Broad Spectrum Proteomics Analysis of the Inferior ...S exposure, respectively. Mass spectrometry data were analyzed by Perseus 29 1.5.5.3 statistical analysis, and gene ontology heat

Cleared, 88PA, Case # 2017-5041, 18 Oct 2017.

1

Running Title: Broad Spectrum Proteomics Analysis of the Inferior Colliculus following Acute 1

Hydrogen Sulfide Exposure 2

Broad Spectrum Proteomics Analysis of the Inferior 3

Colliculus following Acute Hydrogen Sulfide Exposure 4

Dong-Suk Kim1, Poojya Anantharam

1a, Andrea Hoffmann

2, Mitchell L. Meade

3, Nadja Grobe

3, 5

Jeffery M. Gearhart2, Elizabeth M. Whitley

4, Belinda Mahama

1b, Wilson K. Rumbeiha

1* 6

1Veterinary Diagnostic & Production Animal Medicine, Iowa State University, Ames, IA, US 7

2Henry M Jackson Foundation on contract 711HPW/USAFSAM/FHOF, Wright Patterson Air 8

Force Base, Dayton, OH, US 9

3711HPW/RHDJ, Wright Patterson Air Force Base, Dayton, OH, US 10

4Pathogenesis, LLC, Gainesville, FL, US 11

*Corresponding Author’s Contact Information:[email protected], 515-294-0630 12

Key Words: Hydrogen Sulfide, Brain Injury, Proteomic Profiling, Proteomic Analysis, TMT 13

labeled LC-MS/MS, Neurodegeneration 14

Present addresses: 15

aPoojya Anantharam: Medical Countermeasures, MRI Global, Kansas City, MO, US 16

bBelinda Mahama: Neuroscience, Brown University, Rhode Island, Providence, US 17

.CC-BY-NC-ND 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/237370doi: bioRxiv preprint

https://doi.org/10.1101/237370

http://creativecommons.org/licenses/by-nc-nd/4.0/


2

Abstract: Acute exposure to high concentrations of H2S causes severe brain injury and long-term 18

neurological disorders. The mechanisms of H2S-induced neurodegeneration are not known. To 19

better understand the cellular and molecular mechanisms of H2S-induced neurodegeneration we 20

used a broad-spectrum proteomic analysis approach to search for key molecules in H2S-induced 21

neurotoxicity. Mice were subjected to acute whole body exposure of up to750 ppm of H2S. The 22

H2S-treated group showed behavioral motor deficits and developed severe lesions in the inferior 23

colliculus (IC), part of the brainstem. The IC was microdissected for proteomic analysis. Tandem 24

mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative 25

proteomics was applied for protein identification and quantitation. LC-MS/MS was able to 26

identify 598, 562, and 546 altered proteomic changes for day 1 (2 h post H2S exposure), day 2, 27

and day 4 of H2S exposure, respectively. Mass spectrometry data were analyzed by Perseus 28

1.5.5.3 statistical analysis, and gene ontology heat map clustering. Quantitative real-time PCR 29

was used to confirm some of the H2S-dependent proteomics changes. Taken together, acute 30

exposure to H2S induced behavioral motor deficits along with progressive neurodegeneration 31

including disruption of several biological processes in the IC such as cellular morphology, 32

energy metabolism, and calcium signaling. The obtained broad-spectrum proteomics data may 33

provide important clues to elucidate mechanisms of H2S-induced neurotoxicity. 34

35


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Highlights: 36

Mice exposed to H2S recapitulated H2S-induced neurotoxicity manifested in humans. 37

The IC in the mouse brain is the most sensitive to H2S-induced neurodegeneration. 38

Proteomic expressions of key proteins were validated at transcription level. 39

Several biological pathways were dysregulated by H2S exposure. 40

41

42


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1. Introduction 43

Hydrogen sulfide (H2S) is a highly neurotoxic colorless gas with a “rotten egg” odor (Chou et 44

al., 2016). It is as an environmental pollutant that causes occupational hazards in a variety of 45

industrial processes including the oil and gas industry, intensive animal farming operations, 46

sewer and waste water treatment plants, pulp and paper plants, and gas storage facilities, among 47

several others (Chou et al., 2016). It has been estimated that there are more than 1,000 reports of 48

human exposures to H2S each year in the United States (Chou et al., 2016). Besides accidental 49

H2S poisoning in industrial settings, intentional acute exposure to high concentrations of H2S as 50

a means of suicide has been increasingly observed in Western and Asian societies (Morii et al., 51

2010; Reedy et al., 2011). The raw chemical ingredients used to generate H2S in such 52

circumstances are readily accessible in local stores (Morii et al., 2010). More significantly, H2S 53

is listed as a high priority chemical by the US Department of Homeland Security because of its 54

potential to be misused in nefarious acts such as chemical terrorism, particularly in confined 55

spaces such as the underground transit systems. Although H2S is toxic at high concentrations, it 56

is also beneficial at physiologic concentrations. For this reason, there is also tremendous interest 57

in potential therapeutic applications of H2S for treatment of a number of human disease 58

conditions. Proposed pharmacological uses of H2S include ulcer treatment, ischemic reperfusion 59

injury, as an anti-inflammatory for treatment of Crohn’s disease, endotoxin induced 60

inflammation, arterial hypertrophy, visceral pain, Parkinson’s disease and cancer among others 61

(Szabo, 2007; Popov, 2013). Furthermore, H2S was shown to activate nitric oxide (NO) synthesis 62

through induction of endothelial nitric oxide synthase. Similar to NO, H2S serves as a 63

gasotransmitter and signaling molecule in the CNS and regulating vasodilation (Tan et al., 2010). 64


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Like most toxicants, the toxicity of H2S exposure is dose-dependent, but H2S characteristically 65

has a steep-dose response curve (Guidotti, 1996). At concentrations below 30 parts per million 66

(ppm), H2S causes headache, coughing and throat irritation. At 150 ppm H2S induces olfactory 67

fatigue and temporary loss of smell after 5-60 min of exposure (Wasch et al., 1989). 68

