0.34 1.61 0.18

139 Hi 1728 0.73 0.13 0.64 2.12 0.21 0.90 0.75

140 Hi 1728 0.74 0.36 0.62 0.15 0.17 0.79 0.80

141 Hi 1728 0.82 0.07 0.44 0.00 0.10 0.82 0.87

142 Hi 1728 0.56 0.12 0.54 2.91 0.73 0.73 0.71

143 Hi 1728 1.20 0.33 0.60 1.11 0.15 1.37 1.32

144 Hi 1728 0.76 0.12 0.46 0.91 0.08 1.02 0.53



9-HETE EKODE LTB4 20-COOH-LTB420-OH-LTB46-trans-LTB4LTB3 5-HETE 5-HEPE

0.40 8.12 0.32 0.71 0.12 0.20 0.23 1.25 0.36

0.60 8.15 0.32 0.49 0.13 0.29 0.12 1.65 1.48

0.38 5.00 0.33 0.44 0.14 0.38 0.14 0.98 1.22

0.61 9.50 0.35 0.45 0.07 0.25 0.15 1.65 1.51

0.32 8.33 0.38 0.78 0.10 0.23 0.25 1.54 0.87

0.38 4.53 0.34 1.04 0.15 0.73 0.23 1.25 0.66

0.50 15.42 0.36 0.55 0.05 0.44 0.18 1.77 0.69

0.54 21.37 0.38 0.87 0.07 0.85 0.19 1.59 0.85

0.55 12.71 0.38 0.43 0.08 0.00 0.17 3.27 0.78

0.94 11.53 0.42 0.48 0.10 0.29 0.15 2.94 1.13

0.37 4.23 0.35 0.63 0.08 0.03 0.14 1.40 0.34

0.66 5.60 0.37 0.62 0.23 0.31 0.18 1.97 0.85

0.42 9.02 0.43 0.87 0.10 0.24 0.23 2.68 0.64

0.46 8.29 0.41 1.02 0.12 0.00 0.13 2.62 0.71

0.37 12.45 0.38 0.66 0.05 0.00 0.14 2.82 0.89

0.46 7.38 0.35 0.53 0.20 0.20 0.21 3.08 1.02

0.57 13.49 0.38 1.41 0.09 1.16 0.19 3.42 1.56

0.49 15.95 0.36 0.80 0.13 0.69 0.14 1.86 0.72



15-HETE 15(S)-HETrE13-HOTrE 13-HODE 15-HEPE 17-HDoHE 12-HETE 8-HETE 9-HOTrE

1.37 0.30 2.09 24.25 0.40 2.29 19.01 0.55 0.44

2.14 0.62 4.64 74.02 1.25 4.07 33.38 0.88 2.03

1.42 0.40 3.24 46.20 1.23 4.10 8.75 0.51 1.06

1.83 0.63 5.66 77.59 1.57 3.55 33.20 0.81 2.79

1.77 0.54 2.87 34.65 0.88 9.26 32.21 0.64 1.10

1.66 0.33 3.33 27.37 0.48 12.73 13.21 0.47 0.72

1.69 0.72 3.88 51.62 0.70 2.30 20.20 0.83 1.84

2.23 0.81 5.28 70.17 0.96 6.73 27.05 1.33 1.98

5.73 1.08 1.73 51.22 0.44 4.81 111.81 1.45 1.90

4.98 1.02 2.35 71.85 0.50 11.46 64.69 1.25 3.24

1.82 0.42 1.95 19.32 0.31 5.76 26.10 0.54 0.66

2.71 0.38 2.82 33.38 0.77 12.24 14.99 0.75 0.92

3.32 0.65 1.38 25.87 0.29 0.85 8.35 0.77 0.65

3.45 0.71 2.02 41.86 0.48 6.03 28.98 0.94 1.60

3.79 0.82 3.21 54.63 0.49 4.20 32.07 1.05 2.89

3.68 0.88 1.84 37.30 0.50 7.19 39.07 1.01 1.21

4.33 0.95 3.55 59.06 1.57 5.51 77.03 1.14 1.84

1.68 0.71 3.62 42.57 0.58 1.57 44.48 0.90 1.50



9-HODE 12-HEPE 8-HEPE 12-oxo-ETE15-oxo-ETE13-oxo-ODE9-oxo-ODE 20-HETE 14(15)-EpETrE

10.54 5.32 0.22 13.13 0.12 0.30 3.67 10.29 0.87

24.18 15.60 1.61 113.91 0.38 0.41 3.55 6.88 0.77

16.74 4.25 1.52 12.00 0.10 0.29 2.44 6.22 0.50

27.66 15.16 1.44 21.96 0.24 0.59 3.37 7.32 0.79

15.29 10.08 0.24 25.33 0.28 0.30 5.21 6.23 0.75

13.79 3.22 0.25 10.92 0.25 0.30 4.21 5.04 0.99

18.05 5.98 0.41 40.10 0.26 1.61 5.35 14.08 3.25

25.88 7.69 0.46 35.65 0.49 3.21 10.04 20.17 2.85

24.28 28.44 0.25 28.59 0.61 0.54 7.91 11.77 3.28

32.66 20.45 0.49 12.14 0.35 0.91 7.20 16.78 2.13

7.76 5.85 0.14 32.94 0.40 0.30 2.48 4.08 1.45

14.09 4.10 0.41 32.48 0.39 0.44 3.77 5.85 2.41

11.85 2.34 0.27 3.84 0.49 0.41 4.52 7.20 3.14

19.61 7.08 0.41 10.46 0.59 0.33 5.44 10.56 2.66

25.14 8.42 0.36 11.75 0.31 1.22 5.93 13.40 2.76

16.78 9.58 0.64 10.86 1.13 0.39 6.57 4.58 3.05

28.30 26.25 0.80 19.22 0.85 0.45 8.77 12.21 2.96

17.67 13.44 0.48 91.81 0.45 1.62 6.03 8.87 2.59



11(12)-EpETrE8(9)-EpETrE5(6)-EpETrE12(13)-EpOME9(10)-EpOME15(16)-EpODE12(13)-EpODE9(10)-EpODE19(20)-EpDPE

