Metabolomic Signatures of Brainstem in Mice following Acute and Subchronic Hydrogen Sulfide Exposure

Hydrogen sulfide (H2S) is an environmental toxicant of significant health concern. The brain is a major target in acute H2S poisoning. This study was conducted to test the hypothesis that acute and subchronic ambient H2S exposures alter the brain metabolome. Male 7–8-week-old C57BL/6J mice were exposed by whole-body inhalation to 1000 ppm H2S for 45 min and euthanized at 5 min or 72 h for acute exposure. For subchronic study, mice were exposed to 5 ppm H2S 2 h/day, 5 days/week for 5 weeks. Control mice were exposed to room air. The brainstem was removed for metabolomic analysis. Enrichment analysis showed that the metabolomic profiles in acute and subchronic H2S exposures matched with those of cerebral spinal fluid from patients with seizures or Alzheimer’s disease. Acute H2S exposure decreased excitatory neurotransmitters, aspartate, and glutamate, while the inhibitory neurotransmitter, serotonin, was increased. Branched-chain amino acids and glucose were increased by acute H2S exposure. Subchronic H2S exposure within OSHA guidelines surprisingly decreased serotonin concentration. In subchronic H2S exposure, glucose was decreased, while polyunsaturated fatty acids, inosine, and hypoxanthine were increased. Collectively, these results provide important mechanistic clues for acute and subchronic ambient H2S poisoning and show that H2S alters brainstem metabolome.


Introduction
The brain is a highly complex organ that regulates many body functions.To perform these roles, it relies on complex metabolic processes which are energy dependent [1,2].Consequently, the brain is susceptible to toxic environmental chemicals that target mitochondria and impair energy metabolism.An example of such chemicals is hydrogen sulfide (H 2 S), a potent gas produced endogenously and in the environment.Endogenously produced H 2 S plays key roles in brain signaling and is essential for normal brain physiologic functions.However, excessive amounts of H 2 S in the brain, either from aberrant endogenous metabolic pathways (genetic defects) or environmental exposures, leads to neuronal dysfunction, including neurodegeneration [3][4][5][6][7][8][9][10]. Ethylmalonic encephalopathy (OMIM 602473), a life-limiting disease, is caused by a genetic defect in H 2 S metabolism in mitochondria, resulting in chronic excessive H 2 S exposure in the brain [11].
Studies from our laboratory showed that acute exogenous H 2 S exposure induces transcriptomic and proteomic changes in the brainstem, thalamus, and other brain regions [3,[5][6][7][8][9]12,13].A wealth of literature shows that H 2 S impairs mitochondrial function, specifically targeting cytochrome c oxidase (Complex IV) in the electron transport chain, thus inhibiting ATP production [14][15][16][17].Chemical hypoxia and/or energy deficits are linked to a loss of central respiratory drive, convulsions, and death following acute H 2 S exposures [18,19].In cases without a fatal outcome, it is likely that deficits in brain metabolism may cause immediate, intermediate, delayed, and long-term neurotoxicity following acute or chronic H 2 S exposure.It is possible that some of these H 2 S-induced toxicological changes may predispose exposed individuals to other neurological conditions, including neurodegenerative conditions via gene-environment interactions.To our knowledge, despite the well-known impact of H 2 S on cellular metabolism, there are no metabolomic studies on the brainstem upon acute and chronic H 2 S exposures.This is a knowledge gap curtailing our understanding of toxic mechanisms of H 2 S-induced neurotoxicity.
We postulated that metabolic signatures of the brainstem could be observed in an inhalation whole-mouse model because human exposure to H 2 S gas is typically via inhalation.For this project, we focused on the brainstem because this region has been previously shown to undergo neurodegeneration following acute H 2 S exposure and also because it controls vital autonomic functions such as breathing [3].Impaired breathing is widely implicated as a cause of death following acute H 2 S poisoning [18].It was also previously reported that H 2 S preferentially accumulates in the brainstem region [20].Moreover, the exact underlying mechanisms by which H 2 S suppresses breathing, causing death, remain to be elucidated.Therefore, understanding the metabolomic changes in the brainstem following H 2 S exposure will advance our knowledge of the mechanisms underlying H 2 S-induced neurotoxicity, neuropathology, and death.

Animals
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Iowa State University (ISU) and the University of California at Davis (UC Davis).IACUC-18-136, February 2016, and IACUC-21819, October 2020, were approved by ISU and UC Davis, respectively.Care and use of animals were performed in accordance with the IACUC, and animals were treated humanely and handled with care.We also followed the ARRIVE guidelines in the design and execution of this study [21].
Briefly, 7-8-week-old male C57BL/6J mice were purchased from Jackson Laboratories (Sacramento, CA and Bar Harbor, ME, USA).Only male mice were used because previous studies in our laboratory found male mice to be more sensitive to acute H 2 S poisoning than females [9].Mice were randomly assigned to different groups and housed in Laboratory Animal Resources at the College of Veterinary Medicine, ISU, and in the Teaching and Research Animal Care Services facility (TRACS) at the School of Veterinary Medicine, UC Davis, with a 12:12 h light and dark cycle.Room temperature and relative humidity were maintained at 22 • C and 50 ± 10%, respectively.Protein rodent maintenance diet (Teklad HSD Inc., Madison, WI, USA) and drinking water were provided ad libitum.Mice were acclimated for 1 week before the start of the experiments.

Gas Exposure
We conducted two separate experiments to mimic the two real-world scenarios of H 2 S exposure.In the first scenario, we conducted acute H 2 S exposure to mimic situations involving large scale industrial accidents or nefarious acts (Figure 1A).In the second scenario, we conducted subchronic exposure to low level ambient H 2 S to mimic occupational settings (Figure 1B).According to the OSHA guidelines, workers can be exposed to up to 10 ppm H 2 S for 8 h a day (TWA), 5 days a week without negatively impacting their health [22].For this reason, we chose to use H 2 S at a concentration of 5 ppm. Figure 1.A summary of experimental exposure paradigms.(A) Exposure paradigm of single acute exposure to 1000 ppm H2S.This exposure model induces 50% mortality (LCt50).Control mice were exposed to room air.(B) Exposure paradigm of subchronic ambient exposure to 5 ppm H2S.In this model, mice were exposed to 5 ppm H2S for 2 h/day, 5 days/week for 5 weeks.Control mice were exposed to room air.