Concentrations higher than 500 ppm cause headache, dizziness, unconsciousness, and respiratory 69

failure. At high concentration above 1,000 ppm H2S can lead to immediate loss of consciousness, 70

commonly called “knock-down”, and ultimately seizures, and sudden death (Chou et al., 2016). 71

Typically, acute exposure to high concentrations of H2S is associated with high mortality withing 72

a few hours post-exposure (McCabe and Clayton, 1952). Timely rescue of victims can prevent 73

disaster and allow victims to fully recover. Currently, there is no effective antidote to treat 74

victims of H2S gas exposure and the mortality rate remains high (Lindenmann et al.; Herbert, 75

1989; Vicas, 1989; Smith, 1997; Lindenmann et al., 2010). In addition, delayed neurological 76

disorders, which can last for many years, are commonly reported in survivors of acute H2S 77

exposures (Matsuo et al., 1979; Parra et al., 1991; Tvedt et al., 1991a; Tvedt et al., 1991b; 78

Snyder et al., 1995; Kilburn, 2003; Woodall et al., 2005). These delayed neurological sequelae 79

are incapacitating, and require prolonged medical attention that lacks defined medical 80

interventions. 81

The development of effective therapeutics requires a good understanding of the molecular 82

mechanisms and pathways of H2S-induced neurodegeneration and neurological sequelae. These 83

mechanisms remain largely unknown. There is an acute need for countermeasures for treatment 84

of mass civilian casualties of acute H2S poisoning in the field, such as following catastrophic 85

industrial meltdowns or intentional terrorist activities. Elucidating molecular mechanisms 86


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underlying H2S-induced neurotoxicity is essential in developing suitable targeted therapeutics to 87

counter both acute and delayed neurotoxic effects of H2S poisoning in humans. 88

We recently developed a relevant inhalational animal model that exhibits the clinical, 89

pathological, and motor behavioral symptoms of H2S exposed survivors (Anantharam et al., 90

2017). The objective of this study was to build on our previous work and investigate proteomic 91

changes and altered gene expression in a mouse model of acute H2S-exposure to identify novel 92

toxic mechanisms. In prior studies, we discovered that the central inferior colliculus (IC) region 93

of the brainstem is the most sensitive brain region to H2S-induced neurodegeneration 94

(Anantharam et al., 2017). Consequently, we focused on the IC in this proteomic study, although 95

we have also observed histopathological changes in other parts of the brain such as the thalamus 96

and cortex (Anantharam et al., 2017). This study demonstrated, for the first time, that H2S 97

exposure induces significant proteomic changes in the IC, which may play an important role in 98

execution of H2S-induced neurotoxicity. 99

2. Materials and Methods 100

2.1 Chemicals 101

Hydrogen sulfide gas was purchased from Airgas (Radnor, PA). RNeasy mini kit was 102

purchased from Qiagen (Germantown, MD). High Capacity cDNA RT kit were purchased from 103

ThermoFisher Scientific (Waltham, MA). RT² SYBR Green ROX qPCR Mastermix and primers 104

for Gapdh were purchased from Qiagen (Valencia, CA). 105

2.2 Animals and treatment 106

This study was approved by Iowa State University Animal Care and Use Committee. Seven- to 107

eight-week-old male C57 BL/6J mice were housed at room temperature of 20 - 22 °C under a 12-108

h light cycle, and a relative humidity of 35 – 50 %. Protein Rodent maintenance diet (Teklad 109


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HSD Inc, WI, US) and water were provided ad libitum. Prior to exposure on day 1, all mice were 110

acclimated to breathing air for 40 min on each of two days preceding day 1 when mice were first 111

exposed to H2S. Mice were exposed by whole body inhalation exposure either to normal 112

breathing air from a tank under pressure (Control) or to 650 - 750ppm H2S (H2S-treated) once or 113

for short-term daily exposures lasting for up to 6 days. The exposure paradigm is summarized in 114

Fig 1. On day 1, mice were exposed to H2S of 650 - 750 ppm for 40 min only. Those mice 115

terminated on day 1 were euthanized 2 h post H2S-eposure. The remaining groups of H2S-116

exposed mice were exposed daily to 765 ppm H2S for 15 min. only. In this regard, mice 117

euthanized on day 1 received only 1 acute exposure, those euthanized on day 2 received 2 acute 118

exposures of H2S and those euthanized on day 4 received 4 acute exposures to H2S. Negative 119

controls were exposed to normal breathing air daily and euthanized on day 4. Mice were 120

euthanized by decapitation 2 h after the last exposure to H2S on days 1, 2 and 4 for proteomics 121

analysis and quantitative gene expression analysis by real-time PCR and on days 1, 3 and 7 for 122

histopathology evaluation. Following decapitation, brains were immediately removed from the 123

skull. The IC were microdissected on ice and immediately flash frozen using liquid nitrogen, and 124

stored at -80°C until further use. Animals were cared for in accordance with the Institutional 125

Animal Care and Use committee guidelines. 126

2.3 Behavioral assessment 127

The VersaMax open field test was used to assess motor deficits induced by H2S. We used this 128

test because previous studies in the lab had indicated it to be sensitive to acute H2S intoxication 129

in this mouse model (Anantharam et al., 2017). Spontaneous activity was measured using an 130

automated computer device (Model RXYZCM-16; Accuscan, Columbus, OH, USA). The 131

activity chamber’s dimensions are 40 x 40 x 30.5 cm, and it is made of Plexiglas with a Plexiglas 132


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lid. The lid has holes for ventilation. Data was analyzed using VersaMax Analyzer (Model CDA-133

8; Accuscan). Mice were placed in the activity chamber 2 min prior to recording for 10 min, for 134

acclimation. Vertical activity, horizontal activity, and distance travelled were measured. 135

2.4 Histopathology 136

Mice were euthanized 24 h after the last H2S exposure. They were deeply anesthetized with a 137

cocktail of 100 mg/kg bw ketamine and 10 mg/kg bw xylazine. The thoracic cavity was opened 138

to expose the heart and fresh 4 % paraformaldehyde (PFA, pH 7.4) was injected through the left 139

ventricle. After perfusion, the calvarium was opened and brains were post-fixed in 4 % PFA for 140

