Dietary administration of black raspberries modulates arsenic biotransformation and reduces urinary 8-oxo-2'-deoxyguanosine in mice.

Arsenic in drinking water is a worldwide public health problem due to its pathogenic induction of oxidative stress in various organ systems. Phytochemicals present in polyphenolic-rich fruits such as black raspberries (BRBs) have diverse health benefits, including antioxidation and modulation of enzymes in xenobiotic metabolism. We used a mouse model combined with a standardized BRB-rich diet to investigate the impact of BRB consumption on arsenic biotransformation. We observed a significant reduction of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) together with elevated levels of methylation and urinary excretion of arsenic in mice concurrently fed BRBs upon arsenic exposure. Moreover, enzyme expression and liver metabolites involved in arsenic metabolism were found to be different between mice on BRB and control diets with arsenic exposure. These data indicate that BRB consumption affected arsenic biotransformation in vivo likely via alterations in related metabolic enzymes and cofactors, providing evidence on reduction of arsenic toxicity by consumption of BRBs.

Effects of the Artificial Sweetener Neotame on the Gut Microbiome and Fecal Metabolites in Mice.

Although artificial sweeteners are widely used in food industry, their effects on human health remain a controversy. It is known that the gut microbiota plays a key role in human metabolism and recent studies indicated that some artificial sweeteners such as saccharin could perturb gut microbiome and further affect host health, such as inducing glucose intolerance. Neotame is a relatively new low-caloric and high-intensity artificial sweetener, approved by FDA in 2002. However, the specific effects of neotame on gut bacteria are still unknown. In this study, we combined high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS) metabolomics to investigate the effects of neotame on the gut microbiome and fecal metabolite profiles of CD-1 mice. We found that a four-week neotame consumption reduced the alpha-diversity and altered the beta-diversity of the gut microbiome. Firmicutes was largely decreased while Bacteroidetes was significantly increased. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis also indicated that the control mice and neotame-treated mice have different metabolic patterns and some key genes such as butyrate synthetic genes were decreased. Moreover, neotame consumption also changed the fecal metabolite profiles. Dramatically, the concentrations of multiple fatty acids, lipids as well as cholesterol in the feces of neotame-treated mice were consistently higher than controls. Other metabolites, such as malic acid and glyceric acid, however, were largely decreased. In conclusion, our study first explored the specific effects of neotame on mouse gut microbiota and the results may improve our understanding of the interaction between gut microbiome and neotame and how this interaction could influence the normal metabolism of host bodies.

Manganese-induced sex-specific gut microbiome perturbations in C57BL/6 mice.

Overexposure to manganese (Mn) leads to toxic effects, such as promoting the development of Parkinson's-like neurological disorders. The gut microbiome is deeply involved in immune development, host metabolism, and xenobiotics biotransformation, and significantly influences central nervous system (CNS) via the gut-brain axis, i.e. the biochemical signaling between the gastrointestinal tract and the CNS. However, it remains unclear whether Mn can affect the gut microbiome and its metabolic functions, particularly those linked to neurotoxicity. In addition, sex-specific effects of Mn have been reported, with no mechanism being identified yet. Recently, we have shown that the gut microbiome is largely different between males and females, raising the possibility that differential gut microbiome responses may contribute to sex-selective toxicity of Mn. Here, we applied high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS) metabolomics to explore how Mn2+ exposure affects the gut microbiome and its metabolism in C57BL/6 mice. Mn2+ exposure perturbed the gut bacterial compositions, functional genes and fecal metabolomes in a highly sex-specific manner. In particular, bacterial genes and/or key metabolites of neurotransmitter synthesis and pro-inflammatory mediators are significantly altered by Mn2+ exposure, which can potentially affect chemical signaling of gut-brain interactions. Likewise, functional genes involved in iron homeostasis, flagellar motility, quorum sensing, and Mn transportation/oxidation are also widely changed by Mn2+ exposure. Taken together, this study has demonstrated that Mn2+ exposure perturbs the gut microbiome and its metabolic functions, which highlights the potential role of the gut microbiome in Mn2+ toxicity, particularly its sex-specific toxic effects.