Acute Exposure Experiment
For the acute study, we followed our previously published LCt50 model [3,[5][6][7][8][9]12,13] (Figure 1A).Briefly, two cohorts of male mice (n = 10) were exposed to 1000 ppm of H2S for 45 min using a whole-body inhalation exposure chamber, as reported previously [3,[5][6][7][8][9]12,13].This level and length of exposure results in 50% mortality (lethal concentration time 50 [LCt50]) during exposure, and mice typically exhibit dyspnea, convulsions, loss of consciousness, and death in that order [9].For the metabolomic analysis, we used mice that survived the LCt50 exposure.One cohort of surviving mice was sacrificed 5 min post exposure in order to determine the metabolic profile of mice immediately after exposure while the mice were still manifesting clinical effects of acute exposure.The other cohort was sacrificed 72 h post H2S exposure in order to determine metabolic changes at the time when neurodegenerative changes are typically initially observed in this model histologically [3,5,9].For the controls, we used two cohorts of mice exposed once to normal room air (RA) for the same duration as H2S-exposed mice.As was the case for H2S-exposed mice, one cohort of control mice (n = 5) was euthanized at 5 min post RA exposure while the other was euthanized at 72 post RA exposure.All mice that received RA survived.Both H2S gas and RA were introduced to the exposure chamber from pressurized gas cylinders.The concentration of H2S in the exposure chamber was monitored in real time using a H2S sensor (RKI Eagle, RKI Instrument, Union City, CA, USA).Following exposure, mice were euthanized by decapitation and the brainstem (medulla and pons) was immediately further micro-dissected and the pontine reticular nucleus and the gigantocellular reticular nucleus isolated on ice.These tissue samples were immediately stored at −80 °C until metabolomic analysis.

Ambient Exposure Experiment
In this experiment, male mice (n = 5) were exposed to 5 ppm H2S, 2 h/day, 5 days/week for 5 weeks, while the control group of mice (n = 4) were exposed to normal room air (RA).This dosage was chosen based on our prior pilot studies and also because 5 ppm is the midpoint concentration of the OSHA guideline of a time weighted average of 10 ppm of H2S in an 8 h work day, 5 days a week for the construction and shipyard industries [23].Both H2S gas and RA were delivered to the exposure chambers from pressurized gas cylinders.None of the mice in this subchronic experiment died.The experimental design is shown in Figure 1B.

Acute Exposure Experiment
For the acute study, we followed our previously published LCt 50 model [3,[5][6][7][8][9]12,13] (Figure 1A).Briefly, two cohorts of male mice (n = 10) were exposed to 1000 ppm of H 2 S for 45 min using a whole-body inhalation exposure chamber, as reported previously [3,[5][6][7][8][9]12,13].This level and length of exposure results in 50% mortality (lethal concentration time 50 [LCt 50 ]) during exposure, and mice typically exhibit dyspnea, convulsions, loss of consciousness, and death in that order [9].For the metabolomic analysis, we used mice that survived the LCt 50 exposure.One cohort of surviving mice was sacrificed 5 min post exposure in order to determine the metabolic profile of mice immediately after exposure while the mice were still manifesting clinical effects of acute exposure.The other cohort was sacrificed 72 h post H 2 S exposure in order to determine metabolic changes at the time when neurodegenerative changes are typically initially observed in this model histologically [3,5,9].For the controls, we used two cohorts of mice exposed once to normal room air (RA) for the same duration as H 2 S-exposed mice.As was the case for H 2 S-exposed mice, one cohort of control mice (n = 5) was euthanized at 5 min post RA exposure while the other was euthanized at 72 post RA exposure.All mice that received RA survived.Both H 2 S gas and RA were introduced to the exposure chamber from pressurized gas cylinders.The concentration of H 2 S in the exposure chamber was monitored in real time using a H 2 S sensor (RKI Eagle, RKI Instrument, Union City, CA, USA).Following exposure, mice were euthanized by decapitation and the brainstem (medulla and pons) was immediately further micro-dissected and the pontine reticular nucleus and the gigantocellular reticular nucleus isolated on ice.These tissue samples were immediately stored at −80 • C until metabolomic analysis.

Ambient Exposure Experiment
In this experiment, male mice (n = 5) were exposed to 5 ppm H 2 S, 2 h/day, 5 days/week for 5 weeks, while the control group of mice (n = 4) were exposed to normal room air (RA).This dosage was chosen based on our prior pilot studies and also because 5 ppm is the midpoint concentration of the OSHA guideline of a time weighted average of 10 ppm of H 2 S in an 8 h work day, 5 days a week for the construction and shipyard industries [23].Both H 2 S gas and RA were delivered to the exposure chambers from pressurized gas cylinders.None of the mice in this subchronic experiment died.The experimental design is shown in Figure 1B.

Metabolomic Analysis
The micro-dissected brainstem tissue samples were stored at −80 • C before submission to the West Coast Metabolomics Center, UC Davis, for untargeted metabolomics analysis.These metabolomic analyses consisted of three assays: (1) primary metabolism by GC-TOF MS, (2) biogenic amines (hydrophilic compounds) by HILIC-MS/MS, and (3) lipidomics by RPLC-MS/MS.Control samples from mice exposed to RA and samples from H 2 S-exposed mice were processed identically.Detailed procedures of metabolomic analysis is described in the supplementary data [24][25][26][27].

Data Analysis
Raw data peak heights were normalized to the sum of internal standards for primary amine and lipidomics data analyses.Data of primary metabolites were normalized by a vector normalization method by calculating the sum of all peak heights of all identified metabolites.Data were further processed to identify outliers by using a boxplot with 1.5× interquartile range (IQR).Outliers were removed for the definitive analysis.The dataset of metabolomic analysis is publicly available in dryad, an open data publishing platform (https://doi.org/10.5061/dryad.tdz08kq5n)(accessed on 21 December 2023).

Enrichment Analysis
Quantitative metabolite set enrichment analysis (MSEA) was performed using Metabo-Analyst 5.0 (https://www.metaboanalyst.ca)(accessed on 21 December 2023).Metabolites that were significantly altered compared to the RA groups were further analyzed by quantitative MSEA.

Heatmap
Metabolites that were significantly altered compared to the RA groups were summarized and visualized in a heatmap.Concentration changes in metabolites were visualized as a log 2-fold change.Visualization was processed using Python version 3.0 (https://www.python.org)accessed on 21 December 2023.

Statistical Analysis
Data are presented as mean and standard deviation of the mean.ANOVA with post hoc Tukey HSD test was performed for metabolites of acute H 2 S exposure using the statsmodels module in Python (https://www.python.org,version 3.0) accessed on 21 December 2023.Unpaired Student's t-test was performed for the metabolites of the ambient H 2 S exposure study using Excel software of Microsoft 365.p-values ≤ 0.1 were considered statistically significant for this explorative analysis.Statistical analysis of the metabolite set enrichment data was performed in MetaboAnalyst 5.0 (https://www.metaboanalyst.ca)accessed on 21 December 2023.

Exposure to Acute and Ambient Hydrogen Sulfide
Mice exposed acutely to H 2 S for 45 min manifested seizures, dyspnea, ataxia, and knockdown during exposure, as previously reported [3,[5][6][7]9,12,13]. Mice that survived after acute H 2 S exposure and were euthanized at 5 min post H 2 S exposure exhibited weak, ataxic, and labored breathing.Mice euthanized at 72 h post H 2 S exposure were active and moved about normally.However, mice acutely exposed to H 2 S exhibited a reduction in body weight [3].In contrast, mice from the 5 ppm exposure group did not exhibit any abnormal clinical signs or behavior, despite a reduction in body weight 30 days post ambient H 2 S exposure.