0.66 0.42 1.40 14.62 10.65 5.27 0.03 0.36 8.03

1.60 0.37 2.29 33.16 11.98 30.28 0.42 1.08 9.43

0.64 0.47 1.18 27.66 8.11 20.58 0.45 0.58 5.07

0.87 0.63 1.94 34.93 13.14 36.81 0.58 1.16 9.33

0.54 0.75 1.64 14.52 9.34 7.56 0.04 0.53 9.17

1.22 0.28 1.22 13.68 9.19 6.35 0.04 0.61 10.99

1.62 1.19 3.54 153.17 24.10 56.85 2.23 2.85 26.19

2.06 0.88 2.82 168.34 30.37 68.61 2.81 2.77 32.51

2.58 1.07 3.51 76.91 20.27 32.16 1.02 1.72 24.74

1.79 1.40 2.09 104.76 22.74 53.47 1.55 2.28 20.97

1.03 1.00 0.95 19.35 5.99 7.09 0.22 0.41 6.68

1.68 0.97 1.92 65.29 12.91 23.48 0.61 1.40 13.81

1.90 1.42 2.57 58.40 12.55 17.42 0.51 0.43 17.25

2.22 1.82 1.94 84.67 15.21 24.44 1.07 1.35 16.38

1.87 1.20 1.85 97.61 15.09 28.26 1.47 1.43 16.74

2.15 0.84 2.64 80.17 14.43 26.53 0.98 1.66 18.65

2.34 1.05 2.63 98.51 23.70 46.33 0.42 1.53 25.90

1.78 1.11 2.56 97.27 18.79 44.62 1.27 1.52 22.90



16(17)-EpDPE13(14)-EpDPE10(11)-EpDPE17(18)-EpETE14,15-DiHETrE11,12-DiHETrE8,9-DiHETrE5,6-DiHETrE12,13-DiHOME

0.61 0.42 0.96 1.22 1.49 0.71 0.48 0.23 30.27

0.80 0.61 0.89 3.84 3.78 1.86 0.76 0.26 82.73

0.47 0.22 0.65 3.25 1.45 0.63 0.37 0.15 61.96

0.82 0.46 0.96 3.57 2.60 1.15 0.57 0.18 92.64

0.72 0.53 1.00 1.78 1.30 0.63 0.56 0.33 26.57

0.89 0.50 0.77 1.11 1.25 0.61 0.66 0.19 50.07

3.03 0.95 1.68 7.37 2.44 1.73 0.79 0.14 29.41

2.95 0.86 2.31 8.42 5.70 3.67 1.47 0.22 80.93

3.27 1.50 2.48 6.65 2.17 1.46 0.59 0.12 23.45

1.93 1.16 1.93 7.38 2.43 1.62 0.58 0.12 42.72

0.69 0.28 0.69 2.11 0.85 0.46 0.28 0.13 12.18

1.36 0.94 1.25 4.86 0.79 0.54 0.49 0.22 23.14

2.04 0.80 1.49 5.46 1.23 0.99 0.54 0.17 11.95

2.45 0.91 1.76 4.41 2.05 1.75 0.64 0.25 15.20

2.16 0.89 1.40 5.10 2.23 1.63 0.52 0.12 20.53

2.18 1.09 2.01 5.97 1.79 1.37 0.64 0.20 15.90

2.26 1.21 2.09 8.29 1.20 0.80 0.68 0.24 44.00

1.87 0.57 1.00 5.63 1.19 0.70 0.47 0.15 51.71



9,10-DiHOME15,16-DiHODE12,13-DiHODE9,10-DiHODE19,20-DiHDPE16,17-DiHDPE13,14-DiHDPE10,11-DiHDPE7,8-DiHDPE

9.14 6.72 0.58 0.30 29.00 2.58 0.70 0.65 1.45

24.03 21.92 3.87 3.10 42.56 13.40 2.37 1.30 1.66

18.38 15.81 3.13 2.39 20.13 5.74 0.85 0.67 1.24

26.39 22.12 4.15 3.16 27.58 8.33 1.53 0.92 1.51

10.40 8.69 0.58 0.62 27.74 2.76 0.77 0.54 1.66

18.24 10.50 1.11 0.83 18.92 2.03 0.62 0.62 1.89

11.69 3.85 1.16 1.93 33.79 7.63 2.12 0.87 1.20

26.27 8.44 2.72 3.57 65.05 16.53 4.57 2.25 2.09

9.79 2.77 1.06 1.17 33.63 4.89 1.22 0.72 0.74

15.09 4.58 1.61 1.90 32.40 5.68 1.32 0.88 0.71

4.32 3.19 0.81 0.52 12.68 1.77 0.47 0.44 0.50

9.11 4.60 0.91 1.22 12.30 1.80 0.57 0.56 0.46

6.79 0.71 0.46 0.57 18.59 2.88 0.96 0.56 0.49

7.04 2.19 0.71 0.94 22.29 5.90 1.74 0.73 0.39

9.46 2.55 0.71 1.33 24.14 6.21 1.77 0.71 0.56

7.81 2.67 0.84 1.09 23.47 4.54 1.22 0.60 0.87

19.85 5.20 1.86 2.11 18.01 2.60 0.91 0.66 1.05

16.77 5.68 1.62 2.16 17.19 2.82 0.81 0.53 0.72



4,5-DiHDPE17,18-DiHETE14,15-DiHETE11,12-DiHETE11,12-,15-TriHETrE9,12,13-TriHOME9,10,13-TriHOME5,15-DiHETEResolvin E1