Multi-Omics Reveals that Lead Exposure Disturbs Gut Microbiome Development, Key Metabolites, and Metabolic Pathways.

Lead exposure remains a global public health issue, and the recent Flint water crisis has renewed public concern about lead toxicity. The toxicity of lead has been well established in a variety of systems and organs. The gut microbiome has been shown to be highly involved in many critical physiological processes, including food digestion, immune system development, and metabolic homeostasis. However, despite the key role of the gut microbiome in human health, the functional impact of lead exposure on the gut microbiome has not been studied. The aim of this study is to define gut microbiome toxicity induced by lead exposure in C57BL/6 mice using multiomics approaches, including 16S rRNA sequencing, whole genome metagenomics sequencing, and gas chromatography-mass spectrometry (GC-MS) metabolomics. 16S rRNA sequencing revealed that lead exposure altered the gut microbiome trajectory and phylogenetic diversity. Metagenomics sequencing and metabolomics profiling showed that numerous metabolic pathways, including vitamin E, bile acids, nitrogen metabolism, energy metabolism, oxidative stress, and the defense/detoxification mechanism, were significantly disturbed by lead exposure. These perturbed molecules and pathways may have important implications for lead toxicity in the host. Taken together, these results demonstrated that lead exposure not only altered the gut microbiome community structures/diversity but also greatly affected metabolic functions, leading to gut microbiome toxicity.

Nicotine Alters the Gut Microbiome and Metabolites of Gut-Brain Interactions in a Sex-Specific Manner.

As the primary active substance in tobacco, nicotine affects the activity of the central nervous system, and its effects are sex-dependent. There are complex interactions between the gut and brain, and the gut microbiome can influence neuronal activity and host behavior, with diverse chemical signaling being involved. However, it is unclear whether nicotine can affect the normal gut microbiome and associated chemical signaling of the gut-brain axis. Sex is an important factor that shapes the gut microbiome, but the role of sex in the interaction among nicotine, gut bacteria, and related metabolites remains unknown. In this study, we applied high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS) to explore how nicotine exposure affects the gut microbiome and its metabolism in female and male C57BL/6J mice, with a focus on the chemical signaling involved in gut-brain interactions. 16S sequencing results indicated that the community composition of the gut microbiome was differentially perturbed by nicotine in females and males. Differential alterations of bacterial carbohydrate metabolic pathways are consistent with lower body weight gain in nicotine-treated males. Oxidative stress response and DNA repair genes were also specifically enriched in the nicotine-treated male gut microbiome. The fecal metabolome indicated that multiple neurotransmitters, such as glutamate, gamma-aminobutyric acid (GABA), and glycine, were differentially altered in female and male mice. Some neuroactive metabolites, including leucine and uric acid, were also changed. This study demonstrates a sex-dependent effect of nicotine on gut microbiome community composition, functional bacterial genes, and the fecal metabolome.

Sex-Specific Effects of Arsenic Exposure on the Trajectory and Function of the Gut Microbiome.

The gut microbiome is deeply involved in numerous aspects of human health; however, it can be readily perturbed by environmental toxicants, such as arsenic. Meanwhile, the interaction among host, gut microbiome, and xenobiotics is a very complex dynamic process. Previously, we have demonstrated that gut microbiome phenotypes driven by host genetics and bacterial infection affect the responses to arsenic exposure. The role of host sex in shaping the gut microbiome raises the question whether sex plays a role in exposure-induced microbiome responses. To examine this, we used 16S rRNA sequencing and metagenomics sequencing to analyze the changes of the gut microbiome and its associated functional metagenome in both female and male C57/BL6 mice. Our results clearly demonstrated that arsenic exposure perturbed the trajectory and function of the gut microbiome in a sex-specific manner.

Chronic sucralose consumption inhibits farnesoid X receptor signaling and perturbs lipid and cholesterol homeostasis in the mouse livers, potentially by altering gut microbiota functions.