Metabolomic Changes in the Brainstem following Acute and Ambient Hydrogen Sulfide
H 2 S induced significant metabolomic changes in the brainstem.Metabolomic changes in brainstems from mice given a single high dose of acute exposure were different from those of mice repeatedly administered low levels of H 2 S over a month.The results of the changes from the metabolomic analyses included primary metabolites, biogenic amine metabolites, and lipidomic metabolites (summarized in heatmaps for acute and ambient H 2 S exposures; Figures 2 and 3).The Venn diagram shows the number of significantly altered identified metabolites which were increased more than 50% or decreased 33% compared to the room air control group (Figure 4).The Venn diagram represents the number of significantly altered metabolites in common or which were unique at 5 min and at 72 h post acute H 2 S exposure and following subchronic ambient H 2 S exposures.The significantly altered metabolites are listed in Tables 1 and 2 for 5 min and 72 h post acute H 2 S exposure and following subchronic ambient H 2 S exposure, respectively.
Metabolites 2024, 14, x FOR PEER REVIEW 6 of 28     In the Venn diagram, immediate response reflects metabolic changes in brainstems of mice acutely exposed to H2S and euthanized 5 min post acute exposure, while early response reflects changes at 72 h post acute exposure.The results show that both acute and subchronic H2S exposure significantly altered brainstem metabolites.Several metabolites including a phosphatidylinositol (PI 36:5), prostaglandin E1, thiazolidine-4-carboxylic acid, 3-methyhistidine, and phosphatidylethanol 18:1_18:1 (PEtOH 18:1_18:1) were increased both at 5 min post acute H2S and subchronic ambient H2S, while no common metabolites were decreased by both acute and subchronic ambient H2S exposures relative to the RA group (Figure 4B).3-Methylhistidine and PEtOH 18:1_18:1 were increased at all three euthanasia time points.Metabolites which increased in common due to acute H2S exposure both at 5 min and 72 h include caffeic acid, N 8 -acetylspermidine, vanillin-4-sulfate, flavin adenine, 3-methylhistidine, and PEtOH 18:1_18:1, while those which decreased due to acute H2S exposure were N-α-methylhistamine, 2,8-quinolinediol, vanillin, a glycerophospholipid (Cardiolipin 40:4_42:7), adenosine, and behenic acid.Metabolites presented in the heatmap analyses are further listed in Tables 1 and 2 with mass-to-charge ratio and fold change (log2 scale).In the Venn diagram, immediate response reflects metabolic changes in brainstems of mice acutely exposed to H 2 S and euthanized 5 min post acute exposure, while early response reflects changes at 72 h post acute exposure.The results show that both acute and subchronic H 2 S exposure significantly altered brainstem metabolites.Several metabolites including a phosphatidylinositol (PI 36:5), prostaglandin E1, thiazolidine-4-carboxylic acid, 3-methyhistidine, and phosphatidylethanol 18:1_18:1 (PEtOH 18:1_18:1) were increased both at 5 min post acute H 2 S and subchronic ambient H 2 S, while no common metabolites were decreased by both acute and subchronic ambient H 2 S exposures relative to the RA group (Figure 4B).3-Methylhistidine and PEtOH 18:1_18:1 were increased at all three euthanasia time points.Metabolites which increased in common due to acute H 2 S exposure both at 5 min and 72 h include caffeic acid, N 8 -acetylspermidine, vanillin-4-sulfate, flavin adenine, 3-methylhistidine, and PEtOH 18:1_18:1, while those which decreased due to acute H 2 S exposure were N-α-methylhistamine, 2,8-quinolinediol, vanillin, a glycerophospholipid (Cardiolipin 40:4_42:7), adenosine, and behenic acid.Metabolites presented in the heatmap analyses are further listed in Tables 1 and 2 with mass-to-charge ratio and fold change (log 2 scale).

Enrichment Analysis
We performed enrichment analysis using Metaboanalyst 5.0 to investigate whether H 2 S-induced metabolic profiles cause any biological dysregulation and whether H 2 Sinduced metabolic dysregulations share any similarity with any existing pathological signature databases in Metaboanalyst 5.0.While the database did not have brainstem as a tissue, we chose the human cerebral spinal fluid (CSF) dataset as this most closely matched our experimental design.The results showed several matches with human neuropathological conditions which are summarized in Figures 5-7.Briefly, the metabolomic profiles following acute H 2 S poisoning matched with several neurological disorders including seizures, epilepsy, Alzheimer's disease, and Glut-1 deficiency syndrome, among others (Figures 5 and 6).The metabolomic profiles of subchronic ambient H 2 S exposure matched with several pathological conditions including Alzheimer's disease, dementia, Parkinson's disease, seizures, Glut-1 deficiency, and anoxia, among others (Figure 7).

H 2 S Dysregulated Brainstem Neurotransmitters
We analyzed neurotransmitters following both acute and subchronic ambient H 2 S exposures in the brainstem region.Interestingly, excitatory neurotransmitters, namely, aspartate and glutamate, were decreased, while serotonin, an inhibitory neurotransmitter, was increased at 5 min after single acute H 2 S exposure (Figure 8A).Adenosine, which acts both as a central excitatory and as an inhibitory neurotransmitter in the brain, was decreased following both acute and subchronic H 2 S exposures.Subchronic ambient H 2 S exposure decreased serotonin concentration in the brainstem (Figure 8B).Tyrosine, a precursor of catecholamines (excitatory), was also increased at 5 min post acute H 2 S exposure, while tryptophan, a precursor of serotonin, was increased at 72 h post acute H 2 S exposure.Tryptophan was also increased due to subchronic ambient H 2 S exposure (Figure 9B).

H2S Dysregulated Brainstem Neurotransmitters
We analyzed neurotransmitters following both acute and subchronic ambient H2S exposures in the brainstem region.Interestingly, excitatory neurotransmitters, namely, aspartate and glutamate, were decreased, while serotonin, an inhibitory neurotransmitter, was increased at 5 min after single acute H2S exposure (Figure 8A).Adenosine, which acts both as a central excitatory and as an inhibitory neurotransmitter in the brain, was decreased following both acute and subchronic H2S exposures.Subchronic ambient H2S exposure decreased serotonin concentration in the brainstem (Figure 8B).Tyrosine, a precursor of catecholamines (excitatory), was also increased at 5 min post acute H2S exposure, while tryptophan, a precursor of serotonin, was increased at 72 h post acute H2S exposure.Tryptophan was also increased due to subchronic ambient H2S exposure (Figure 9B).

