Nicotine Alters the Gut Microbiome and Metabolites of GutâBrain

Article pubs.acs.org/crt

Nicotine Alters the Gut Microbiome and Metabolites of Gut−Brain Interactions in a Sex-Specific Manner Liang Chi,† Ridwan Mahbub,‡ Bei Gao,† Xiaoming Bian,† Pengcheng Tu,† Hongyu Ru,§ and Kun Lu*,† †

Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ‡ Department of Environmental Health Science, University of Georgia, Athens, Georgia 30602, United States § Department of Population Health and Pathobiology, North Carolina State University, Raleigh, North Carolina 27606, United States S Supporting Information *

ABSTRACT: 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 sexdependent effect of nicotine on gut microbiome community composition, functional bacterial genes, and the fecal metabolome.

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INTRODUCTION As the primary active substance in tobacco, nicotine is primarily delivered to the lung and fast absorbed through the alveoli but can also be absorbed through the skin and gastrointestinal tract, especially for those who use smokeless tobacco products like skin patches and chewing tobacco.1−3 Nicotine is known to have multiple positive and negative psychological and physiological effects on the human body including improved metabolic rate, influence on appetite, and regulation of body weight.4−6 The interactions between nicotine and the nervous system are of great interest because nicotine can easily pass the blood−brain barrier and influence neural activities to modify human behavior.7 It is known that nicotine can trigger the release of dopamine in the brain, and thus, it is highly related to the reward system.8,9 In addition, nicotine can modulate the levels of multiple neurotransmitters, such as serotonin, norepinephrine, gamma-aminobutyric acid (GABA) and glutamic acid, which can further influence human cognition, learning, and memory.10−12 Microbiota in the gastrointestinal tract play a critical role in food fiber digestion, xenobiotic biotransformation, and immune system development.13,14 In recent years, the gut−brain axis has attracted great interest, and accumulating evidence indicates that gut microbiota deeply influence central nervous system © 2017 American Chemical Society

(CNS) activities and host behavior, with chemical signaling of the gut−brain axis being involved. Animals with neuron diseases such as depression and anxiety are generally associated with gut bacteria alterations, while oral administration of Lactobacillus rhamnosus could alter GABA receptor expression in key CNS stress-related brain regions and influence anxietylike behaviors in mice.15 Bacterially derived products, especially neurotransmitters, inflammatory-promoting mediators and neuroactive metabolites, serve as key chemical signals of the gut−brain axis16−18 and play an important role in cross-talk between bacteria and the CNS.19 However, the gut microbiome is highly dynamic and can also be influenced by a series of environmental factors such as antibiotics and heavy metals.20−23 The perturbation of commensal gut microbiota can influence normal gut−brain communication and change CNS activities and host behaviors. For example, antimicrobial-induced gut bacteria perturbation increased the hippocampal expression of brain-derived neurotropic factors and affected the exploratory behavior of mice.24 Administration of some antibiotics administration has been found to alter animal behavior, presumably by disturbing the normal interactions of gut Received: June 8, 2017 Published: October 16, 2017 2110

DOI: 10.1021/acs.chemrestox.7b00162 Chem. Res. Toxicol. 2017, 30, 2110−2119

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Chemical Research in Toxicology bacteria and the nervous system.25 However, it is unclear whether oral administration of nicotine can disturb the gut microbiome and whether nicotine exposure can influence chemical signaling involved in gut−brain interactions. Previous studies have revealed that the effects of nicotine are sex-dependent.26 For example, early studies indicated that male animals were more sensitive than females to the nicotineinduced suppression of Y-maze activity and the discriminative stimulus effects of nicotine, while females generally showed more responsiveness to the locomotor-activating effects of nicotine than males.9,27−29 Animal studies also demonstrated that nicotine differentially regulated nicotinic acetylcholine receptors in female and male rat brains,30 which are related to its sex-specific effect on the learning abilities of rats.31 Meanwhile, sex is also an important factor that determines interactions between gut bacteria and exposure to xenobiotics.32 Our previous research demonstrated that exposure to arsenic and organophosphate can differentially disturb the normal bacterial community structures of male and female mice.33,34 However, the potential role of sex on the cross-talk between nicotine and gut bacteria is unexplored. In the present study, we analyzed the effects of 3-month oral administration of nicotine exposure on the gut microbiome and its metabolic profiles in female and male C57BL/6 mice. Both 16S rRNA gene sequencing and metagenomics sequencing have been applied to measure nicotine-induced bacterial compositional changes and functional gene alterations of the gut microbiome, respectively. We applied gas chromatography−mass spectrometry (GC−MS) to investigate how the metabolomics profiles of gut bacteria are affected in nicotineexposed mice. Our present research characterizes the sexspecific effects of nicotine exposure on the gut microbiome for the first time and highlights nicotine’s effects on altering chemical signaling implicated in the gut bacteria−brain interactions.