Sucralose has raised concerns regarding its safety and recent studies have demonstrated that sucralose consumption can disrupt the normal gut microbiome and alter metabolic profiles in mice. However, the extent to which this perturbation affects the functional interaction between the microbiota and the host, as well as its potential impact on host health, remains largely unexplored. Here, we aimed to investigate whether chronic sucralose consumption, at levels within the Acceptable Daily Intake (ADI), could disturb key gut microbial functions and lead to adverse health effects in mice. Following six-month sucralose consumption, several bacterial genera associated with bile acid metabolism were decreased, including Lactobacillus and Ruminococcus. Consequently, the richness of secondary bile acid biosynthetic pathway and bacterial bile salt hydrolase gene were decreased in the sucralose-treated gut microbiome. Compared to controls, sucralose-consuming mice exhibited significantly lower ratios of free bile acids and taurine-conjugated bile acids in their livers. Additionally, several farnesoid X receptor (FXR) agonists were decreased in sucralose-treated mice. This reduction in hepatic FXR activation was associated with altered expression of down-stream genes, in the liver. Moreover, the expression of key lipogenic genes was up-regulated in the livers of sucralose-treated mice. Changes in hepatic lipid profiles were also observed, characterized by lower ceramide levels, a decreased PC/PE ratio, and a mildly increase in lipid accumulation. Additionally, sucralose-consumed mice exhibited higher hepatic cholesterol level compared to control mice, with up-regulation of cholesterol efflux genes and down-regulation of genes associated with reverse cholesterol transport. In conclusion, chronic sucralose consumption disrupts FXR signaling activation and perturbs hepatic lipid and cholesterol homeostasis, potentially by diminishing the bile acid metabolic capacity of the gut microbiome. These findings shed light on the complex interplay between sucralose, the gut microbiota, and host metabolism, raising important questions about the safety of its long-term consumption.

A Black Raspberry-Rich Diet Protects From Dextran Sulfate Sodium-Induced Intestinal Inflammation and Host Metabolic Perturbation in Association With Increased Aryl Hydrocarbon Receptor Ligands in the Gut Microbiota of Mice.

Dietary modulation of the gut microbiota recently received considerable attention, and ligand activation of aryl hydrocarbon receptor (AHR) plays a pivotal role in intestinal immunity. Importantly, black raspberry (BRB, Rubus occidentalis) is associated with a variety of beneficial health effects. We aim to investigate effects of a BRB-rich diet on dextran sulfate sodium (DSS)-induced intestinal inflammation and to determine whether its consequent anti-inflammatory effects are relevant to modulation of the gut microbiota, especially its production of AHR ligands. A mouse model of DSS-induced intestinal inflammation was used in the present study. C57BL/6J mice were fed either AIN-76A or BRB diet. Composition and functions of the gut microbiota were assessed by 16S rRNA sequencing and comparative metagenome analysis. Metabolic profiles of host and the gut microbiome were assessed by serum and fecal metabolomic profiling and identification. BRB diet was found to ameliorate DSS-induced intestinal inflammation and host metabolic perturbation. BRB diet also protected from DSS-induced perturbation in diversity and composition in the gut microbiota. BRB diet promoted AHR ligand production by the gut microbiota, as revealed by increased levels of fecal AHR activity in addition to increased levels of two known AHR ligands, hemin and biliverdin. Accordingly, enrichment of bacterial genes and pathways responsible for production of hemin and biliverdin were found, specific gut bacteria that are highly correlated with abundances of hemin and biliverdin were also identified. BRB dietary intervention ameliorated intestinal inflammation in mice in association with promotion of AHR ligand production by the gut microbiota.

Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure.

Campylobacter jejuni is a significant cause of human gastroenteritis worldwide, and all strains express an N-glycan that is added to at least 80 different proteins. We characterized 98 C. jejuni isolates from infants from 7 low- and middle-income countries and identified 4 isolates unreactive with our N-glycan-specific antiserum that was raised against the C. jejuni heptasaccharide composed of GalNAc-GalNAc-GalNAc(Glc)-GalNAc-GalNAc-diNAcBac. Mass spectrometric analyses indicated these isolates express a hexasaccharide lacking the glucose branch. Although all 4 strains encode the PglI glucosyltransferase (GlcTF), one aspartate in the DXDD motif was missing, an alteration also present in ∼4% of all available PglI sequences. Deleting this residue from an active PglI resulted in a nonfunctional GlcTF when the protein glycosylation system was reconstituted in E. coli, while replacement with Glu/Ala was not deleterious. Molecular modeling proposed a mechanism for how the DXDD residues and the structure/length beyond the motif influence activity. Mouse vaccination with an E. coli strain expressing the full-length heptasaccharide produced N-glycan-specific antibodies and a corresponding reduction in Campylobacter colonization and weight loss following challenge. However, the antibodies did not recognize the hexasaccharide and were unable to opsonize C. jejuni isolates lacking glucose, suggesting this should be considered when designing N-glycan-based vaccines to prevent campylobacteriosis.