H 2 S Dysregulated Energy Homeostasis
We analyzed to determine changes in lipid metabolism in the brainstem following acute and subchronic H 2 S exposures.Immediately following acute H 2 S exposure, BCAAs were significantly increased more than two-fold (Figure 10A).Interestingly, subchronic ambient H 2 S also significantly increased BCAAs, albeit less than acute exposure (Figure 10B).
Behenic acid concentration, a very long-chain fatty acid, was significantly decreased more than two-fold following acute H 2 S exposure (Figure 11A).Following subchronic ambient H 2 S exposure, major fatty acids in the brainstem including oleic acid, arachidonic acid (AA), and docosahexaenoic acid (DHA) were significantly increased compared to control (Figure 11B).
Cellular respiration was also dysregulated following both acute and subchronic ambient H 2 S exposures.Glucose concentration was significantly elevated at 5 min post acute H 2 S exposure, while it decreased following ambient H 2 S exposure (Figure 12).Elevated glucose concentration can affect multiple biological pathways.The polyol pathway converts glucose to sorbitol and then to fructose.Sorbitol and fructose concentrations were also significantly increased at 5 min post acute H 2 S exposure.Lactate is formed via glycolysis and was also increased following acute H 2 S exposure, but it was decreased following subchronic ambient H 2 S exposure.However, ribulose 5-phosphate and UDP-GlcNAc, intermediates of the pentose phosphate pathway (PPP) and hexosamine biosynthetic pathways (HBP), respectively, were not significantly altered.Succinate, fumarate, and malate which are intermediates of the Krebs cycle were also increased at 5 min post acute H 2 S exposure.

H2S Dysregulated Energy Homeostasis
We analyzed to determine changes in lipid metabolism in the brainstem following acute and subchronic H2S exposures.Immediately following acute H2S exposure, BCAAs were significantly increased more than two-fold (Figure 10A).Interestingly, subchronic ambient H2S also significantly increased BCAAs, albeit less than acute exposure (Figure 10B).Behenic acid concentration, a very long-chain fatty acid, was significantly decreased more than two-fold following acute H2S exposure (Figure 11A).Following subchronic ambient H2S exposure, major fatty acids in the brainstem including oleic acid, arachidonic acid (AA), and docosahexaenoic acid (DHA) were significantly increased compared to control (Figure 11B).
Cellular respiration was also dysregulated following both acute and subchronic ambient H2S exposures.Glucose concentration was significantly elevated at 5 min post acute H2S exposure, while it decreased following ambient H2S exposure (Figure 12).Elevated glucose concentration can affect multiple biological pathways.The polyol pathway converts glucose to sorbitol and then to fructose.Sorbitol and fructose concentrations were also significantly increased at 5 min post acute H2S exposure.Lactate is formed via glycolysis and was also increased following acute H2S exposure, but it was decreased following subchronic ambient H2S exposure.However, ribulose 5-phosphate and UDP-GlcNAc, intermediates of the pentose phosphate pathway (PPP) and hexosamine biosynthetic pathways (HBP), respectively, were not significantly altered.Succinate, fumarate, and malate which are intermediates of the Krebs cycle were also increased at 5 min post acute H2S exposure.

H 2 S Dysregulated Inosine Metabolism
Inosine and its metabolites (hypoxanthine and xanthine) were increased by subchronic ambient H 2 S exposure (Figure 13), indicating an increased catabolism of purines under these conditions.Allantoin, an oxidation product of urate and usually considered a marker of oxidative stress, was the only metabolite increased at 5 min post acute H 2 S exposure.In contrast, inosine, hypoxanthine, and xanthine were all increased following subchronic ambient H 2 S exposure along with guanine and guanosine which are converted to xanthine (Figure 13).chronic ambient H2S exposure (Figure 13), indicating an increased catabolism of purines under these conditions.Allantoin, an oxidation product of urate and usually considered a marker of oxidative stress, was the only metabolite increased at 5 min post acute H2S exposure.In contrast, inosine, hypoxanthine, and xanthine were all increased following subchronic ambient H2S exposure along with guanine and guanosine which are converted to xanthine (Figure 13).

Discussion
Hydrogen sulfide is widely reported to impact a broad spectrum of physiological functions.It is also reported to be a highly toxic compound with steep dose-response relationships.Acute high dose exposures are linked to acute death and to permanent neurobehavioral abnormalities and neurodegeneration [28][29][30][31], while the impacts of chronic low level H2S exposure in humans remain ambiguous.Acute H2S exposure impairs breathing and this is widely cited as the cause of H2S-induced acute death.The brainstem