24-48 h before removal from the skull. Brains were processed in paraffin, sectioned at 5 microns, 141

and stained with hematoxylin and eosin, and examined microscopically. Neurons were stained 142

with NeuN antibody (ab177487, Abcam, Cambridge, MA) using an indirect immunostaining 143

protocol. Diaminobenzidine was used as chromogen. Stained sections were examined using a 144

Nikon Eclipse Ci-L microscope equipped with a DS-Fi2 camera. For image analysis for the 145

quantification of neurons, NeuN-positive cells (sites of DAB chromogen deposition) were 146

enumerated in each of five 400X photomicrographs of the IC from mice exposed to H2S or 147

breathing air, and the mean number of NeuN-positive cells/mouse were compared between 148

groups. 149

2.5 Mass Spectrometry Analysis 150

Samples were processed according to a previously published method with some modifications 151

(Meade et al., 2015). Briefly, IC tissues were placed in 100µl of urea lysis buffer and 152

homogenized with a handheld pestle homogenizer. Protein samples were further reduced and 153

alkylated. Small aliquots of each sample were taken to measure protein concentration using the 154

Bradford assay from Bio-Rad (Hercules, CA). The remaining samples were diluted and trypsin-155


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digested overnight, followed by desalting using a C18 peptide trap from Michrom (Auburn, CA). 156

The desalted samples were vacufuged prior to individual sample labeling using TMT-6plex 157

labels according to the manufacturers’ instructions (Thermo FisherScientific, Waltham, MA). 158

Labeled samples of each exposure group were combined with the control for sample comparison 159

(Table 1). Peptides were separated on a Waters BEH C18 capillary column prior to online 160

analysis using a 240-min linear increasing gradient of acetonitrile with 0.1% formic acid. 161

Following elution from the column, ions were generated using 2.6 kV on a taper tip in a New 162

Objective nanosource and entered into an LTQ-OrbitrapVelos mass spectrometer (Thermo 163

Fisher, San Jose, CA). A full scan was taken in the LTQ, followed by data-dependent MS/MS 164

analysis of the top 6peaks. MS/MS analysis included collision-induced dissociation (CID) in the 165

LTQ for structural information and higher-energy collisional dissociation (HCD) in the Orbitrap 166

for quantitation. MS/MS data were aligned and quantitated using MaxQuant1.5.4.1 (Cox and 167

Mann, 2008) Analytics Platform with PTXQC (Bielow et al., 2016) quality control data 168

management. Peptide alignment was executed with the mouse Uniprot protein database 169

UP000000589_10090.fasta; enzyme: trypsin; carboxymethyl (C), and oxidation (M), FDR <1% 170

based on peptide q-value under standard settings (see supplemental data). The secondary 171

computational analysis was executed using Perseus 1.5.5.3 (Tyanova et al., 2016) for statistical 172

rendering, and web-based software Morpheus for heatmap rendering. Prior to analysis of 173

experimental samples, small aliquots of individually labeled control samples were analyzed to 174

determine individual variations in controls. Due to low variability in the controls, a pooled 175

control setting was used. Sample analysis included modification of the MaxQuant protein output 176

list by normalization of individual TMT reporter ion intensities by division through the median 177

intensity followed by determination of fold TMT expression ratios by division of individual 178


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experimental TMT values (126,127,128,129,and 130) through the TMT value (131) of the 179

pooled control. 180

Group

Total Mice/

Animal ID

TMT

Label

Treatment

Exposure/

Dose

Euthanasia

GA

Control

(n=5)

1,2,3,4,5,

pooled

131

40 min day 1

15 min day 2

15 min day 3

15 min day 4

+ 2 h recovery

Inhalation

/

air

Day 4

GB

1

exposure

(n=5)

1

2

3

4

5

126

127

128

129

130

40 min day 1

+ 2 h recovery

Inhalation

/

650 - 700

ppm H2S

Day 1

GC

2

exposures

(n=5)

1

2

3

4

5

126

127

128

129

130

40 min day 1

15 min day 2

+ 2 h recovery

Inhalation

/

650 – 700

ppm H2S

Day 2

GD

4

(n=4)

1

126

40 min day 1

15 min day 2

Inhalation

/

Day 4


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exposures 2

3

5

127

128

130

15 min day 3

15 min day 4

+ 2 h recovery

650 – 700

ppm H2S

Table 1. Animal exposure paradigm for mass spectrometry and distribution of TMT sample 181

labeling. GA= group A; GB= group B; GC=group C; GD= group D. (two column table) 182

2.6 Perseus Statistical Analysis 183

The previously modified Maxquant protein list was entered into the Perseus 1.5.5.3 184

software program followed by annotating Maxquant defined Swissprot protein accession 185

numbers with mouse gene identifiers. Statistical analysis included one-sample t-tests of fold 186

expression values by considering the deviation of samples from 1 fold expression (no change in 187

TMT reporter ion intensities vs. control intensity values) using p<0.05 as a criterion for 188

significance. Scatter Plots were established by plotting one-sample t-test difference fold protein 189

expression vs. –log one-sample t-test p value fold protein expression according to the associated 190

Perseus tool set. 191

2.7 Morpheus Heatmap Rendering 192

The Perseus processed protein list containing mouse gene identifiers, protein names and gene 193

ontology (GO) biological process, one-sample t-test, levels of significantly and non-significantly 194

modulated proteins was entered into the web-based Morpheus heatmap-rendering tool 195

(https://software.broadinstitute.org/morpheus/). A gradient coloring scheme was applied to 196

display upregulated (above 1.2 fold expression (in log2 display above 0.263), red color) vs. 197

downregulated (below 0.83 fold expression (in log2 display below -0.263) blue color) protein 198

nodes. White color nodes represent changes of non-modulated nodes to control levels (between 199