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BIO Laboratories, Carlsbad, CA) according to the manufacturer’s instructions. The resultant DNA was quantified by Nanodrop and stored at −80 °C until further analysis. Purified DNA (1 ng) was used to amplify the V4 region of 16S rRNA from bacteria using the universal primers 515 (5′-GTGCCAGCMGCCGCGGTAA) and 806 (5′-GGACTACHVGGGTWTCTAAT). The resultant DNA products were barcoded and quantified with a Qubit 2.0 Fluorometer using a Qubit dsDNA HS Assay kit (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions and were pooled for sequencing. Sequencing was performed on an Illumina Miseq at the Georgia Genomics Facility to generate pair-end 250 × 250 (PE250, v2 kit) reads. The raw mate-paired fastq files were merged and qualityfiltered using Geneious 8.0.5 (Biomatters, Auckland, New Zealand) with the error probability limit set as 0.01. The data were analyzed using quantitative insights into microbial ecology (QIIME, version 1.9.1). UCLUST was used to obtain operational taxonomic units (OTUs) with 97% sequence similarity. The data were assigned at five different levels: phylum, class, order, family, and genus. Analysis of Metagenomics Data. For metagenomics sequencing, DNA (10 ng/μL) from individual mouse was fragmented using the Bioruptor UCD-300 sonication device, followed by sequencing library construction using the Kapa Hyper Prep Kit according to the manufacturer’s instructions. The resulting DNA was pooled, quantified, and sequenced at the Georgia Genomics Facility using an Illumina NextSeq High Output Flow Cell. The raw fastq files were imported into the MG-RAST metagenomics analysis server (version 3.5) with MG-RAST ID: Control (male), 4616834.3, 4616844.3, 4616856.3, 4616870.3, and 4616877.3; Nicotine treatment (male), 4616833.3, 4616845.3, 4616851.3, 4616860.3, and 4616867.3; Control (female), 4616838.3, 4616849.3, 4616854.3, 4616861.3, and 4616876.3; Nicotine treatment (female), 4616829.3, 4616853.3, 4616859.3, 4616862.3, and 4616874.3.36 Sequences were assigned to the M5NR Subsystems database for functional analysis with a maximum e-value cutoff 10−5, 75% minimum identity cutoff, and minimum alignment length cutoff of 35. Gas Chromatography−Mass Spectrometry Metabolomics Profiling. Metabolites were extracted from fecal samples of individual mouse using methanol and chloroform as described previously.21 Briefly, 20 mg of feces was vortexed with 1 mL of a methanol/ chloroform/water solution (2:2:1) for 1 h, followed by centrifugation at 3200g for 15 min. The resultant upper phase and lower phase were transferred to an HPLC vial, dried for approximately 4 h in a SpeedVac, and derivatized using N,O bis(trimethylsilyl)trifluoroacetamide (BSTFA). The derivatized samples were analyzed using an Agilent Technologies 6890N Network GC System/5973 Mass-Selective Detector (Agilent Technologies, Santa Clara, CA) with an Agilent J&W GC column (30 m length; 0.250 mm diameter (narrow bore); film thickness 0.25 μm) (Agilent Technologies, Santa Clara, CA) under the following conditions: initial oven temperature was set at 60 °C for 2 min, ramped to 320 °C at 8 °C/min, and held at 320 °C for 10.5 min. Two microliters of sample solution were injected with helium as the carrier gas at a flow rate of 0.8 mL/min. The temperature of the injector, ion source, and MS Quadrupole were set at 275 °C, 230 °C, and 150 °C, respectively. The mass spectrometer was operated in full scan mode from 50−600 m/z. The XCMS Online tool was used to pick up and align peaks and calculate the accumulated peak intensity. To identify the metabolite represented by a particular feature, retention time and m/z data from the XCMS Online output were used to filter the total ion chromatogram. The compounds were identified after matching with the NIST MS database. Statistical Analysis. Principal coordinate analysis (PCoA) was used to compare the gut microbiome profiles between the control and treatments, while a nonparametric t test was conducted by Metastats that is integrated with Mothur software37 to determine statistically significant changes (p < 0.05) to the gut-microbial community composition between treatments and the control, as previously described.38 To generate differences in the metabolic profiles of the control and nicotine group, a two-tail Welch’s t test (p < 0.05) was used.