Discussion
Hydrogen sulfide is widely reported to impact a broad spectrum of physiological functions.It is also reported to be a highly toxic compound with steep dose-response relationships.Acute high dose exposures are linked to acute death and to permanent neurobehavioral abnormalities and neurodegeneration [28][29][30][31], while the impacts of chronic low level H 2 S exposure in humans remain ambiguous.Acute H 2 S exposure impairs breathing and this is widely cited as the cause of H 2 S-induced acute death.The brainstem is an important brain region which regulates autonomous functions including breathing [32].Despite the significance of this compound in health and disease, the lack of data on the impact of H 2 S on the brain metabolism is striking.This is the first comprehensive metabolomic study of acute high dose H 2 S exposure and subchronic low dose H 2 S exposure on the brain.For the subchronic low dose study, we used 5 ppm H 2 S to expose mice only for 2 h per day Monday through Friday for 5 weeks.As a reference, the OSHA TWA exposure guidelines for an 8 h workday, 5 days a week, is 10 ppm.The results showed that both acute and subchronic ambient H 2 S significantly altered many classes of metabolites including organic oxygen compounds, organic acids, phenylpropanoids, polyketides, organoheterocyclic compounds, benzenoids, nucleosides, nucleotides, organic nitrogen compounds, lipids, and lipid-like molecules in the brain.Importantly, metabolic changes induced by H 2 S exposure were different depending on whether it was a high dose single exposure or subchronic low dose exposure.Some metabolites, however, were increased both by acute and by subchronic exposures, suggesting that such compounds are sensitive indicators of H 2 S exposure.
3-Methylhistidine, a derivative of histidine and methionine amino acids, and phosphatidylethanol 18:1_18:1 (PEtOH 18:1_18:1), a glycerophosphoethanol, were both significantly increased following both single H 2 S exposure and subchronic ambient H 2 S exposure.The functions of these two metabolites in the brainstem are not clear at the moment.Vanillin was decreased, while vanillin-4-sulfate was increased by acute H 2 S exposure.Vanillin has been linked to colorectal cancer.The roles of vanillin and vanillin-4-sulfate and the role of H 2 S exposure in altering vanillin and vanillin-4-sulfate remain to be determined.However, these metabolites may serve as potential biomarkers of H 2 S poisoning.
Several metabolites were altered at 5 min and 72 h following single acute H 2 S exposure.N 8 -acetyl spermidine was increased by 0.8-fold following acute H 2 S exposure.It is a polyamine derived from spermidine by deacetylation and is reported to play a role in regulating cell signaling and gene expression, among other functions [33].It was previously reported that increases in N 8 -acetyl spermidine are caused by inhibiting N 8acetylspermidine deacetylase.This compound is linked to the differentiation of neurons and to elevated dopamine [33].Interestingly, acute H 2 S exposure increases dopamine concentration in the brainstem [3].N 8 -acetyl spermidine is also considered a biomarker for ischemic cardiomyopathy [34].Interestingly, H 2 S is a cytochrome c oxidase inhibitor and causes chemical ischemia [35].The exact role of N 8 -acetyl spermidine remains to be studied, but it may signal brain ischemia.
Flavin adenine, a derivative of riboflavin or vitamin B2, is also known as flavin adenine dinucleotide (FAD), and it plays a critical role as a cofactor of many enzymes.FAD was increased more than 1-fold at 5 min and 0.5-fold after 72 h following single acute exposure and is therefore a potential biomarker of acute H 2 S exposure in the brainstem.Since FAD plays a significant role as a cofactor in brain energy metabolism, it is possible that a sudden increase in FAD in the brainstem may indicate an imbalance in the redox cellular state (favoring FAD over FADH).FADH is not detectable by any of the mass spectrometers used in this study.
Adenosine and behenic acid were decreased 1-fold and 2-fold at 5 min and 72 h post acute H 2 S exposure, respectively.Adenosine is a nucleoside which plays many important roles in energy transfer, in signal transduction, as a neurotransmitter, and as a potent vasodilator [36].H 2 S is well known to reduce ATP generation by inhibiting cytochrome c oxidase in the electron transport chain.Adenosine 5 ′ -monophosphate was significantly decreased by about 1.5-fold following subchronic ambient H 2 S exposure.A decrease in adenosine concentration may limit several biological functions in which it is involved.Behenic acid, in contrast, is a very long-chain saturated fatty acid (VLCFA) and was decreased by more than 1.5-fold at 5 min and 72 h post acute H 2 S exposure, but not by subchronic H 2 S exposure.The reduction in behenic acid concentration may negatively impact energy production and homeostasis, among other effects.These results suggest that both acute and subchronic H 2 S exposure alter brainstem metabolism but in different ways.More research is needed to understand the implications of these results on brain function.
The brainstem has been reported to be particularly vulnerable to acute H 2 S poisoning.Following exogenous exposure, H 2 S was reportedly found in its highest concentrations in this brain region [20].This brain region is also recognized as the center of regulation for key autonomic functions [37,38].We have also previously shown that the brainstem is susceptible to H 2 S-induced neurodegeneration [3].In addition, we showed that the inhibition of central respiratory drive (from the brainstem) is a key cause of death in our mouse model of acute H 2 S poisoning [3].Clinical effects of acute H 2 S poisoning characterized by seizures and apnea, among others, were not seen in mice subchronically exposed to 5 ppm H 2 S for 1 month, indicating that the mechanisms involved were different.In this study, the only clinical effects noted following subchronic H 2 S exposure in mice was a loss in body weight.
The results of the metabolite enrichment analysis summarized in Figures 5-7 are very interesting.When the metabolomic profiles of acute and subchronic H 2 S exposure in mice are compared to existing databases of metabolomics of the cerebrospinal fluid from patients suffering from different neurological conditions, a pattern of similarities and differences emerges.At 5 min post exposure, the metabolomic enrichment pattern of acute H 2 S poisoning matches with many human neurological disorders including early-onset encephalopathy and cortical myoclonus, leukemia, propionic acidemia, anoxia, Rett syn-drome, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), Glut-1 deficiency syndrome, different seizure disorders, and schizophrenia, among others.In this mouse model, we have reported convulsions and neurodegeneration [3,[7][8][9]12,13].AD and PD are neurodegenerative diseases, while epilepsy matched with convulsions in our mouse model.We have also reported a significant increase in dopamine and serotonin concentrations in this model [3,9,12].Dopamine is a modulatory neurotransmitter which is both excitatory and inhibitory depending on which receptors it binds to.Serotonin is predominantly inhibitory and has been linked to schizophrenia.The summary of neurotransmitter changes in Figure 8 suggests that acute and subchronic H 2 S exposure tilts neurotransmitters toward inhibitory and excitatory effects, respectively.It is probable that the loss of central respiratory drive is linked predominantly to the neurotransmitter status observed following acute H 2 S exposure in this study.
H 2 S exposure is widely reported to cause hypoxic neuronal injury, which is consistent with its mechanism of action of inhibiting cytochrome c oxidase activity [35].Reduced cytochrome c oxidase activity is also reported in Alzheimer's disease [39], and chronic neurodegenerative diseases are characterized by brain energy deficits [1,2].The metabolomic profiles at 5 min post acute H 2 S exposure matched a metabolic profile of neurological disorders observed at 72 h min post acute exposure, including epilepsy, aseptic meningitis, Alzheimer's disease, schizophrenia, and different seizure disorders.
We were surprised to see that subchronic H 2 S exposure to 5 ppm, which is within the OSHA guidelines of 10 ppm for an 8 h workday, caused metabolic derangement in the brainstem.This is a significant finding which needs confirmation because of the health implications in the workplace.Strikingly, the metabolome of subchronic ambient H 2 S also matched with many human neurological disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia, which was similar to what we observed after single acute H 2 S exposure, with some exceptions.Notably, the metabolome of subchronic ambient H 2 S exposure additionally matched that of the CSF from patients with dementia, Canavan disease, alcoholism, and multiple sclerosis.Common themes of both acute and subchronic low dose H 2 S exposure revolve around metabolomic similarities between the metabolome of H 2 S exposure and that of seizure disorders and neurodegenerative conditions, especially Alzheimer's disease, Parkinson's disease, neuroinflammation, ataxia, and hypoxic-induced injury.This possibly signals that environmental H 2 S exposure shifts the brain metabolism, which may predispose or increase susceptibility to these debilitating disease conditions.Indeed, this mouse model with acute H 2 S exposure exhibited seizures, knockdown, and acute death [3,[6][7][8][9]12,13].These results signal that H 2 S-induced metabolomic changes play important roles in H 2 S-induced physiological symptoms including seizures and loss of consciousness.Notably, it is interesting that patients predisposed to seizures or neurofibromatosis are very sensitive to acute H 2 S poisoning [40].Also, H 2 S aggravated seizure-like events in a rat seizure model [41].Regarding the discovery of similarities in the metabolomes of acute and subchronic H 2 S exposure to those of the CSF of patients with Alzheimer's disease, there is meagre literature on this topic.It is well known that the interaction of genes and the environment plays important roles in many neurological disorders including Alzheimer's disease and Parkinson's disease.Considering that H 2 S is an environmental pollutant of occupational concern and that H 2 S exposure may be closely linked to many neurological disorders with big health and economic burdens in society, more work should be focused on this area to investigate the potential role of ambient H 2 S exposure in AD and/or other neurological conditions summarized in Figures 5-7.
The effects of acute H 2 S and subchronic ambient H 2 S exposures on neurotransmitters were investigated.Acute H 2 S exposure decreased aspartic acid, glutamic acid, and adenosine, while serotonin was increased.Serotonin was significantly decreased by subchronic ambient H 2 S exposure.Aspartic acid and glutamic acid are excitatory neurotransmitters, while serotonin is inhibitory [42,43].Adenosine acts both as an excitatory and inhibitory neurotransmitter.We previously reported that dopamine and serotonin were significantly increased in the brainstem at 5 min post acute H 2 S [3], which is consistent with the findings in this study.Tryptophan is a precursor to serotonin, while phenylalanine and tyrosine are precursors to dopamine.In this study, tyrosine was significantly increased at 5 min post acute exposure in the brainstem, while tryptophan was increased by subchronic ambient H 2 S exposure.Other neurotransmitters including acetylcholine, histidine, serine, noradrenaline, γ-aminobutyric acid (GABA), glycine, and histamine were not significantly altered in this study.
Glutamate and aspartate are the main excitatory neurotransmitters in the brain and are released in a Ca 2+ -dependent manner upon electrical stimulation.Serotonin is an inhibitory neurotransmitter and has an impact on a wide range of behavioral effects such as appetite, aggression, memory, fear, and depression.It is also involved in other central nervous system (CNS) functions such as respiration, body temperature, motor control, and bowel movement [44].It is well known that acute exposure to high concentrations of H 2 S induces breathing suppression, a loss of consciousness, and acute death in humans.However, the exact mechanisms behind these effects of acute H 2 S-induced toxicity are not known.A decrease in glutamate and aspartate coupled with an increase in serotonin following acute H 2 S exposure in the brainstem may play important roles in H 2 S-induced inhibition of breathing and/or loss of consciousness, considering that the brainstem regulates essential vital functions including breathing activity and consciousness [32].Interestingly, we recently reported that sodium sulfide, a H 2 S donor, suppressed neuronal activity in vitro by suppressing Ca 2+ oscillation in primary cortical neurons [6].An electrical stimulus releases glutamate from synaptic vesicles to the synaptic cleft.The released glutamate is taken up to glial cells by glutamate-aspartate transporters and converted to glutamine, which is then transported back to the neuron.Glutaminase in mitochondria converts glutamine to glutamate and aspartate.Acute H 2 S exposure induced increased glutamine at 5 min post acute exposure, indicating that glutamate-glutamine cycling may be dysregulated.A decrease in aspartate may influence the conversion of glutamine to glutamate, leading to a decrease in glutamate upon acute H 2 S exposure.Serotonin was also reported to inhibit glutamate release and the action of released glutamate [43].In addition, an acute surge of serotonin induces focal seizures [45].
Interestingly, subchronic ambient H 2 S exposure significantly decreased serotonin concentration.The current OSHA guideline sets the recommended airborne exposure limit (REL) to 10 ppm for hydrogen sulfide in the workplace [22].To the best of our knowledge, it has not been previously reported that subchronic ambient H 2 S exposure dysregulates serotonin.Therefore, this finding is novel.There are many serotonin receptors.It should be interesting to investigate the health impact of decreased serotonin in subchronic ambient H 2 S exposure.
Glutamate not only acts as a neurotransmitter but also as an anaplerotic component in tricarboxylic acid (TCA) cycle in energy balance.Glutamate is converted to α-ketoglutarate.In addition to the altered glutamate and glutamine, fumarate and malate were increased by 0.5-fold compared to the RA control mice at 5 min post acute H 2 S exposure.Intermediates of the TCA cycle including succinate, fumarate, and malate were increased at 5 min post acute H 2 S exposure, while succinate and fumarate were decreased following ambient H 2 S exposure.
Glucose is a key compound in glycolysis.Glucose concentration was significantly increased at 5 min post acute H 2 S exposure compared to the RA control group but decreased following ambient H 2 S exposure.An increase in brain glucose during acute H 2 S exposure may suggest that it is spared following the inhibition of cytochrome c oxidase by H 2 S. Elevated glucose concentrations can affect multiple biological pathways including the polyol pathway, glycolysis, the PPP, and HBP.Intermediates of the polyol pathway and glycolysis were increased, while the PPP and HBP were not significantly altered at 5 min post acute H 2 S exposure, indicating acute H 2 S exposure significantly dysregulates energy balance.An accumulation of succinate may be caused by inverse activity of succinate dehydrogenase converting fumarate to succinate [46].Lactate concentration remained elevated following acute H 2 S exposure, which is in line with previous findings that the treatment of sodium hydrosulfide induced elevation in lactate.It was shown that acute H 2 S exposure-induced inhibition of cytochrome c oxidase in the brain, including the brainstem, remained up to 72 h [3].It is plausible that the inhibition of cytochrome c oxidase subsequently affected the ETC and TCA cycle following acute H 2 S exposure, which could not meet the energy demand of the CNS.An accumulation of succinate was shown to produce ROS and induce static epilepticus in a kainic acid rat model [46].Although the exact effects of ambient H 2 S exposure on decreased glucose levels needs to be confirmed, ambient H 2 S exposure decreased the glucose level compared to the RA control group.
Fatty acids have multiple important roles in the nervous system, from serving as structural components of membranes, energy sources, signaling molecules, cellular differentiation, and apoptosis to contributing to pathological conditions such as aging and neurodegenerative diseases [47].The regulation of fatty acids is tightly controlled and in different ways in different brain regions.Behenic acid is a VLCFA.VLCFAs are preferentially metabolized in the peroxisome.The statistically significant reduction in behenic acid observed in this study may indicate altered peroxisome fatty acid beta oxidation.Subchronic ambient H 2 S strikingly increased metabolites of both the mono-unsaturated fatty acid (MUFA) and poly-unsaturated fatty acid (PUFA) synthesis pathways in the brainstem, including oleic acid, arachidonic acid (AA), and docosahexaenoic acid (DHA) in the brainstem compared to the RA control group.Arachidonic acid, which was also increased, is pro-inflammatory and is also linked to neurodegenerative diseases such as AD [48].A deficiency in DHA has been associated with neurodegenerative disorders [49].We have also previously reported that acute H 2 S poisoning causes neuroinflammation and neurodegeneration [3,9,12,13].
Subchronic ambient H 2 S also dysregulated inosine metabolism and increased inosine, hypoxanthine, and xanthine.Inosine is metabolized to hypoxanthine, xanthine, and finally to uric acid [50].Inosine was shown to play important roles in neuroprotection, among other roles, presumably via anti-inflammatory and antioxidant properties [51].AMP was shown to be metabolized to IMP, hypoxanthine, xanthine, and uric acid to enhance ATP production during acute energy consumption [50].The administration of inosine was shown to induce hypoglycemia [52].Indeed, subchronic ambient H 2 S decreased glucose concentration more than 1-fold compared to the RA control group.Hypoxanthine and its metabolites were shown to be increased in post mortem tissues over a time span of several hours [53].In this study, brainstem regions were immediately dissected and flash-frozen.Moreover, the changes in hypoxanthine and its metabolites were compared to those of the RA control group which were handled identically to brain tissues from H 2 S exposed mice.This finding shows that subchronic ambient H 2 S dysregulates energy homeostasis.More work is needed on this topic to determine whether subchronic ambient H 2 S exposure causes a brain energy deficit.Brain energy deficit has been cited a contributing factor in neurodegeneration [1,2].Potentially, this suggests that ambient H 2 S exposure may be an environmental factor predisposing to neurodegeneration.
This study has many potential applications.Understanding the basic mechanisms of H 2 S-induced neurotoxicity will lead to development of therapeutic drugs for the treatment of victims of acute and subchronic H 2 S poisoning.Hydrogen sulfide is considered a chemical weapon [54,55], and currently, there are no FDA-approved drugs for treating victims of acute H 2 S poisoning.Also, environmental pollutants, including those that affect mitochondria, play a role in inducing neurodegeneration.The results of this study will likely contribute to our knowledge on the role that H 2 S plays in neurodegeneration and other neurological diseases shown in Figures 5-7.In addition, this work may guide the development of novel diagnostic and forensic biomarkers of acute and subchronic H 2 S poisoning.
This study, though yielding interesting results, had some limitations.For example, this study only consisted of male, adult mice.Future studies should include female mice as well so as to determine whether there may be sex differences in brain metabolism following H 2 S exposure.As has been reported [9], male mice are more sensitive to acute H 2 S poisoning than females.Therefore, including female mice in this study may shed light on the toxic mechanisms of H 2 S. Another limitation of this study was that the acute study only lasted up to 72 h post H 2 S exposure.In previous studies, we have shown neurodegeneration to be fully manifested on day 7 in the brainstem and other brain regions [3,5,[7][8][9].Also, in human victims of single acute H 2 S poisoning accidents, a plethora of long-term debilitating neurological sequalae including neuropsychiatric disturbance, sleep disorders, headaches, memory and cognition deficits, and persistent vegetative states are reported [25][26][27][28]56].Therefore, future studies should investigate metabolomic changes in the brainstem several months later to determine the metabolomic profiles of delayed neurotoxic effects.Finally, concentrations of tissue metabolites, especially brain tissue, can be influenced by and vary in sample collection and sample preparation procedures [57,58].It has been shown that concentrations of brain tissue metabolites will decrease or increase if the post mortem autolysis is not prevented by procedures like microwave fixation before brain dissection [57,58].In this study, control and H 2 S exposed mice were decapitated and the brain was rapidly removed and micro-dissected on ice over a 2-3 min time period before they were immediately stored at −80 • C until analysis.All experiments had suitable control samples and all samples were handled similarly, and comparisons were made between the RA control and H 2 S exposed mice.Thus, whereas the absolute concentrations of metabolites would likely be different if samples were microwave fixed before brain microdissection, the outcome of the study should be the same.However, future metabolomic studies will be performed on microwave-fixed mice to completely stop enzymatic activity during tissue collection and sample preparation.