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0.83 and 1.2 fold expression). The heatmap was hierarchically clustered by Euclidean distance 200

using row average linkage and grouping of rows by GO biological process. 201

2.8 Validation of gene expression via quantitative real-time RT-PCR 202

After mice were exposed to H2S, tissues were dissected and immediately stored at -80 °C till 203

analysis. Total RNA was extracted from frozen tissues using the RNeasy® Plus Mini kit with 204

treatment of DNase I according to the manufacturer’s protocol. Validated primers for Gapdh 205

(Qiagen, #PPR57734E) were used as the housekeeping gene controls. The threshold cycle (Ct) 206

was calculated from the instrument software, and fold change in gene expression was calculated 207

using the Ct method as described earlier (Kim et al., 2016). The following primers were used 208

to check the quantitative transcriptional level of Prkab1, Vim, and Ahsa1; 5’-209

TCCGATGTGTCTGAGCTGTC-3’ and 5’-CCCGTGTCCTTGTTCAAGAT-3’ for Prkab1 210

(Bandow et al., 2015), 5’-TCCACACGCACCTACAGTCT-3’ and 5’-211

CCGAGGACCGGGTCACATA-3’ for Vim (Ulmasov et al., 2013), 5’-212

CAGAGGGGCACTTTGCCACCA-3’ and 5’-CACGGCCTTCCATGCACAGCT-3' for Ahsa1. 213

2.9 Statistical analysis 214

Data were analyzed using Prism 4.0 (GraphPad Software, San Diego, CA). Non-paired 215

Student’s t-test was used when two groups were being compared. Differences were considered 216

statistically significant for p-values <0.05. Data are represented as the mean ± S.E.M. of at least 217

two separate experiments performed at least in triplicate. 218

3. Results 219

3.1 Acute exposure to H2S induces motor behavioral deficits and seizures in C57 black mice. 220

Locomotor activities of mice were assessed on days 2, 4, and 6. Results indicated that the 221

horizontal and vertical activities were decreased by more than 50% and were statistically 222


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different in mice exposed to acute H2S compared to the breathing air group (Fig. 2 A and B). 223

Total distance traveled was also decreased by more than 50% and was also statistically different 224

in H2S-exposed mice compared to controls (Fig. 2 C). More importantly, mice exposed to H2S 225

exhibited severe seizure activity. On day 1, on average, seizure activity was observed starting at 226

15 min of H2S exposure. More than 50% of mice were shown to have seizure activity at 40 min 227

of exposure to H2S. Mice exposed to H2S more than once exhibited seizure activity after only 5 228

min of H2S exposure. In these groups of mice, at 10 min of exposure to H2S, 40% of mice 229

exhibited seizure activity on day 6. Collectively, these results indicate increased susceptibility of 230

mice to H2S after each successive H2S exposures, suggesting that the toxic effects of H2S 231

exposure are cumulative (Fig. 2D). 232

233

Figure 1. Acute exposure paradigm of hydrogen sulfide on C57 black mice. 234


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Mice were exposed to 765 ppm of hydrogen sulfide in a chamber for 40 min either once only 235

(day 1) and for 15 min on the following days up to day 7. Mice were sacrificed 2 h post-H2S 236

exposure on specified days of the study. Negative control mice were exposed to breathing air 237

from a cylinder daily up to day 7. Separate groups of mice were sacrificed on days 1, 3, and 7 for 238

immunohistochemistry. Groups of mice for proteomics studies were sacrificed on days 1, 2 and 4. 239

(One column figure) 240

241

Figure 2.Acute exposure to hydrogen sulfide induced motor behavioral deficits. 242

C57 black mice were exposed to H2S as shown in Fig. 1. Locomotor activity was measured using 243

an automated VersaMax locomotor activity monitor during 10 min time test on days 2, 4, and 6. 244

Horizontal activity (A), vertical activity (B), and total distance traveled (C) were analyzed 245


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between groups. Seizure activity was analyzed on a time scale (D). Asterisks (*, p < 0.05; **, p < 246

0.01) indicate statistically significant differences between H2S and breathing air negative control 247

groups. (One and half column figure) 248

249

3.2 Acute hydrogen sulfide exposure induces brain damage 250

Motor behavioral deficits induced by exposure to H2S may be a sign of injury to the central 251

nervous system (CNS). Therefore, histopathology was performed to identify the effects of H2S 252

exposure in the IC. Mice were exposed to H2S as designated in Fig. 1 and sacrificed at multiple 253

time points (day1, 3, or 7) for this portion of study. Microscopic examination of PFA-perfused 254

brains revealed H2S-induced neurodegeneration and loss of neurons in the IC (Figure 3A). By 255

day 7, mice exposed to H2S exhibited severe damage to the IC, with necrosis, vacuolar change, 256

and infiltration by neuroglial cells. In order to further characterize loss of neurons, IC tissues 257

were analyzed by immunohistochemical staining for the neuron specific marker, NeuN. On day 7, 258

there was a marked loss of neurons in the IC of H2S-exposed mice, compared with the breathing 259

air group. Enumeration of neurons in the IC revealed approximately 70 % loss of IC neurons by 260

day 7 of H2S exposure (Fig. 3 B and C) with infiltration by glial cells. In H2S exposed mice, 261

neurons in the midbrain adjacent to the IC appeared unaffected morphologically and glial cell 262

numbers and activation state were not altered. These results demonstrate selective loss of 263

neurons of the IC. 264


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265

Figure 3. Neurodegeneration and necrosis in the IC of mice exposed to 650-750 ppm H2S in 266

acute short-term repeated inhalation exposures over 7 days. Note the loss of neurons and 267

development of clear vacuoles in the neuropil (arrow) on day 7 in mice exposed to H2S and the 268

minimal morphologic effects to earlier H2S exposures or breathing air (A). Inhalation of H2S 269

resulted in marked and selective loss of neurons in the IC (1000X magnification images, B), with 270

retention of neurons in the regions surrounding the IC (200X magnification image). Computer 271

aided image analysis of NeuN immunostained sections reveals marked loss of neurons in the IC 272

of mice exposed to H2S (p=0.001, Students t test). Representative photomicrographs of mice 273

exposed to breathing air or H2S, hematoxylin and eosin (A) and NeuN immunohistochemistry 274