2.38 7.49 0.94 0.25 0.33 13.25 7.34 0.10 23.47

1.26 13.74 3.95 0.74 0.35 16.60 7.80 0.10 18.65

0.80 9.77 2.60 0.47 0.53 15.18 5.70 0.10 19.46

1.51 10.69 3.78 0.70 0.16 13.55 4.87 0.10 15.55

1.66 8.81 1.16 0.25 0.63 43.42 23.38 0.11 19.51

1.11 5.10 0.76 0.25 0.97 36.83 18.61 0.14 27.33

3.22 7.36 2.99 0.72 0.15 2.09 0.00 0.11 10.84

5.78 14.87 5.17 1.20 0.77 16.25 6.05 0.10 7.64

3.84 6.70 1.80 0.53 0.65 16.74 5.90 0.11 12.25

4.56 6.97 1.89 0.60 0.45 5.44 0.47 0.12 14.25

0.83 2.98 0.74 0.19 0.27 3.44 0.00 0.09 10.18

2.21 3.44 0.97 0.26 0.59 17.31 8.33 0.12 16.61

3.13 4.08 1.02 0.35 0.54 14.51 9.87 0.13 18.25

3.67 5.86 1.71 0.53 0.54 14.01 4.90 0.10 16.52

2.50 5.76 2.06 0.55 0.14 13.32 4.81 0.10 9.24

7.42 5.80 1.56 0.37 1.03 14.14 15.92 0.16 17.85

3.46 5.79 1.54 0.41 0.85 6.16 3.89 0.20 19.18

1.54 5.03 1.25 0.31 0.27 6.19 0.08 0.11 9.17



LXA4

0.09

0.26

0.11

0.08

0.09

0.08

0.16

0.44

0.09

0.10

0.09

0.25

0.10

0.24

0.05

0.21

0.48

0.31



Origin # Group 6-keto-PGF1aPGE2 PGD2 PGF2a PGD1 15-deoxy-PGJ211-HETE

115 Vehicle 0.612786 0.218459 0.121927 0.208148 0.013287 0.035531 0.053011

116 Vehicle 0.956194 0.386346 0.158625 0.320304 0.023161 0.039398 0.06692

117 Vehicle 1.376529 0.85117 0.325032 0.396892 0.028867 0.035373 0.099083

118 Vehicle 1.365408 0.562445 0.256395 0.284891 0.029533 0.029159 0.08331

119 Vehicle 0.939118 0.878885 0.273148 0.604412 0.021071 0.032862 0.089597

120 Vehicle 2.660328 1.045844 0.289158 0.883455 0.039807 0.036129 0.160688

133 Low 1728 2.86382 1.51714 0.768938 0.505077 0.725672 0.255651 0.51054

134 Low 1728 0.402519 0.366602 0.163822 0.139071 0.021203 0.044107 0.072778

135 Low 1728 1.134424 1.38821 0.869759 1.243642 0.058021 0.131235 0.343799

136 Low 1728 0.929851 0.933749 0.372035 0.910885 0.027194 0.024349 0.184279

137 Low 1728 0.223406 0.704127 0.177762 0.282541 0.038602 0.032414 0.1122

138 Low 1728 0.344297 1.251326 0.295564 1.743336 0.023832 0.029072 0.255544

139 Hi 1728 0.64107 0.880575 0.357194 0.793268 0.023041 0.028822 0.126731

140 Hi 1728 1.302977 1.243945 0.411332 1.124204 0.031967 0.027863 0.26551

141 Hi 1728 1.259654 0.8524 0.422008 0.280382 0.036814 0.033938 0.217729

142 Hi 1728 0.998102 0.444168 0.171561 0.182186 0.013455 0.023324 0.134411

143 Hi 1728 0.249087 1.387744 0.326129 1.500634 0.027874 0.016343 0.280939

144 Hi 1728 0.29534 1.327986 0.293624 1.360559 0.029014 0.033823 0.311188



9-HETE EKODE LTB4 20-COOH-LTB420-OH-LTB46-trans-LTB4LTB3 5-HETE 5-HEPE

0.067853 0.380647 0.064116 0.049453 0.017601 0.20438 0.01587 0.085581 0.014185

0.048073 0.312643 0.0592 0.052563 0.034477 0.023874 0.022486 0.091649 0.022387

0.065941 0.595679 0.071417 0.050735 0.01997 0.177846 0.017253 0.153374 0.020635

0.064296 0.336425 0.072547 0.059757 0.020525 0.062872 0.021029 0.142306 0.016216

0.059516 0.457214 0.058296 0.057807 0.023078 0.04886 0.023905 0.119605 0.029003

0.078542 1.181789 0.059505 0.06126 0.011836 0.045036 0.019524 0.166964 0.034562

0.203744 1.353911 0.207195 1.349274 0.265559 0.355086 0.13916 0.392215 0.073583

0.04139 0.397768 0.047511 0.087986 0.013445 0.029179 0.016266 0.082697 0.016078

0.421167 2.933581 0.231385 0.272204 0.132343 0.380552 0.155808 0.735233 0.138383

0.121184 0.659799 0.07201 0.059682 0.01517 0.178055 0.016679 0.255135 0.040245

0.095262 3.555115 0.037803 0.075867 0.00669 0.071538 0.013188 0.204857 0.054505

0.106914 0.36582 0.051144 0.058514 0.017505 0.099096 0.025318 0.18142 0.032932

0.090926 0.820914 0.068098 0.075709 0.024407 0.205261 0.017799 0.221306 0.024457

0.127235 0.787516 0.07306 0.076836 0.019652 0.09641 0.023756 0.306961 0.037674

0.104315 0.513432 0.049146 0.058433 0.015089 0.091768 0.017592 0.324443 0.036582

0.056135 0.836944 0.051618 0.055703 0.029163 0.154589 0.024834 0.160157 0.04197

0.11675 1.234484 0.0555 0.059743 0.023945 0.219686 0.022178 0.140093 0.061704

0.0978 0.748396 0.064443 0.070246 0.019641 0.120162 0.028746 0.096758 0.029898



15-HETE 15(S)-HETrE13-HOTrE 13-HODE 15-HEPE 17-HDoHE 12-HETE 8-HETE 9-HOTrE

0.376804 0.064036 0.125627 1.804169 0.12202 1.428552 1.809009 0.070388 0.018619

0.609455 0.13631 0.119471 1.592272 0.17491 1.462823 3.354617 0.089598 0.016675

0.956516 0.216492 0.133204 1.917558 0.291631 2.65702 4.304629 0.125338 0.019957

0.811332 0.205847 0.15818 1.815429 0.253952 2.422955 4.652585 0.149875 0.019636

0.674998 0.113426 0.125057 1.515146 0.237936 1.65332 3.877494 0.138802 0.017756

0.74353 0.111381 0.150987 2.292522 0.398245 2.751634 3.106782 0.090213 0.026262

3.017167 0.769511 0.473171 6.271757 0.963781 8.277269 21.01629 0.651107 0.069111

0.658962 0.1678 0.140871 1.332826 0.193647 1.94745 2.969319 0.086764 0.018962

1.907105 0.384686 0.490881 7.835754 0.664926 5.837052 21.66928 0.570486 0.076797

1.486609 0.33769 0.146631 2.254071 0.514386 4.373995 7.696472 0.238249 0.019057

0.979411 0.087056 0.098176 1.699852 0.367844 4.473531 3.136645 0.116592 0.022583

0.81092 0.09114 0.12553 2.105628 0.568129 6.759525 6.298741 0.187822 0.022248

1.241961 0.215197 0.11222 1.976468 0.307661 3.777612 7.141128 0.226007 0.025342

1.880459 0.27821 0.112721 2.344623 0.537548 6.130197 14.40448 0.305041 0.021092

1.097876 0.255628 0.091389 1.523802 0.252627 2.309894 5.857338 0.24912 0.018512

0.762676 0.119812 0.101421 1.356356 0.28393 1.850825 3.494148 0.125863 0.019253

1.03486 0.137089 0.14389 2.590617 0.527202 4.893099 4.53335 0.165671 0.021714

0.921204 0.123242 0.140178 1.54157 0.526415 3.808423 3.672426 0.134066 0.017526



9-HODE 12-HEPE 8-HEPE 12-oxo-ETE15-oxo-ETE13-oxo-ODE9-oxo-ODE 20-HETE 14(15)-EpETrE