Effect of epicardial fat pad ablation on acute atrial electrical remodeling and inducibility of atrial fibrillation.

BACKGROUND: Atrial electrical remodeling (AER) is the underlying mechanism of atrial fibrillation (AF). The present study investigated the impact of epicardial fat pad (FP) ablation on acute AER (AAER) and inducibility of AF. METHODS AND RESULTS: AAER was performed in 28 mongrel dogs through 4-h rapid atrial pacing (RAP). Before RAP, 14 dogs (ablation group) underwent FP ablation, and the other 14 (control group) underwent a sham procedure. The atrial effective refractory period (ERP) and vulnerability window (VW) of AF were measured with and without bilateral cervical vagosympathetic nerve stimulation (VNS) at the high right atrium, ostium of the coronary sinus (CS) and distal CS before and after every hour of RAP. In the control group, ERP was markedly shortened in the first 2 h of RAP and then stabilized. AF was only slightly induced. After RAP, the time course of ERP with and without VNS was similar. VNS significantly shortened ERP and increased VW before and after RAP. In the ablation group, ERP was significantly prolonged after FP ablation. Moreover, neither VNS nor RAP shortened the ERP or increased the VW. AF could not be induced (VW=0). CONCLUSIONS: RAP resulted in AAER, which may be mediated and aggravated by autonomic activity. Epicardial FP ablation generated denervation, which not only abolishes AF inducibility but also prevents RAP-mediated AAER.

Metabolite Profiling of the Gut Microbiome in Mice with Dietary Administration of Black Raspberries.

Mounting evidence has linked gut microbiome to health benefits of various functional foods. We previously reported that administration of a diet rich in black raspberry (BRB) changed the composition and diverse functional pathways in the mouse gut microbiome. To further characterize the functional profile in the gut microbiome of mice on BRB diet, in this follow-up study, we examined the metabolome differences in the gut microbiome driven by BRB consumption via targeted and untargeted metabolomic approaches. A distinct metabolite profile was observed in the gut microbiome of the mice on BRB diet, likely resulting from a combination of microbiome functional changes and unique precursors in BRBs. A number of functional metabolites, such as tetrahydrobiopterin and butyrate that were significantly increased in the gut microbiome may be linked to the beneficial health effects of BRB consumption. These findings suggest the important role of the gut microbiome in the health effects of BRBs and provide a connection among the health benefits of functional foods and the gut microbiome.

Gut Microbiome Toxicity: Connecting the Environment and Gut Microbiome-Associated Diseases.

The human gut microbiome can be easily disturbed upon exposure to a range of toxic environmental agents. Environmentally induced perturbation in the gut microbiome is strongly associated with human disease risk. Functional gut microbiome alterations that may adversely influence human health is an increasingly appreciated mechanism by which environmental chemicals exert their toxic effects. In this review, we define the functional damage driven by environmental exposure in the gut microbiome as gut microbiome toxicity. The establishment of gut microbiome toxicity links the toxic effects of various environmental agents and microbiota-associated diseases, calling for more comprehensive toxicity evaluation with extended consideration of gut microbiome toxicity.

Campylobacter Abundance in Breastfed Infants and Identification of a New Species in the Global Enterics Multicenter Study.

Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide and is associated with high rates of mortality and growth stunting in children inhabiting low- to middle-resource countries. To better understand the impact of breastfeeding on Campylobacter infection in infants in sub-Saharan Africa and South Asia, we examined fecal microbial compositions, bacterial isolates, and their carbohydrate metabolic pathways in Campylobacter-positive infants <1 year of age from the Global Enterics Multicenter Study. Exclusively breastfed infants with diarrhea exhibited high Campylobacter abundances, and this negatively correlated with bacterial carbohydrate metabolism. Although C. jejuni and Campylobacter coli are prevalent among these infants, the second most abundant Campylobacter species was a new species, which we named "Candidatus Campylobacter infans." Asymptomatic Campylobacter carriers also possess significantly different proportions of specific gut microbes compared to diarrheal cases. These findings provide insight into Campylobacter infections in infants in sub-Saharan Africa and South Asia and help inform strategies aimed at eliminating campylobacteriosis in these areas.IMPORTANCECampylobacter is the primary cause of bacterial diarrhea in the United States and can lead to the development of the postinfectious autoimmune neuropathy known as Guillain-Barré syndrome. Also, drug-resistant campylobacters are becoming a serious concern both locally and abroad. In low- and middle-income countries (LMICs), infection with Campylobacter is linked to high rates of morbidity, growth stunting, and mortality in children, and breastfeeding is important for infant nutrition, development, and protection against infectious diseases. In this study, we examined the relationship between breastfeeding and Campylobacter infection and demonstrate the increased selection for C. jejuni and C. coli strains unable to metabolize fucose. We also identify a new Campylobacter species coinfecting these infants with a high prevalence in five of the seven countries in sub-Saharan Africa and South Asia examined. These findings indicate that more detailed studies are needed in LMICs to understand the Campylobacter infection process in order to devise a strategy for eliminating this pathogenic microbe.

The gastrointestinal pathogen Campylobacter jejuni metabolizes sugars with potential help from commensal Bacteroides vulgatus.

Although the gastrointestinal pathogen Campylobacter jejuni was considered asaccharolytic, >50% of sequenced isolates possess an operon for L-fucose utilization. In C. jejuni NCTC11168, this pathway confers L-fucose chemotaxis and competitive colonization advantages in the piglet diarrhea model, but the catabolic steps remain unknown. Here we solved the putative dehydrogenase structure, resembling FabG of Burkholderia multivorans. The C. jejuni enzyme, FucX, reduces L-fucose and D-arabinose in vitro and both sugars are catabolized by fuc-operon encoded enzymes. This enzyme alone confers chemotaxis to both sugars in a non-carbohydrate-utilizing C. jejuni strain. Although C. jejuni lacks fucosidases, the organism exhibits enhanced growth in vitro when co-cultured with Bacteroides vulgatus, suggesting scavenging may occur. Yet, when excess amino acids are available, C. jejuni prefers them to carbohydrates, indicating a metabolic hierarchy exists. Overall this study increases understanding of nutrient metabolism by this pathogen, and identifies interactions with other gut microbes.

Subchronic low-dose 2,4-D exposure changed plasma acylcarnitine levels and induced gut microbiome perturbations in mice.

The gut microbiota critically confers various health benefits, whereas environmental chemicals can affect its constitution and functionality thereby increasing disease risk. In the present study, we aim to evaluate the toxic effects of a wildly-used herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) on the gut microbiome and host using an occupationally relevant dose. A mouse model was used combined with metagenomic sequencing and metabolomic profiling to examine the alterations induced by subchronic low-dose 2,4-D exposure in fecal and plasma samples. The metagenomics results revealed a distinct gut microbial community with profound changes in diverse microbial pathways including urea degradation, amino acid and carbohydrate metabolism in 2,4-D-treated mice. Moreover, the metabolomics results revealed that the metabolic profiles in treatment group were differentiated from control group in both fecal and plasma samples. Toxic effects on the host of 2,4-D at an occupationally relevant dose were observed indicated by decreased acylcarnitine levels in plasma. These findings indicated that 2,4-D can cause toxicity and substantially impact the gut microbiome in mice at occupationally relevant doses, inferring that the relationship between environmental contaminants and microbiota is largely underestimated calling for more comprehensive consideration of the toxicity of occupational exposures.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Characterization of the Functional Changes in Mouse Gut Microbiome Associated with Increased Akkermansia muciniphila Population Modulated by Dietary Black Raspberries.