(B), 400X magnification (A) and 1000X and 200X magnification (B). Neurons in the IC region 275


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of the H2S exposed mice on day 7 were enumerated and compared to breathing air control (C). 276

(Two column figure) 277

3.3 Changes in the Broad Spectrum Proteome following acute H2S Exposure 278

Proteomic profiles of the IC were determined using TMT peptide labeling coupled with 279

LC/MS/MS analysis by comparison of the breathing air negative control group and the H2S 280

exposed group is described in the methods section. Mass spec was able to identify 598, 562, and 281

546 altered proteins for days1, 2, and 4 of H2S exposure, respectively. Subsequent analyses 282

showed alterations of protein expressions (Fig. 4, heatmap) with 36, 12 and 13 proteins being 283

significantly downregulated (below 0.83 fold) and 8, 6, and 12 proteins being significantly 284

upregulated (above 1.2 fold) compared to the control for days 1, 2 and 4 of H2S exposure, 285

respectively. A single (day 1) H2S exposure, in which mice were euthanized only 2 h post a 286

single exposure, demonstrated the highest range of fold protein expression changes compared to 287

the control with the majority of proteins being in the downregulation cluster. This was followed 288

by day 2 exposures with equal distribution of upregulated and downregulated proteins, while day 289

4 manifested the lowest range of fold changes with the least changes in the proteomic profile 290

(Fig. 5 A, B, C, D, scatter plots). 291


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292

Figure 4.Heatmap of IC changes in proteomic profile following H2S exposure 293


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Morpheus rendered Heatmap of determined changes in protein expression on days 1, 2 and 4. 294

H2S exposure using hierarchical clustering by Euclidean distance, row average linkage and 295

grouping of rows by gene ontology for biological process (GOBP). Heatmap displays fold 296

expression values of five (day 1 and day 2) or four (day 4) IC tissue samples, and average fold 297

expression values, mouse gene identifiers (gene names), protein names, and GOBP. “+” 298

indicates t-test (of fold expression values) significantly modulated proteins; grey not identified, 299

white non regulated, red upregulated (above 1.2 fold expression vs. control), blue downregulated 300

(below 0.83 fold expression vs. control) converted into a log2 data display. (Two column figure) 301

Gene Ontology Term Day1 Day2 Day4

actin cytoskeleton organization 3 4 2

adhesion 1 0 1

anion transport 1 0 0

axon guidance 3 0 1

blood 1 2 1

cell cycle 1 0 1

cellular transport 1 0 1

DNA metabolism 1 2 0

energy metabolism 6 3 5

glucose metabolism 3 3 1

immune response 1 1 1

microtubule organization 1 1 2

mitochondrial transport 1 0 0

oxidoreductase 1 0 0

protein degradation 1 0 0


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protein folding 3 1 0

RNA metabolism 7 2 3

signaling 6 2 2

vesicle transport 3 3 2

fatty acid steroid metabolism 0 1 1

mitochondrial proteolipid 0 1 1

synaptic transmission 0 1 1

tight junction component 0 1 0

carbohydrate transport 0 0 1

lipid metabolism 0 0 1

phosphatidylethanolamine biosynthesis 0 0 1

translation 0 0 1

lysosomal proton pump 0 1 1

302

Table 2. Functional annotation of significantly modulated protein expression in the IC following 303

H2S exposure on days 1, 2, and 4. Table displays total number of proteins associated with GO-304

term definition. A gradient scheme was applied to better indicate number distribution with lower 305

numbers in light orange and higher numbers in dark orange. Note that day 1 represents mice 306

exposed once and euthanized 2 h post-exposure. (One and half column table) 307

We further analyzed H2S-dependent alterations in the proteome using Perseus annotation for 308

gene ontology (GO) biological pathways that were summarized into general categories when 309

applicable by integrating gene cards information (www.genecards.org). We found that several 310

gene ontology functions were affected after H2S exposure (Table 2). Acute exposure to H2S on 311

day 1 resulted in alteration of RNA, energy and glucose metabolism, and changes in signaling 312


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pathways, axon guidance, actin cytoskeleton reorganization, and vesicle transport. On day 2 313

changes in gene ontology included glucose and energy metabolism, actin cytoskeleton 314

organization and vesicle transport. Changes in energy and RNA metabolism were most abundant 315

on day 4. Interestingly, there was an upregulation of cytoplasmic interacting protein Cyfip2 and 316

nuclear hormone receptor and transcriptional repressor family Nr1d2 that were observed at all 317

three time points. There was also a downregulation of anti-apoptotic and immunogenic protein 318

Bcl2 associated athanogene Bag5 on day 1, suggesting potential induction of apoptosis. This is 319

further supported by an observed downregulation of core histones H3f3a, and Hist1h3a that 320

might result in a DNA damage response. Evidence of potential immunogenic changes was given 321

by an upregulation in complement binding protein C1qbp on day 3 and a downregulation in anti-322

inflammatory protein macrophage migration inhibition factor Mif on day 1. These latter changes 323

suggest inflammation is involved in H2S-induced neurotoxicity. 324

325


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Figure 5.Overall distribution of IC changes in proteomic profile following H2S exposure 326

A)Scatter Plot to display overall distribution of one-sample t-test differences in protein 327

expression profiles vs. –Log one-sample t-test p values following day 1 (red), day 2 (blue), and 328

day 4 (green) H2S exposure. Scatter Plots of one-sample t-test fold protein expression differences 329

vs. –Log one-sample t-test p values from B) day 1, C) day 2, and D) day 4 of H2S exposure. 330

One-sample t-test of significantly modulated proteins in the H2S exposure group vs. the control 331

according to fold expression values are displayed with official mouse gene symbols in red. (Two 332

column figure) 333

334

3.4 Validation of proteomic changes of genes after H2S exposure 335

To confirm the observed changes in the IC proteomic profile following acute exposure to H2S, 336

several genes were analyzed by quantitative RT-PCR. Mice were sacrificed 2 h after each 337

designated time point to measure cellular response right after H2S exposure. Protein kinase 338