0.506759 0.177585 0.016738 5.053253 0.089183 0.031301 0.421337 0.064741 0.036176

0.44733 0.254517 0.034789 6.801508 0.102704 0.026052 0.421684 0.057025 0.018426

0.593705 0.263127 0.027603 16.73455 0.276489 0.024667 0.360299 0.061534 0.035012

0.583685 0.17519 0.022024 6.924372 0.136575 0.035898 0.461238 0.085732 0.018827

0.529352 0.247036 0.018614 13.40917 0.276041 0.04308 0.450581 0.091285 0.062836

0.735448 0.418384 0.014631 19.50895 0.395822 0.025603 0.495794 0.11297 0.047425

1.808979 1.171672 0.058912 49.55249 0.557718 0.132862 1.49623 0.6558 0.166199

0.39058 0.190149 0.009892 6.219621 0.104136 0.028382 0.308093 0.240448 0.034332

2.517467 3.522689 0.162334 133.8143 0.736505 0.204568 2.648898 0.322609 0.184179

0.732051 0.707024 0.029148 19.73892 0.430841 0.030066 0.470443 0.178505 0.072144

0.66243 0.421464 0.033924 45.48099 1.630235 0.027737 0.997295 0.084338 0.128147

0.686124 0.923791 0.042923 10.71194 0.282822 0.044479 0.773994 0.297386 0.103081

0.629772 0.325799 0.022644 17.49105 0.302893 0.030147 0.501278 0.096702 0.069343

0.676412 1.430798 0.033453 14.53737 0.329418 0.032562 0.494861 0.082505 0.096057

0.649809 0.432682 0.025293 21.69519 0.242256 0.027228 0.380428 0.06276 0.083443

0.394605 0.391906 0.048427 18.31002 0.373319 0.037715 0.550057 0.150164 0.06517

0.696256 0.493772 0.026045 43.04296 0.726815 0.053316 0.979083 0.26616 0.169921

0.443011 0.325604 0.026796 23.85318 0.415747 0.039573 0.624891 0.289281 0.118303



11(12)-EpETrE8(9)-EpETrE5(6)-EpETrE12(13)-EpOME9(10)-EpOME15(16)-EpODE12(13)-EpODE9(10)-EpODE19(20)-EpDPE

0.041856 0.064592 0.062325 0.132566 0.100761 0.055698 0.00729 0.007179 0.399098

0.061845 0.073668 0.043012 0.313277 0.11285 0.262864 0.006066 #VALUE! 0.260731

0.079855 0.118755 0.087836 0.320366 0.136424 0.249152 0.01395 0.010821 0.341708

0.043195 0.084106 0.072949 0.418468 0.15323 0.386644 0.011131 0.011869 0.347628

0.123656 0.132043 0.107122 0.198011 0.153576 0.084174 0.002277 0.006641 0.494202

0.117164 0.19915 0.198466 0.203671 0.213165 0.110033 0.004829 0.004585 0.479328

0.162218 0.171603 0.111555 2.942377 0.679201 1.64346 0.227788 0.241215 2.131312

0.050612 0.045873 0.033091 0.904818 0.153856 0.680872 0.051 0.008856 0.555556

0.485403 0.528362 0.537523 2.370386 0.958909 1.01712 0.093826 0.086045 3.377893

0.119985 0.261291 0.216571 0.844317 0.257884 0.546855 0.028812 0.023514 0.676558

0.209549 0.068085 0.249691 0.406008 0.279245 0.14856 0.007068 0.010466 0.710918

0.171177 0.052935 0.190185 0.575995 0.286494 0.143367 0.019866 0.00976 0.58486

0.123663 0.027133 0.157767 0.323585 0.182284 0.098966 0.008801 0.014879 0.286667

0.163308 0.054232 0.233707 0.589147 0.227661 0.211434 0.01647 0.017377 0.35312

0.131229 0.057022 0.155431 0.801629 0.184821 0.371828 0.033894 0.01227 0.414469

0.063811 0.188263 0.168489 0.383995 0.161252 0.151211 0.012013 0.013562 0.369882

0.226185 0.042592 0.285887 0.900976 0.266535 0.418422 0.024677 0.027461 1.146981

0.214473 0.071321 0.156759 0.789114 0.262072 0.419188 0.02711 0.033371 0.950195



16(17)-EpDPE13(14)-EpDPE10(11)-EpDPE17(18)-EpETE14,15-DiHETrE11,12-DiHETrE8,9-DiHETrE5,6-DiHETrE12,13-DiHOME

0.021211 0.024815 0.036158 0.02427 0.037437 0.027521 0.025654 0.007101 0.190061

0.025916 0.034267 0.040998 0.067664 0.052968 0.034246 0.026904 0.009141 0.662454

0.028658 0.020596 0.027127 0.049325 0.041985 0.026447 0.010531 0.011225 0.447108

0.014538 0.016119 0.030824 0.05143 0.038847 0.027481 0.011603 0.008714 0.895221

0.056538 0.035783 0.069629 0.056381 0.035232 0.024825 0.008464 0.006544 0.126563

0.051297 0.046281 0.107162 0.096688 0.038858 0.028712 0.014163 0.006217 0.282352

0.167116 0.027507 0.165292 0.32892 0.106941 0.174439 0.021345 0.064413 0.923862

0.025296 0.021918 0.041806 0.078094 0.068911 0.04555 0.015444 0.007517 0.453376

0.222644 0.060153 0.26173 0.53203 0.18215 0.154913 0.155967 0.069662 1.260703

0.037592 0.046271 0.086302 0.088507 0.038033 0.032184 0.019611 0.01161 0.071094

0.065578 0.088451 0.125355 0.140146 0.029423 0.015595 0.008783 0.011316 0.041966

0.045941 0.102557 0.141823 0.237828 0.01996 0.018603 0.017214 0.010908 0.07874

0.033654 0.014264 0.094435 0.14038 0.026709 0.023486 0.016296 0.013539 #VALUE!

0.060661 0.06216 0.073265 0.112609 0.032704 0.026799 0.023727 0.005962 #VALUE!