Conclusions
In summary, the results of this study identified 3-methylhistidine and PEtOH18:1_18:1 as potential biomarkers of H 2 S exposure, because they were significantly increased in the brainstems of mice exposed to H 2 S both acutely and subchronically.Twelve metabolites were altered by acute H 2 S exposure including N-acetylspermidine, adenosine, behenic acid, flavin adenine, vanillin, vanillin-4-sulfate, caffeic acid, and behenic acid, which were identified as potential biomarkers of acute H 2 S exposure.Acute H 2 S decreased glutamate and aspartate, while serotonin was increased, suggesting that overall acute H 2 S shifts neurotransmitters predominantly toward inhibitory status.Serotonin was decreased by subchronic ambient H 2 S exposure and so was adenosine, suggesting that chronic H 2 S exposure may predispose neurological excitation.Glucose and fructose were increased in acute H 2 S poisoning, while glucose was significantly decreased in subchronic ambient H 2 S. MUFAs and PUFAs were increased in subchronic ambient H 2 S exposure.Overall, the results of this study provide evidence that both acute and subchronic H 2 S exposure alters neurotransmitters and energy metabolism in the brainstem.This has opened novel mechanisms underlying H 2 S-induced neurotoxicity.Surprisingly, and more importantly, this study shows that current OSHA guidelines (10 ppm for 40 h/work week) may not be adequately protecting worker health, as a subchronic exposure of mice to 5 ppm H 2 S for only a few hours per day significantly altered neurotransmitters and energy metabolism.