AMP-activated non-catalytic subunit beta 1 (Prkab1, also called Ampk) is known to correlate 339

with calcium fluctuations as a measure of cellular calcium response (Yong et al., 2010). Prkab1 340

mRNA expression was analyzed as a means to measure the calcium-dependent cellular response 341

following H2S exposure. Prkab1mRNA expression was upregulated following H2S exposure on 342

day 1, 2 and 4 which is in line with the mass spec observed upregulation of protein Kinase 343

cAMP-activated catalytic subunit alpha (Prkca) and calcium activated protein kinase C beta 344

(Prkacb) on exposure day 1. To confirm potential H2S exposure dependent pro-inflammatory and 345

ischemic effects, mRNA expression of Vimentin (Vim) was measured. Vim mRNA expression 346

demonstrated a steady increase throughout all days of H2S exposure (Fig. 6 B). This is further 347


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supported by the increased protein expression of Vim on exposure day 1, while mass spec lacked 348

detection of Vim in the other samples. 349

Due to a mass-spec observed downregulation in the activator of 90 kDa heat shock protein and 350

chaperone ATPase homlog1 (Ahsa1) on day 1 and 2, mRNA expression of Ahsa1 was examined 351

by RT-PCR (Fig. 6 C). Similar to the mass spec observed protein expression, mRNA expression 352

of Ahsa1 demonstrated a significant 30-40 % downregulation on days 1 and 2 of H2S exposure, 353

compared to the breathing air control group. 354

355

Figure 6.Validation of expression changes of three genes at the mRNA level in IC after H2S 356

exposure. The transcriptional level of three genes (A; Prkab1, B; Vim, C; Ahsa1) was measured 357

by quantitative PCR. Samples were normalized with the reference gene Gapdh. Data are 358

represented as the mean ± S.E.M. Note that only a single acute exposure to H2S was needed to 359

cause upregulation of gene expression of PkAb1 and Vim or downregulation of Ahsa1 gene 360

expression. (Two column figure) 361

362

4. Discussion 363

Acute H2S exposure often leads to neurological sequelae among human survivors, suggesting a 364

trigger of neurodegeneration cascade following acute H2S exposure (Matsuo et al., 1979; Tvedt 365


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et al., 1991a; Tvedt et al., 1991b; Kilburn, 1993; Snyder et al., 1995; Woodall et al., 2005). 366

However, the exact cellular and molecular mechanisms underlying delayed neurological 367

disorders after acute H2S exposure are poorly understood. Few studies have been done to 368

characterize mechanisms of H2S exposure-induced neurotoxicity in survivors of acute H2S 369

poisoning. We recently developed an inhalational mouse model which recapitulates H2S-induced 370

neurotoxicity observed in humans (Anantharam et al., 2017). In this model, mice exposed to H2S 371

showed clinical and pathological characteristics as shown in human patients. In case studies of 372

human H2S inhalation exposure, H2S concentrations > 500 ppm were reported to rapidly induce 373

severe neurotoxicity and temporary paralysis of breathing, leading to immediate collapse 374

(Guidotti, 1996; Chou et al., 2016). Survivors of acute high H2S exposures (> 500 ppm) 375

developed chronic neurological problems such as loss of memory, loss of control of facial 376

muscles, persistent headaches, and neurodegeneration. These patients also exhibited motor 377

behavioral deficits such as spastic gait (Wasch et al., 1989; Tvedt et al., 1991a; Tvedt et al., 378

1991b; Callender et al., 1993). Brain scan images of patients accidently exposed to H2S 379

suggested a dysregulation of basal ganglia region that governs motor behavior (Schneider et al., 380

1998; Nam et al., 2004). This is consitent with the observed motor deficits in our RotaRod 381

studies that are in agreement with clinical phenotypes of H2S exposed human patients. According 382

to Wasch et al., the brainstem was demonstrated to accumulate the highest concentration of H2S 383

compared to other brain regions in the rat (Wasch et al., 1989). We have previously identified 384

and herein confirmed that the IC, a brainstem region, is highly sensitive to H2S-induced necrosis. 385

The relative sensitivity of the IC to H2S-induced injury reflects the unique elements and cell 386

populations in this region of the brain. We observed histologic features of neurodegeneration, 387

neuronal death, and reactive gliosis is in the IC starting on day 3 with massive cell necrosis and 388


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glial scarring in the IC on day 7. Immunohistochemistry with neuron specific antibody (NeuN) 389

revealed severe neuronal loss in the IC with H2S exposure. Loss of neuronal cells was more than 390

70 % compared to breathing air control group in IC. In contrast, brain stem regions adjacent to 391

the IC were shown to be relatively unaffected, indicating unique susceptibility of the IC region to 392

H2S toxicity. The function of the IC is to integrate auditory and other sensory signals and is 393

reportedly a region with high metabolic rate requirements (Ridgway et al., 2006; Houser et al., 394

2010) while it is also the most highly vascularized brain region (Gonzalez-Lima et al., 1997). 395

H2S is a systemic metabolic toxicant that reportedly interferes with ATP synthesis. It is 396

reasonable to infer that brain regions with high blood supply and metabolic rates such as the IC 397

are more vulnerable to H2S-induced toxicity. 398

One of the hypotheses underlying H2S-induced neurotoxicity is that H2S causes hypoxia-399

dependent neurodegeneration following H2S induced hypotension and pulmonary edema-400

dependent oxygen deprivation in inhalation victims of H2S poisoning (Nicholls and Kim, 1982; 401