0.036672 0.006496 0.063949 0.090012 0.053879 0.055658 0.034971 0.059845 0.039946

0.064775 0.046141 0.0629 0.101919 0.031915 0.03108 0.011912 0.012658 0.047634

0.079699 0.098018 0.111727 0.180696 0.032565 0.028888 0.01283 0.01963 0.218392

0.120387 0.053201 0.084314 0.207787 0.031846 0.023024 0.006369 0.012683 0.311589



9,10-DiHOME15,16-DiHODE12,13-DiHODE9,10-DiHODE19,20-DiHDPE16,17-DiHDPE13,14-DiHDPE10,11-DiHDPE7,8-DiHDPE

0.176349 0.091326 0.043101 0.011931 0.640611 0.059075 0.008788 0.012117 0.011751

0.334853 0.218458 0.107922 0.062382 0.704312 0.130096 0.02624 0.021612 0.013643

0.202828 0.210427 0.056162 0.043432 0.528366 0.107808 0.022895 0.01545 0.013647

0.42587 0.24123 0.079569 0.06593 0.574224 0.085517 0.023219 0.007078 0.011893

0.164588 0.112834 0.049701 0.018985 0.590848 0.07207 0.014267 0.013744 0.009219

0.134358 0.099411 0.039348 0.018408 0.518187 0.073935 0.025467 0.026033 0.030779

0.64366 0.043979 0.140122 0.059886 1.478486 0.200424 0.034745 0.03104 0.07511

0.352286 0.064319 0.040246 0.055391 0.66695 0.140037 0.04639 0.02007 0.029883

1.947561 0.102318 0.204985 0.222567 2.96003 0.3506 0.081946 0.069232 0.147295

0.200016 0.039401 0.034811 0.052269 0.620168 0.06184 0.020449 0.018268 0.016787

0.064107 0.060872 0.04193 0.022371 0.360402 0.052802 0.013219 0.027591 0.022149

0.211005 0.059107 0.051712 0.028239 0.365433 0.041813 0.007245 0.009919 0.024697

0.163622 0.015277 0.038709 0.016563 0.337395 0.036007 0.011664 0.017103 0.016164

0.124041 0.030307 0.058596 0.021846 0.397673 0.074608 0.020674 0.014191 0.027706

0.201951 0.025296 0.044684 0.033654 0.515453 0.095253 0.042232 0.026571 0.016943

0.21013 0.032831 0.066216 0.024123 0.530208 0.053335 0.017223 0.01128 0.015336

0.301452 0.058656 0.033106 0.03509 0.533379 0.051818 0.024029 0.02635 0.02388

0.223922 0.040527 0.039201 0.036278 0.442819 0.049657 0.013149 0.019381 0.018399



4,5-DiHDPE17,18-DiHETE14,15-DiHETE11,12-DiHETE11,12-,15-TriHETrE9,12,13-TriHOME9,10,13-TriHOME5,15-DiHETEResolvin E1