Gut microbiome plays an essential role in host health through host-gut microbiota metabolic interactions. Desirable modulation of beneficial gut bacteria, such as Akkermansia muciniphila, can confer health benefits by altering microbiome-related metabolic profiles. The purpose of this study is to examine the effects of a black raspberry-rich diet to reshape the gut microbiome by selectively boosting A. muciniphila population in C57BL/6J mice. Remarkable changes of the mouse gut microbiome were revealed at both compositional and functional levels with an expected increase of A. muciniphila in concert with a profound impact on multiple gut microbiome-related functions, including vitamin biosynthesis, aromatic amino acid metabolism, carbohydrate metabolism, and oxidative stress. These functional alterations in the gut microbiome by an easily accessed freeze-dried black raspberry-supplemented diet may provide novel insights on the improvement of human health via gut microbiome modulation.

Draft Genome Sequences of Nine Campylobacter hyointestinalis subsp. lawsonii Strains.

With increasing reports of Campylobacter hyointestinalis species associated with human diseases, more genome sequences are required to understand the virulence mechanisms of this emerging pathogen. Here, we describe the genome sequences of nine C. hyointestinalis subsp. lawsonii strains.

The organophosphate malathion disturbs gut microbiome development and the quorum-Sensing system.

The gut microbiome has tremendous potential to impact health and disease. Various environmental toxicants, including insecticides, have been shown to alter gut microbiome community structures. However, the mechanism that compositionally and functionally regulates gut microbiota remains unclear. Quorum sensing is known to modulate intra- and interspecies gene expression and coordinate population responses. It is unknown whether quorum sensing is disrupted when environmental toxicants cause perturbations in the gut microbiome community structure. To reveal the response of the quorum-sensing system to environmental exposure, we use a combination of Illumina-based 16S rRNA gene amplicon and shotgun metagenome sequencing to examine the impacts of a widely used organophosphate insecticide, malathion, on the gut microbiome trajectory, quorum sensing system and behaviors related to quorum sensing, such as motility and pathogenicity. Our results demonstrated that malathion perturbed the gut microbiome development, quorum sensing and quorum sensing related behaviors. These findings may provide a novel mechanistic understanding of the role of quorum-sensing in the gut microbiome toxicity of malathion.

Sex-Specific Effects of Organophosphate Diazinon on the Gut Microbiome and Its Metabolic Functions.

BACKGROUND: There is growing recognition of the significance of the gut microbiome to human health, and the association between a perturbed gut microbiome with human diseases has been established. Previous studies also show the role of environmental toxicants in perturbing the gut microbiome and its metabolic functions. The wide agricultural use of diazinon, an organophosphate insecticide, has raised serious environmental health concerns since it is a potent neurotoxicant. With studies demonstrating the presence of a microbiome-gut-brain axis, it is possible that gut microbiome perturbation may also contribute to diazinon toxicity. OBJECTIVES: We investigated the impact of diazinon exposure on the gut microbiome composition and its metabolic functions in C57BL/6 mice. METHODS: We used a combination of 16S rRNA gene sequencing, metagenomics sequencing, and mass spectrometry-based metabolomics profiling in a mouse model to examine the functional impact of diazinon on the gut microbiome. RESULTS: 16S rRNA gene sequencing revealed that diazinon exposure significantly perturbed the gut microbiome, and metagenomic sequencing found that diazinon exposure altered the functional metagenome. Moreover, metabolomics profiling revealed an altered metabolic profile arising from exposure. Of particular significance, these changes were more pronounced for male mice than for female mice. CONCLUSIONS: Diazinon exposure perturbed the gut microbiome community structure, functional metagenome, and associated metabolic profiles in a sex-specific manner. These findings may provide novel insights regarding perturbations of the gut microbiome and its functions as a potential new mechanism contributing to diazinon neurotoxicity and, in particular, its sex-selective effects. Citation: Gao B, Bian X, Mahbub R, Lu K. 2017. Sex-specific effects of organophosphate diazinon on the gut microbiome and its metabolic functions. Environ Health Perspect 125:198-206; http://dx.doi.org/10.1289/EHP202.