Dorman et al., 2002; Miyazato et al., 2013; Rumbeiha et al., 2016). However, the proximate 402

molecular pathways by which these events lead to neurodegeneration are unknown. In this study, 403

we used proteomic analysis to define cellular and molecular mechanisms of H2S induced 404

neurodegeneration during early stages of injury. We demonstrated that H2S exposure induced 405

significant alteration of protein expression in the IC in various biological pathways, including 406

cellular morphology, energy metabolism, and calcium signaling. It was most interesting that a 407

single H2S exposure exerted the most significant changes, suggesting a single exposure, as 408

commonly occurs during single accidental H2S exposures, is sufficient to trigger such proteomic 409

changes. 410


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Energy failure is another hypothesis of H2S-induced neurotoxicity because it exerts its cellular 411

toxicity by inhibition of cytochrome C oxidase which plays a crucial role in mitochondrial ATP 412

synthesis (Nicholls and Kim, 1982; Dorman et al., 2002). In this study, expression levels of 413

cytochrome C oxidase-related proteins were not changed, while cytochrome C oxidase subunit 6 414

C (Cox6c) and mitochondrial cytochrome C oxidase subunit 2 (mt-Co2) were decreased on day 4 415

and 1, respectively, supporting potential impairment of energy production following H2S 416

exposure. Several other proteins related to energy production were also altered. For instance, 417

ATP synthase subunits Atp5j2 and ATP5l together with cytochrome C oxidases 6C were 418

downregulated on days 2, and 4, respectively. In addition, citric acid cycle proteins malic 419

enzyme (Me1) and phosphoglycerate mutase (Pgam2) were downregulated on day 1. Further 420

studies are warranted to check whether Coxy6c and mt-Co2 are the bona fide targets of H2S 421

toxicity. Collectively, these results suggest hypoxia, resulting in low oxygen tissue delivery, and 422

impaired ATP synthesis arising from dysregulation of cytochrome C related proteins, likely work 423

in concert leading to neuronal death in the IC. 424

The IC undergoes severe degenerative changes during H2S exposure including widespread 425

neuronal loss, which is regionally selective. Long-term exposure to H2S was demonstrated to 426

result in neuronal structural damage by demyelination (Solnyshkova, 2003). Interestingly, 427

claudin-11 (Cldn11), a major tight junction component in neuronal myelin structures of the 428

central nervous system (CNS) was downregulated following day 1 and 2 of H2S exposure 429

supporting potential H2S induced neurodegeneration (Gow et al., 1999; Tiwari-Woodruff et al., 430

2001). Besides, mass spectrometry detected alterations in proteins related to cellular 431

morphological changes included regulators of actin cytoskeleton and microtubule organization. 432

Dynamine-3 (Dnm3), a GTP binding protein associated with the microtubule system was 433


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27

downregulated on day 1. Dynein Axonemal Heavy Chain 6 (Dnah6), a component of 434

microtubule-associated motor system, was upregulated on day 4. Moreover, a couple of adhesion 435

proteins were altered, e.g., vimentin (Vim) was upregulated on day 1 while prelaminin-A/C 436

(Lmna) was upregulated on day 4 of H2S exposure. Dihydropyrimidinase-related protein Dpysl4, 437

and neurofilament light polypeptide Nefl were downregulated on day 1 together with a 438

downregulation of myelin proteolipid protein Plp1 throughout the study period. With regards to 439

other cytoskeletal proteins, Tuba1b and Tubb6 were downregulated on day 4. These changes 440

may impact cell integrity and function in the IC, leading to neuronal death. 441

H2S exposure has previously been shown to induce dysregulation of calcium concentrations 442

(Garcia-Bereguiain et al., 2008). It was further reported by others that exposure to H2S may 443

affect regulation of calcium (Nagai et al., 2004; Garcia-Bereguiain et al., 2008; Yong et al., 444

2010). In this study, the calcium binding protein neurogranin (Nrgn) demonstrated a non-445

significant upregulation on day 1. However, we found a significant upregulation in cAMP-446

dependent protein kinase that matched the expression pattern of Prka1 mRNA supporting 447

potential activation of calcium-dependent signaling. These results agree with previous reports 448

that demonstrated H2S-dependent calcium and cyclic-AMP signaling through Prka (Kimura, 449

2000; Yong et al., 2010). Collectively, these results suggest calcium dysregulation as a potential 450

mechanism of H2S-induced neurotoxicity. 451

Changes in immunogenic biomarkers represent one of earliest responses to cytotoxicity. 452

Evidence of potential H2S-dependent immunogenic changes was given by a downregulation in 453

anti-inflammatory protein macrophage migration inhibition factor Mif on day 1 and an 454

upregulation in the reactive oxygen species (ROS) response and complement binding protein 455

C1qbp on day 4. Immunogenic changes in MIF have been previously observed (Roger et al., 456


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28

2003). ROS response and complement binding protein C1qbp has been previously reported 457

(McGee and Baines, 2011). In addition, there was an upregulation of cytoplasmic interacting 458

protein (Cyfip2) and nuclear hormone receptor and transcriptional repressor family (Nr1d2) that 459

were observed throughout the exposure period. Cyfip2 was previously shown by others to have a 460

p53-response element in its promoter region and to be one of the direct targets of p53 (Jackson et 461

al., 2007). Besides, the anti-apoptotic and immunogenic protein Bcl2 associated athanogene 462

(Bag5) was downregulated on day 1. Endoplasmic reticulum stress-induced downregulation of 463

Bag5 has previously reported (Bruchmann et al., 2013; Gupta et al., 2016). Further evidence of 464

potential cell stress-dependent impairment in protein folding was shown by an RT-PCR and 465

mass spec detected downregulation in Ahsa1 that plays a role as chaperone and activating heat 466

shock protein 90 (Okayama et al., 2014; Tripathi et al., 2014). These results indicate that 467

inflammation may play an important role in H2S-induced neurotoxicity. 468

H2S-induced neurotoxicity has been suggested to resemble the injury caused by ischemic 469

hypoxic conditions (Doujaiji and Al-Tawfiq, 2010; Rumbeiha et al., 2016). Previous studies 470

have suggested neurofilament Nefl, and collapsin Response Mediator Protein 3 (Dpysl4) as 471

biomarkers of hypoxia and cerebral ischemia (Hou et al., 2006; Lian et al., 2015). Dpysl4 is 472

cleaved by activated calpain reaction in response to cerebral ischemia (Hou et al., 2006). Both 473