0.159971 0.139166 0.009551 0.002954 0.070323 0.233561 0.451246 0.027029 2.400569

0.152152 0.19187 0.045334 0.015585 0.213228 0.535334 #VALUE! 0.021164 2.872838

0.12093 0.193139 0.026531 0.014882 0.256382 1.382336 0.261222 0.020862 1.455278

0.100924 0.179596 0.029363 0.013078 0.324716 1.29722 0.900576 0.023463 2.518766

0.179379 0.16433 0.015365 0.00766 0.163037 0.713734 0.099865 0.019555 2.370417

0.129011 0.120548 0.011539 0.009465 0.525087 1.665844 0.403568 0.013161 1.142784

0.549976 0.291188 0.054472 0.035656 0.781394 4.671805 11.61565 0.076897 74.26371

0.273097 0.176065 0.034852 0.012649 0.075892 0.16127 #VALUE! 0.011872 1.557754

1.658988 0.528423 0.069361 0.057379 1.092276 2.026949 1.914944 0.101326 20.73341

0.223264 0.112439 0.021794 0.010719 0.712472 1.727044 0.679307 0.026155 1.440972

0.189016 0.12334 0.02062 0.00823 0.455702 1.898317 0.905803 0.014498 1.832499

0.311859 0.098184 0.013421 0.004135 0.266897 1.1724 1.447146 0.022295 3.263252

0.199075 0.088202 0.011337 0.008337 0.584571 0.559811 0.789125 0.026089 2.690301

0.22517 0.0916 0.018109 0.008977 0.597867 0.785996 0.409221 0.031179 2.507613

0.318555 0.106999 0.022126 0.010737 0.684737 0.703331 0.515799 0.022352 1.556333

0.197042 0.121676 0.012225 0.004944 0.104209 1.558571 1.568063 0.017877 2.864938

0.376026 0.120857 0.011832 0.009783 0.194804 1.150577 1.342292 0.017404 2.98628

0.243658 0.134026 0.016757 0.008029 0.261296 1.415231 0.907535 0.021038 3.458414



LXA4

0.011358

0.024215

0.034912

0.01612

0.029532

0.032401

0.091628

0.007697

0.069406

0.029676

0.147651

0.034244

0.028164

0.021972

0.05251

0.03644

0.045767

0.038891



Original # Group 6-keto-PGF1aTXB2 PGE2 PGD2 PGF2a PGE1 PGD1

115 OVA+Vehicle 3663.073 376.0273 7863.451 564.8455 845.29 196.12 1308.33

116 OVA+Vehicle 3039.464 236.4339 4659.682 549.9922 525.07 103.83 673.14

117 OVA+Vehicle 2657.946 468.2563 7669.167 449.8852 681.60 170.06 1201.32

118 OVA+Vehicle 3671.499 461.7956 9177.225 979.0713 1033.54 228.45 1264.31

133 Low 1728 5897.612 705.8463 9783.181 741.215 1342.91 229.92 1536.85

134 Low 1728 4331.293 647.3931 8215.391 498.1015 1277.92 176.35 932.15

135 Low 1728 4812.217 403.6614 8130.968 613.7957 921.30 181.70 1086.05

136 Low 1728 4159.56 671.5802 9708.752 721.8982 1075.63 231.89 1382.13

139 Hi 1728 3963.685 347.6451 6075.597 469.4399 612.20 140.79 893.62

140 Hi 1728 3486.334 349.9203 5752.099 483.2927 478.98 138.54 997.28

141 Hi 1728 5365.061 613.5994 10500.13 745.0418 1042.81 200.49 1386.26

142 Hi 1728 5962.055 880.4157 12574.05 1147.543 1879.40 272.15 1639.63



PGE3 PGD3 PGJ2 PGB2 11-HETE 9-HETE EKODE LTB4 LTB5

238.84 11.56 14.86 9.09 1020.90 32.89 44.86 6.83 2.10

221.21 14.46 9.36 3.92 744.34 0.00 35.74 2.05 2.09

221.46 7.48 13.74 8.33 1320.04 163.85 59.27 3.05 5.28

299.87 13.26 16.02 8.99 1737.99 98.42 50.00 7.89 2.19

340.67 15.00 15.10 9.30 2849.31 200.06 44.92 9.33 3.91

496.77 18.09 18.73 11.76 2046.41 113.14 74.53 3.31 2.28

630.48 24.01 19.16 8.64 2100.74 153.53 70.45 2.46 2.60

387.82 0.56 15.96 11.63 2809.20 206.80 50.51 4.47 1.87

297.66 12.28 12.60 7.82 2537.36 118.87 56.81 1.12 2.32

254.22 8.63 12.13 8.27 2085.85 118.16 36.17 2.98 1.61

515.02 12.80 22.19 11.76 2852.78 137.53 87.42 9.61 4.16

615.77 10.21 33.60 18.33 5282.73 212.02 79.81 21.25 8.71



6-trans-LTB45-HETE 5-HEPE 15-HETE 15(S)-HETrE13-HOTrE 13-HODE 15-HEPE 17-HDoHE

33.37 84.79 6.44 2100.64 390.01 68.65 3559.39 260.84 2026.29

9.85 61.65 9.11 1852.28 276.96 114.86 2543.12 439.64 1207.59

73.96 50.83 4.23 2511.49 556.59 157.61 4711.99 338.41 2408.57

25.77 127.48 10.93 2422.06 324.09 44.37 2008.38 302.39 1261.70

52.07 81.47 5.36 5908.71 1090.68 197.31 6232.62 1145.34 5064.08

21.33 68.96 6.55 3772.53 544.94 143.75 3632.39 1099.99 3157.10

28.19 43.79 6.05 6269.63 812.54 213.13 4879.48 3559.33 5546.76

27.07 64.19 6.46 5727.57 700.34 111.18 3349.83 1144.56 5768.76

24.36 87.98 7.81 7285.98 915.43 98.05 3818.37 2029.40 7234.27

26.91 54.05 4.15 4992.70 753.41 106.70 3929.21 1099.88 5404.31

71.48 140.87 7.37 5737.73 797.16 121.24 3166.56 1611.62 6336.93

175.25 217.87 13.74 10869.59 1334.92 133.02 4014.07 2528.11 8828.08



12-HETE 8-HETE 9-HOTrE 9-HODE 12-HEPE 8-HEPE 5-oxo-ETE 12-oxo-ETE15-oxo-ETE

15818.56 342.67 4.46 2432.42 1186.43 7.58 16.23 3574.90 66.60

12258.27 210.13 3.33 1428.60 2075.46 13.90 30.03 2042.81 27.22

46555.84 535.76 5.23 2373.57 4553.51 7.88 69.90 13195.46 80.33

22712.36 331.88 3.31 1958.54 2149.10 7.37 48.45 5548.21 73.22

56969.27 734.02 5.89 3315.98 4229.02 6.78 38.83 9755.20 124.48

33380.23 353.87 4.32 2500.21 4017.44 6.31 20.76 6685.23 65.77

35555.42 463.36 4.81 2594.42 5591.80 17.05 14.90 5763.28 78.97

40773.99 487.25 3.21 2275.65 3463.70 8.71 37.38 6533.13 123.55

32405.77 708.58 3.02 1785.55 1994.75 14.00 12.51 5864.63 119.00

26797.13 514.63 2.91 2194.78 1662.00 6.01 15.45 6901.99 109.25

38831.14 476.13 2.85 1866.91 2837.79 10.71 40.53 10270.68 130.94

76010.82 1203.41 3.77 1889.34 5844.13 13.98 59.60 13420.11 203.62



13-oxo-ODE9-oxo-ODE 20-HETE 14(15)-EpETrE11(12)-EpETrE8(9)-EpETrE5(6)-EpETrE12(13)-EpOME9(10)-EpOME

7.18 72.66 25.31 11.09 20.97 21.05 29.87 25.55 20.83

3.38 20.13 10.42 9.46 15.86 9.31 18.80 17.25 12.70

14.20 46.19 36.59 12.67 21.37 51.72 28.52 21.35 21.77

5.96 37.40 21.05 17.41 33.07 24.69 54.78 20.76 22.32

19.02 43.67 0.00 10.60 17.73 57.99 29.08 31.18 18.73

9.00 21.26 7.46 12.15 23.37 28.19 35.46 27.69 18.30

6.78 29.62 6.87 11.65 18.90 24.67 27.61 20.30 16.77

16.53 40.73 17.94 10.67 22.17 38.09 18.52 26.52 15.66

10.04 59.92 12.51 15.00 20.18 23.07 18.85 34.12 23.76

6.48 51.94 3.65 9.64 20.65 16.80 15.76 27.59 17.57

11.41 54.79 1.39 16.78 35.21 20.59 51.43 29.91 19.93

13.74 42.99 10.13 10.44 35.73 118.63 41.17 22.28 17.17



15(16)-EpODE9(10)-EpODE19(20)-EpDPE16(17)-EpDPE13(14)-EpDPE10(11)-EpDPE7(8)-EpDPE 17(18)-EpETE14,15-DiHETrE