MATERIALS AND METHODS

Animals and Nicotine Exposure. Seven-week-old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were housed in the University of Georgia animal facility for a week before exposure and throughout the experiment in static microisolator cages with Bed-O-Cob combination bedding under environmental conditions of 22 °C, 40− 70% humidity, and a 12:12 h light/dark cycle. Before experimentation, all mice consumed tap water ad libitum and were provided with standard pelleted rodent diet before and during experimentation. During the experimental period, mice were weighed (initial male body weight = 23.8 ± 1.4 g; initial female body weight = 18.3 ± 1.15 g; n = 10) and randomly assigned into either the control group or a 60 mg/L nicotine treatment group (n = 10). Mice were weighed at the end of the experiment before necropsy. The animals were treated humanely, and every effort was made to alleviate suffering. The animal protocol was approved by the University of Georgia Institutional Animal Care and Use Committee. At the start of the experiment, 98% nicotine (Pfaltz and Bauer, Inc., Waterbury, CT) was diluted in tap water and administered to mice (∼8 weeks of age) in their drinking water bottles for 13 weeks; the mice were allowed to consume this water ad libitum. A concentration of 60 mg/L was previously found to produce a steadystate nicotine plasma concentration in mice, comparable to that observed in chronic cigarette smokers.35 Drinking water with nicotine was prepared fresh once a week. Control mice (∼8 weeks of age) continued to receive tap water in their drinking water bottles, which they consumed ad libitum. 16S rRNA Sequencing. Mouse fecal pellets from individual mouse were collected for 16S rRNA analysis at 13 weeks and stored under dry ice before being transferred to −80 °C until further analysis. DNA was isolated from fecal pellets using a PowerSoil DNA Isolation Kit (MO 2111


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Figure 1. (A) Unweighted UniFrac-based PCoA analysis of gut microbiome (beta diversity) via 16S rRNA sequencing. Gut microbiome compositions in males and females were significantly altered after nicotine exposure. (B) Nicotine exposure differentially altered gut microbiota at family level (F, female; M, male; Family 1, an unassigned family in order Bacillales; Family 2, Christensenellaceae; Family 3, Mogibacteriaceae; Family 4, F16; Family 5, Anaeroplasmataceae; Family 6, an unassigned family in order RF39; Family 7, Turicibacteraceae; Family 8, Dehalobacteriaceae; Family 9, Peptococcaceae; ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, N.S. p > 0.05).

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RESULTS

nicotine-induced sex-dependent changes in gut bacteria, with several bacterial components decreased and increased for female and male mice, respectively. For example, in treated female mice, Christensenellaceae, Anaeroplasmataceae, F16, and unassigned families in the orders Bacillales and RF39 were significantly reduced. Families such as F16, Turicibacteraceae, and Peptococcaceae largely increased in male animals under nicotine treatment, while Dehalobacteriaceae was decreased. Nicotine Exposure Perturbed Carbohydrate Metabolism Pathways of Gut Microbiome and Specifically Decreased Body Weight Gain in Male Mice. There was no statistically significant difference in the baseline body weight between control groups and nicotine-treated groups for either male or female mice. After 13 weeks, nicotine-treated male mice

Nicotine Altered Gut Microbiome of Mice in SexSpecific Manner. We first examined the effects of nicotine on the mouse gut microbiome profiles. The unweighted UniFracbased PCoA analysis (Figure 1A) shows that the gut microbiome in nicotine-treated mice was clearly distinct from that of control mice. Significant differences between male and female mice were evident regardless of treatment, highlighting the role of sex in shaping the gut microbiome. Nicotine-treated samples cluster more closely than the corresponding controls, also suggesting the effects of nicotine on perturbing gut microbiome community. As shown in Figure 1B, changes in the taxonomic compositions of gut bacteria communities indicate 2112


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Figure 2. (A) Nicotine exposure significantly reduced body weight gain in male mice, but not female mice; (B) metagenomics data indicated that short-chain fatty acid biosynthesis genes differentially changed in female and male mice with nicotine exposure. (F, female; M, male; ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, N.S. p > 0.05).