Figure 1 .
Figure 1.A summary of experimental exposure paradigms.(A) Exposure paradigm of single acute exposure to 1000 ppm H 2 S.This exposure model induces 50% mortality (LCt 50 ).Control mice were exposed to room air.(B) Exposure paradigm of subchronic ambient exposure to 5 ppm H 2 S. In this model, mice were exposed to 5 ppm H 2 S for 2 h/day, 5 days/week for 5 weeks.Control mice were exposed to room air.

Figure 2 .
Figure 2. Heatmap analysis of significantly altered metabolites following a single acute H2S or RA exposure.Concentrations of significantly altered metabolites from primary metabolites, biogenic amines, and lipidomic metabolites are visualized in a heatmap by fold change (log2 scale).Each metabolite was compared to the average concentration of RA control group.Classification of individual metabolites is shown on the right side of the heatmap.

Figure 2 .
Figure 2. Heatmap analysis of significantly altered metabolites following a single acute H 2 S or RA exposure.Concentrations of significantly altered metabolites from primary metabolites, biogenic amines, and lipidomic metabolites are visualized in a heatmap by fold change (log 2 scale).Each metabolite was compared to the average concentration of RA control group.Classification of individual metabolites is shown on the right side of the heatmap.

Figure 3 .
Figure 3. Heatmap analysis of significantly altered metabolites following subchronic ambient H2S or RA exposure.Concentrations of significantly altered primary organic metabolites, hydrophilic compounds including biogenic amines, and lipidomic metabolites are visualized in a heatmap by

Figure 3 .
Figure 3. Heatmap analysis of significantly altered metabolites following subchronic ambient H 2 S or RA exposure.Concentrations of significantly altered primary organic metabolites, hydrophilic compounds including biogenic amines, and lipidomic metabolites are visualized in a heatmap by fold change (log 2 scale).Each metabolite was compared to the average concentration of RA control group.Classification of individual metabolites is shown on the right side of the heatmap.

Figure 4 .
Figure 4. Venn diagrams summarizing numbers of significantly altered metabolites following acute and ambient H2S exposure.Immediate (5 min) and early (72 h) responses to acute H2S and response to ambient H2S exposure in brainstem are shown.(A) Increased metabolites compared to RA control group.(B) Decreased metabolites compared to RA control group.

Figure 4 .
Figure 4. Venn diagrams summarizing numbers of significantly altered metabolites following acute and ambient H 2 S exposure.Immediate (5 min) and early (72 h) responses to acute H 2 S and response to ambient H 2 S exposure in brainstem are shown.(A) Increased metabolites compared to RA control group.(B) Decreased metabolites compared to RA control group.