3.52 0.00 6.00 3.53 3.02 3.35 30.35 3.12 1.46

12.37 0.00 8.91 0.53 1.20 0.71 22.63 2.79 0.90

10.13 0.00 10.01 3.62 2.46 3.08 106.05 3.36 1.00

10.17 0.00 7.72 4.24 2.54 7.18 76.94 2.96 1.54

15.20 0.00 3.31 1.37 1.49 3.47 68.79 3.64 0.80

17.17 0.08 7.19 1.49 2.12 5.15 48.92 2.77 1.17

10.43 0.25 6.20 2.42 1.83 3.23 30.53 4.01 0.88

11.50 0.54 11.82 3.17 3.07 4.00 73.68 5.15 0.77

11.16 0.41 6.99 3.01 1.31 2.83 36.05 6.16 0.59

10.43 0.39 5.83 1.91 1.74 3.14 58.57 2.98 0.51

12.33 0.00 7.01 2.34 3.60 5.92 107.66 3.26 0.93

6.93 0.00 6.75 6.13 5.34 2.59 40.54 3.44 1.21



11,12-DiHETrE12,13-DiHOME9,10-DiHOME15,16-DiHODE12,13-DiHODE9,10-DiHODE19,20-DiHDPE16,17-DiHDPE13,14-DiHDPE

1.55 16.49 15.84 3.62 1.62 0.46 30.04 64.27 0.48

0.61 12.86 5.95 5.03 1.85 0.12 15.54 44.17 0.55

1.47 12.77 7.60 4.50 1.35 0.51 15.46 82.14 0.71

0.89 19.31 9.81 4.52 1.72 0.82 23.92 27.73 0.51

0.74 7.08 7.84 2.46 1.31 0.21 26.34 109.25 1.06

0.77 8.83 6.65 1.80 0.86 0.24 25.48 44.41 1.04

0.36 4.87 5.06 2.68 0.94 0.00 27.95 86.79 0.93

1.03 8.53 6.36 2.78 1.22 0.47 31.15 61.07 0.58

0.64 7.08 4.22 1.38 1.22 0.00 22.09 64.75 0.54

0.42 4.55 3.47 1.51 0.82 0.00 24.14 65.88 0.49

0.67 5.64 5.40 2.29 1.26 0.37 29.05 72.01 1.02

1.00 5.45 5.43 2.02 1.52 0.00 41.96 103.28 1.00



10,11-DiHDPE7,8-DiHDPE4,5-DiHDPE17,18-DiHETE14,15-DiHETE5,6-DiHETE 11,12-,15-TriHETrE9,12,13-TriHOME9,10,13-TriHOME

1.52 0.89 3.41 4.40 2.40 0.08 50.11 862.46 330.16

1.36 0.01 1.81 4.30 0.64 0.37 62.46 130.37 45.78

1.51 0.40 2.00 1.40 9.21 0.44 63.74 308.25 98.25

1.24 0.56 2.10 3.03 1.77 1.62 70.93 262.63 101.96

1.68 0.53 4.06 2.52 12.67 0.43 91.26 294.11 101.75

1.47 0.05 2.76 3.22 2.54 0.00 102.27 156.62 57.11

1.65 0.50 2.11 1.91 3.73 0.21 118.65 215.20 53.65

1.39 0.65 3.09 2.55 5.62 0.29 100.05 193.58 60.45

1.74 0.57 2.68 1.89 2.59 0.20 70.48 169.84 54.46

1.14 0.32 2.45 2.66 4.72 0.11 60.44 189.06 61.97

1.34 0.28 1.88 3.67 5.20 1.01 115.05 214.98 77.68

2.00 0.81 2.90 3.80 11.13 2.80 339.45 402.64 132.09



8,15-DiHETE5,15-DiHETELXA4

106.65 17.77 7.44

35.76 8.33 5.91

104.08 19.46 5.09

49.54 9.74 4.76

94.28 43.15 4.06

32.00 11.08 4.16

55.03 16.69 5.35

33.88 16.15 3.76

57.23 18.34 3.56

70.32 24.25 2.61

87.18 32.85 6.44

175.70 59.92 11.99



Table E2. The epoxides to diols ratios supported the engagement of sEHI in plasma, BALF and lung homegenates.

Plamsa

Origin # Group

14(15)EpETrE/1

4,15DiHETrE

11,12

EpETrE/11,12

DiHETrE

8,9EpETrE/8,9

DiHETrE

5,6 EpETrE/5,6

DiHETrE

115 Vehicle 0.58 0.93 0.88 6.23

116 Vehicle 0.20 0.86 0.48 8.69

117 Vehicle 0.35 1.02 1.30 8.09

118 Vehicle 0.30 0.76 1.10 10.69

119 Vehicle 0.58 0.86 1.33 4.90

120 Vehicle 0.79 1.98 0.43 6.35

133 Low 1728 1.33 0.94 1.50 25.34

134 Low 1728 0.50 0.56 0.60 13.05

135 Low 1728 1.51 1.77 1.82 29.82

136 Low 1728 0.88 1.11 2.41 17.02

137 Low 1728 1.71 2.23 3.55 7.10

138 Low 1728 3.05 3.12 1.97 8.57

139 Hi 1728 2.56 1.92 2.61 15.30

140 Hi 1728 1.30 1.27 2.85 7.66

141 Hi 1728 1.24 1.14 2.33 15.66

142 Hi 1728 1.71 1.56 1.32 12.92

143 Hi 1728 2.46 2.92 1.53 10.91

144 Hi 1728 2.19 2.54 2.35 16.73

BALF

Origin # Group

14(15)EpETrE/1

4,15DiHETrE

11,12

EpETrE/11,12

DiHETrE

8,9EpETrE/8,9

DiHETrE

5,6 EpETrE/5,6

DiHETrE

115 Vehicle 0.97 1.52 2.52 8.78

116 Vehicle 0.35 1.81 2.74 4.71

117 Vehicle 0.83 3.02 11.28 7.83

118 Vehicle 0.48 1.57 7.25 8.37

119 Vehicle 1.78 4.98 15.60 16.37

120 Vehicle 1.22 4.08 14.06 31.92

133 Low 1728 1.55 0.93 8.04 1.73

134 Low 1728 0.50 1.11 2.97 4.40

135 Low 1728 1.01 3.13 3.39 7.72

136 Low 1728 1.90 3.73 13.32 18.65

137 Low 1728 4.36 13.44 7.75 22.06

138 Low 1728 5.16 9.20 3.08 17.44

139 Hi 1728 2.60 5.27 1.67 11.65

140 Hi 1728 2.94 6.09 2.29 39.20

141 Hi 1728 1.55 2.36 1.63 2.60

142 Hi 1728 2.04 2.05 15.81 13.31



143 Hi 1728 5.22 7.83 3.32 14.56

144 Hi 1728 3.71 9.32 11.20 12.36

Lung Homegenates

Original # Group

14(15)EpETrE/1

4,15DiHETrE

11,12

EpETrE/11,12

DiHETrE

8,9EpETrE/8,9

DiHETrE

5,6 EpETrE/5,6

DiHETrE

115 OVA+Vehicle 7.62 13.57

116 OVA+Vehicle 10.48 26.07

117 OVA+Vehicle 12.63 14.51

118 OVA+Vehicle 11.31 37.02

133 Low 1728 13.18 23.82

134 Low 1728 10.40 30.23

135 Low 1728 13.16 52.03

136 Low 1728 13.87 21.46

139 Hi 1728 25.39 31.74

140 Hi 1728 19.05 49.28

141 Hi 1728 18.06 52.38

142 Hi 1728 8.64 35.79



Table E2. The epoxides to diols ratios supported the engagement of sEHI in plasma, BALF and lung homegenates.