Nefl and Dpysl4 were downregulated during H2S exposure. In addition, mass spec detected 474

downregulation of phosphoglycerate mutase 2 (Pgam2), an ischemia biomarker, on day 1. This 475

biomarker is suggested to correlate with the ischemic conditions (Li et al., 2012). Vimentin (Vim) 476

has been shown as another biomarker for ischemia (Li et al., 2008). Mass spec also detected a 477

steady increase in protein and mRNA expression of the inflammatory and ischemia biomarker 478

Vim for the entire duration of the study. These data support ongoing progression of H2S-479


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29

dependent ischemia-like conditions during acute H2S exposures, further supporting the role of 480

hypoxia as a mechanism of H2S-induced neurotoxicity. 481

482

5. Conclusion 483

In this study we examined the early effects of acute H2S exposure on brains of mice following 484

either a single H2S exposure or up to 4 short-term acute exposures. Taken together, acute 485

exposure to H2S induced neurotoxicity, which manifested as progressive behavioral deficits. The 486

IC was the most sensitive brain region, confirming our previous observations. Results point to 487

demonstrated modulation of several proteome-based biological pathways in the IC. Specifically, 488

results of proteomic and gene expression studies suggest calcium dysregulation, immune 489

mediated inflammatory response, pro-apoptosis mechanisms, and ischemia-like cytotoxicity to 490

be involved in H2S-induced neurotoxicity. This proteomic data provided important clues on 491

mechanisms of H2S-induced neurological toxicity. Further research is recommended to better 492

understand the singular or collective role of these potential mechanisms in H2S-induced mortality, 493

neurotoxicity, and evolution of neurological sequelae. 494

495

496


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Figure 1. Exposure paradigm of hydrogen sulfide on C57 black mice. 497

Mice were exposed to 765 ppm of H2S in a chamber for 40 min either once only (day 1) and for 498

15 min on the following days up to day 7. Mice were sacrificed 2 h post-H2S exposure for each 499

designated time point. Negative control mice were exposed to breathing air from a cylinder daily 500

up to day 7. Separate groups of mice were sacrificed on days 1, 3, and 7 for 501

immunohistochemistry. Groups of mice for proteomics studies were sacrificed on days 1, 2 and 4. 502

503

Figure 2. Exposure to hydrogen sulfide induced motor behavioral deficits. 504

C57 black mice were exposed to H2S as shown in Fig. 1. Locomotor activity was measured using 505

an automated VersaMax locomotor activity monitor during 10 min time test on days 2, 4, and 6. 506

Horizontal activity (A), vertical activity (B), and total distance traveled (C) were analyzed 507

between groups. Seizure activity was analyzed on a time scale (D). Asterisks (*, p < 0.05; **, p < 508

0.01) indicate significant differences between H2S and breathing air negative control groups. 509

510

Figure 3. Neurodegeneration and necrosis in the inferior colliculus of mice exposed to 650-750 511

ppm H2S in short-term repeated acute inhalation exposures over 7 days. Note the loss of neurons 512

and development of clear vacuoles in the neuropil (arrow) at day 7 in mice exposed to H2S and 513

the minimal morphologic effects to earlier H2S exposures or breathing air (A). Inhalation of H2S 514

resulted in marked and selective loss of neurons in the IC (1000X magnification images, B), with 515

retention of neurons in the regions surrounding the IC (200X magnification image). Computer 516

aided image analysis of NeuN immunostained sections reveals marked loss of neurons in the IC 517

of mice exposed to H2S (p=0.001, Students t test). Representative photomicrographs of mice 518


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31

exposed to breathing air or H2S, hematoxylin and eosin (A) and NeuN immunohistochemistry 519

(B), 400X magnification (A) and 1000X and 200X magnification (B). Neurons in the IC region 520

of the H2S exposed mice on day 7 were enumerated and compared to breathing air control (C). 521

522

Figure 4. Heatmap of IC changes in proteomic profile following H2S exposure 523

Morpheus rendered Heatmap of determined changes in protein expression following 1, 2 and 4 524

day H2S exposure using hierarchical clustering by Euclidean distance, row average linkage and 525

grouping of rows by gene ontology for biological process (GOBP). Heatmap displays fold 526

expression values of five (day 1 and day 2) or four (day 4) analyzed IC tissue samples, and 527

average fold expression values, mouse gene identifiers (gene names), protein names, and GOBP. 528

“+” indicates t-test (of fold expression values) significantly modulated proteins; white non 529

regulated, red upregulated (above 1.2 fold expression vs. control), blue downregulated (below 530

0.83 fold expression vs. control) converted into a log2 data display. 531

532

Figure 5. Overall distribution of IC changes in proteomic profile following H2S exposure 533

A) Scatter Plot to display overall distribution of one-sample t-test differences in protein 534

expression profiles vs. –Log one-sample t-test p values following day 1 (red), day 2 (blue), and 535

day 4 (green) H2S exposure. Scatter Plots of one-sample t-test fold protein expression differences 536

vs. –Log one-sample t-test p values from B) day 1, C) day 2, and D) day 4 H2S exposure. One-537

sample t-test of significantly modulated proteins in the H2S exposure group vs. the control 538

according to fold expression values are displayed with official mouse gene symbols in red. 539


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32

Figure 6. Validation of expression change of three genes at the mRNA level in IC after H2S 540

exposure. The transcriptional level of three genes (A; Prkab1, B; Vim, C; Ahsa1) was measured 541

by quantitative PCR. Samples were normalized with the reference gene Gapdh. Data are 542

represented as the mean ± S.E.M. Note that only a single acute exposure to H2S caused 543

upregulation of gene expression of PkAb1 and Vim or downregulation of Ahsa1 gene expression. 544

545

546

Acknowledgment: The authors do not have any conflict of interest. This work was partially 547

supported by the Iowa State University College of Veterinary Medicine Seed grant, Startup funds 548

and incentive account funds for Rumbeiha. 549

550

551

552


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33

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Broad Spectrum Proteomics Analysis of the Inferior ...S exposure, respectively. Mass spectrometry data were analyzed by Perseus 29 1.5.5.3 statistical analysis, and gene ontology heat