Table 1 .
Significantly altered metabolites at 5 min post acute H 2 S exposure compared to RA control group.Common metabolite name, mass-to-charge ratio, fold change (log 2 scale), and statistical significance are listed.ANOVA with post hoc Tukey HSD test was performed for statistical significance.Asterisks indicate significant differences compared to RA group.p * < 0.1, ** < 0.05, *** < 0.01, and **** < 0.001.

Figure 5 .
Figure 5. Disease-based enrichment analysis of significantly altered metabolites of immediate response to H2S exposure (5 min post single acute H2S exposure) for primary metabolites, biogenic amines, and lipidomic metabolites.

Figure 5 .
Figure 5. Disease-based enrichment analysis of significantly altered metabolites of immediate response to H 2 S exposure (5 min post single acute H 2 S exposure) for primary metabolites, biogenic amines, and lipidomic metabolites.

Metabolites 2024 , 28 Figure 6 .
Figure 6.Disease-based enrichment analysis of significantly altered metabolites of early response 72 h post single acute H2S exposure for primary metabolites, biogenic amines, and lipidomic metabolites.

Figure 6 .
Figure 6.Disease-based enrichment analysis of significantly altered metabolites of early response 72 h post single acute H 2 S exposure for primary metabolites, biogenic amines, and lipidomic metabolites.

Figure 7 .
Figure 7. Disease-based enrichment analysis of significantly altered metabolites of subchronic response to repeated H2S exposure for 5 weeks for primary metabolites, biogenic amines, and lipidomic metabolites.

Figure 7 .
Figure 7. Disease-based enrichment analysis of significantly altered metabolites of subchronic response to repeated H 2 S exposure for 5 weeks for primary metabolites, biogenic amines, and lipidomic metabolites.

Metabolites 2024 , 28 Figure 8 .
Figure 8. Neurotransmitter changes following H2S exposure.(A) Fold change in neurotransmitters following acute 1000 ppm H2S exposure.(B) Fold change in neurotransmitters following subchronic ambient H2S exposure.H2S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H2S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H2S exposure groups to test for statistical significance.Asterisks indicate significant differences compared to RA control group.p * < 0.1, ** < 0.05, and **** < 0.001.

Figure 8 .
Figure 8. Neurotransmitter changes following H 2 S exposure.(A) Fold change in neurotransmitters following acute 1000 ppm H 2 S exposure.(B) Fold change in neurotransmitters following subchronic ambient H 2 S exposure.H 2 S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with hoc Tukey HSD test was performed for acute H 2 S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H 2 S exposure groups to test for statistical significance.Asterisks indicate significant differences compared to RA control group.p * < 0.1, ** < 0.05, and **** < 0.001.

Figure 8 .
Figure 8. Neurotransmitter changes following H2S exposure.(A) Fold change in neurotransmitters following acute 1000 ppm H2S exposure.(B) Fold change in neurotransmitters following subchronic ambient H2S exposure.H2S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H2S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H2S exposure groups to test for statistical significance.Asterisks indicate significant differences compared to RA control group.p * < 0.1, ** < 0.05, and **** < 0.001.

Figure 9 .
Figure 9. Altered aromatic amino acids following H2S exposure.(A) Fold change in aromatic amino acids following acute 1000 ppm H2S exposure.(B) Fold change in aromatic amino acids following subchronic ambient H2S exposure.H2S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H2S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H2S exposure groups to test for statistical significance.Asterisks indicate statistically significant differences compared to RA control group.p ** < 0.05 and *** < 0.01.

Figure 9 .
Figure 9. Altered aromatic amino acids following H 2 S exposure.(A) Fold change in aromatic amino acids following acute 1000 ppm H 2 S exposure.(B) Fold change in aromatic amino acids following subchronic ambient H 2 S exposure.H 2 S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H 2 S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H 2 S exposure groups to test for statistical significance.Asterisks indicate statistically significant differences compared to RA control group.p ** < 0.05 and *** < 0.01.

Figure 10 .
Figure 10.Fold change in branched-chain amino acids following H2S exposure.(A) Fold change in BCAAs following acute H2S exposure.(B) Fold change in BCAAs following subchronic ambient H2S exposure.H2S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H2S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H2S exposure groups for statistical significance.Asterisks indicate significant differences compared to RA control group.p * < 0.1, *** < 0.01, and **** < 0.001.

Figure 10 .
Figure 10.Fold change in branched-chain amino acids following H 2 S exposure.(A) Fold change in BCAAs following acute H 2 S exposure.(B) Fold change in BCAAs following subchronic ambient H 2 S exposure.H 2 S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H 2 S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H 2 S exposure groups for statistical significance.Asterisks indicate significant differences compared to RA control group.p * < 0.1, *** < 0.01, and **** < 0.001.Metabolites 2024, 14, x FOR PEER REVIEW 18 of 28

Figure 11 .
Figure 11.Altered fatty acids following H2S exposure.(A) Decreased behenic acid, a very long-chain fatty acid (VLCFA), following acute H2S exposure.(B) Major unsaturated fatty acids in brain were increased following subchronic ambient H2S exposure.H2S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H2S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H2S exposure groups for statistical significance.Asterisks indicate significant differences compared to RA control group.p ** < 0.05, *** < 0.01, and **** < 0.001.

Figure 11 .
Figure 11.Altered fatty acids following H 2 S exposure.(A) Decreased behenic acid, a very long-chain fatty acid (VLCFA), following acute H 2 S exposure.(B) Major unsaturated fatty acids in brain were increased following subchronic ambient H 2 S exposure.H 2 S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H 2 S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H 2 S exposure groups for statistical significance.Asterisks indicate significant differences compared to RA control group.p ** < 0.05, *** < 0.01, and **** < 0.001.

Figure 13 .
Figure 13.Altered inosine and metabolites following subchronic ambient H2S exposure.H2S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H2S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H2S exposure groups for statistical significance.Asterisks indicate significant differences compared to RA control group.p *** < 0.01 and **** < 0.001.

Figure 13 .
Figure 13.Altered inosine and metabolites following subchronic ambient H 2 S exposure.H 2 S groups were compared to RA control group.Values are presented as mean ± standard deviation.ANOVA with post hoc Tukey HSD test was performed for acute H 2 S exposure groups, while unpaired Student's t-test was performed for subchronic ambient H 2 S exposure groups for statistical significance.Asterisks indicate significant differences compared to RA control group.p *** < 0.01 and **** < 0.001.

Funding:
This research was funded by the Iowa state University internal funds for W.K.R. and start-up fundings by the University of California, Davis for W.K.R. Institutional Review Board Statement: This study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of California at Davis (UC Davis).The animal study protocol was approved by IACUC with IACUC-18-136 on 25 September 2018 at ISU and with IACUC-21819 on at UC Davis on 19 October 2020.Informed Consent Statement: Not applicable.

Table 2 .
Significantly altered metabolites following subchronic ambient H 2 S exposure compared to RA control group.Common metabolite name, mass-to-charge ratio, fold change (log 2 scale), and statistical significance are listed.Unpaired Student's t-test was performed for statistical significance.