12,13

EpOME/12,13

DiHOME

9,10EpOME/9,1

0 DiHOME

15,16

EpODE/15,16Di

HODE

12,13

EpODE/12,13

DiHODE

9,10

EpODE/9,10

DiHODE

19,20

EpDPE/19,20

DiHDPE

0.48 1.16 0.78 0.05 1.20 0.28

0.40 0.50 1.38 0.11 0.35 0.22

0.45 0.44 1.30 0.14 0.24 0.25

0.38 0.50 1.66 0.14 0.37 0.34

0.55 0.90 0.87 0.07 0.85 0.33

0.27 0.50 0.60 0.03 0.73 0.58

5.21 2.06 14.76 1.92 1.48 0.77

2.08 1.16 8.13 1.03 0.78 0.50

3.28 2.07 11.60 0.96 1.47 0.74

2.45 1.51 11.69 0.96 1.20 0.65

1.59 1.38 2.23 0.28 0.78 0.53

2.82 1.42 5.10 0.67 1.15 1.12

4.89 1.85 24.58 1.12 0.75 0.93

5.57 2.16 11.17 1.50 1.43 0.74

4.75 1.59 11.08 2.06 1.07 0.69

5.04 1.85 9.96 1.17 1.53 0.79

2.24 1.19 8.91 0.22 0.73 1.44

1.88 1.12 7.86 0.78 0.70 1.33

12,13

EpOME/12,13

DiHOME

9,10EpOME/9,1

0 DiHOME

15,16

EpODE/15,16Di

HODE

12,13

EpODE/12,13

DiHODE

9,10

EpODE/9,10

DiHODE

19,20

EpDPE/19,20

DiHDPE

0.70 0.57 0.61 0.17 0.60 0.62

0.47 0.34 1.20 0.06 #VALUE! 0.37

0.72 0.67 1.18 0.25 0.25 0.65

0.47 0.36 1.60 0.14 0.18 0.61

1.56 0.93 0.75 0.05 0.35 0.84

0.72 1.59 1.11 0.12 0.25 0.93

3.18 1.06 37.37 1.63 4.03 1.44

2.00 0.44 10.59 1.27 0.16 0.83

1.88 0.49 9.94 0.46 0.39 1.14

11.88 1.29 13.88 0.83 0.45 1.09

9.67 4.36 2.44 0.17 0.47 1.97

7.32 1.36 2.43 0.38 0.35 1.60

#VALUE! 1.11 6.48 0.23 0.90 0.85

#VALUE! 1.84 6.98 0.28 0.80 0.89

20.07 0.92 14.70 0.76 0.36 0.80

8.06 0.77 4.61 0.18 0.56 0.70



4.13 0.88 7.13 0.75 0.78 2.15

2.53 1.17 10.34 0.69 0.92 2.15

12,13

EpOME/12,13

DiHOME

9,10EpOME/9,1

0 DiHOME

15,16

EpODE/15,16Di

HODE

12,13

EpODE/12,13

DiHODE

9,10

EpODE/9,10

DiHODE

19,20

EpDPE/19,20

DiHDPE

1.55 1.32 0.97 0.20

1.34 2.13 2.46 0.57

1.67 2.86 2.25 0.65

1.08 2.28 2.25 0.32

4.40 2.39 6.17 0.13

3.13 2.75 9.55 0.28

4.17 3.31 3.88 0.22

3.11 2.46 4.14 0.38

4.82 5.63 8.06 0.32

6.06 5.06 6.93 0.24

5.30 3.69 5.39 0.24

4.09 3.16 3.42 0.16



16,17

EpDPE/16,17

DiHDPE

13,14

EpDPE/13,14

DiHDPE

10,11

EpDPE/10,11

DiHDPE

7,8 EpDPE/7,8

DiHDPE

17,18

EpETE/17,18Di

HETE

0.24 0.60 1.47 0.16

0.06 0.26 0.69 0.28

0.08 0.25 0.96 0.33

0.10 0.30 1.04 0.33

0.26 0.70 1.86 0.20

0.44 0.80 1.24 0.22

0.40 0.45 1.94 1.00

0.18 0.19 1.03 0.57

0.67 1.22 3.43 0.99

0.34 0.88 2.19 1.06

0.39 0.60 1.54 0.71

0.76 1.66 2.24 1.41

0.71 0.83 2.64 1.34

0.42 0.52 2.43 0.75

0.35 0.50 1.98 0.89

0.48 0.90 3.36 1.03

0.87 1.32 3.16 1.43

0.66 0.70 1.87 1.12

16,17

EpDPE/16,17

DiHDPE

13,14

EpDPE/13,14

DiHDPE

10,11

EpDPE/10,11

DiHDPE

7,8 EpDPE/7,8

DiHDPE

17,18

EpETE/17,18Di

HETE

0.36 2.82 2.98 0.17

0.20 1.31 1.90 0.35

0.27 0.90 1.76 0.26

0.17 0.69 4.35 0.29

0.78 2.51 5.07 0.34

0.69 1.82 4.12 0.80

0.83 0.79 5.33 1.13

0.18 0.47 2.08 0.44

0.64 0.73 3.78 1.01

0.61 2.26 4.72 0.79

1.24 6.69 4.54 1.14

1.10 14.16 14.30 2.42

0.93 1.22 5.52 1.59

0.81 3.01 5.16 1.23

0.38 0.15 2.41 0.84

1.21 2.68 5.58 0.84



1.54 4.08 4.24 1.50

2.42 4.05 4.35 1.55

16,17

EpDPE/16,17

DiHDPE

13,14

EpDPE/13,14

DiHDPE

10,11

EpDPE/10,11

DiHDPE

7,8 EpDPE/7,8

DiHDPE

17,18

EpETE/17,18Di

HETE

0.05 6.24 2.21 34.01 0.71

0.01 2.18 0.52 1901.52 0.65

0.04 3.45 2.04 268.38 2.41

0.15 5.01 5.81 137.43 0.98

0.01 1.41 2.06 130.34 1.45

0.03 2.03 3.51 951.10 0.86

0.03 1.97 1.95 60.67 2.09

0.05 5.33 2.86 112.99 2.02

0.05 2.41 1.62 63.75 3.25

0.03 3.53 2.75 184.29 1.12

0.03 3.53 4.41 387.72 0.89

0.06 5.34 1.30 50.14 0.91



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