showed much lower body weight gain than controls (p < 0.05), as seen in Figure 2A. No significant difference was observed in female mice. In addition, sequencing data indicated that multiple carbohydrate metabolism pathways were significantly altered in nicotine-treated female and male mice, as shown in Figures S1 and S2. Specifically, we found that acetate synthesis genes, including acetate kinase and phosphate acetyltransferase, were significantly increased in nicotine-treated male mice, while butyrate synthesis genes such as butyrate kinase, 3-hydroxybutyryl-CoA dehydratase, and acetoacetyl-CoA reductase increased in females (Figure 2B). These results highlight a sex-specific effect of nicotine exposure on the carbohydrate metabolism of the gut microbiome as well as on body weight gain in the host. Nicotine Exposure Specifically Enriched Oxidative Stress Response and DNA Repair Genes in Male Mice. In addition to carbohydrate metabolism-related genes, we also found that multiple oxidative stress response genes increased in male animals exposed to nicotine (Figure 3A) according to metagenomics sequencing data. Superoxide dismutase (SOD) is an important antioxidant enzyme that can transform highly toxic superoxide (O2−) to relatively less toxic hydrogen peroxide (H2O2).39 Likewise, hydroxyacyl-glutathione hydrolase, known as glyoxalase II, is a component of the glyoxalase system, another antioxidant system related to GSH metabolism.40 Here, manganese superoxide dismutase and the gene for glyoxalase II were significantly increased in nicotine-exposed male mice. Other genes related to oxidative stress response including gamma-glutamyl transpeptidase, redox-sensitive transcriptional regulator, and transcriptional regulator (Crp/Fnr family) were also increased (Figure 3A). However, no oxidative

stress response-related genes were significantly altered in female nicotine-treated mice. Oxidative stress is generally associated with DNA damage. Corresponding to the widespread increase in oxidative stress response genes, metagenomics data showed that multiple DNA repair-related genes were significantly increased in nicotineexposed male mice. Genes including error-prone repair protein UmuD, excinuclease ABC subunit A, uracil-DNA glycosylase (family 1), DNA recombination protein RmuC, and G:T/U mismatch-specific uracil/thymine DNA-glycosylase were significantly increased, as shown in Figure 3B. The specific enrichment of oxidative stress response and DNA repair genes indicated that nicotine exposure induced differential effects on the gut microbiome in female and male mice. Nicotine Exposure Perturbed the Fecal Metabolome. In addition to the perturbation of the gut bacteria community components and functional pathways, we also found that the fecal metabolome was also significantly altered, as shown in Figure 4A and C. The change patterns differ in male and female mice, with most metabolites up-regulated in males. Partial leastsquares-discriminant analysis (PLS-DA) analysis also showed that the fecal metabolomes of nicotine-exposed mice were clearly separated from control mice (Figure 4B,D). Many perturbed metabolites, with diverse structures and biological functions, were identified. Dramatically, we found the abundances of multiple amino acids, which function as neurotransmitters or are precursors of neurotransmitter synthesis, were differentially altered in female and male fecal samples by nicotine treatment (Figure 5). In nicotine-exposed female mice, phenylalanine, tyrosine, glutamic acid, GABA, serine, and glycine significantly increased. For male mice, 2113


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Figure 3. Metagenomics sequencing indicated that nicotine exposure significantly enriched (A) oxidative stress response genes and (B) DNA repair genes in the gut microbiome of male mice. (F, female; M, male; ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, N.S. p > 0.05).

differential alterations of multiple carbohydrate degradation and fermentation pathways, nicotine exposure reduced body weight gain in male mice but not female mice. Moreover, oxidative stress response genes and DNA repair genes were widely and specifically enriched in nicotine-exposed males. Fecal metabolomics data reveal that the concentrations of multiple neurotransmitters and metabolites with neuron regulation functions, such as glutamic acid, GABA, glycine, uric acid, and xanthurenic acid, were differentially altered in female and male mice. In conclusion, this study demonstrated that oral administration of nicotine caused different effects on the gut microbiome in female and male mice; these effects perturbed the metabolites that play a role in gut−brain communication. The present study first characterizes sex-specific effects of nicotine exposure on the gut microbiome and highlights the effect of nicotine on altering chemical signaling of gut−brain interactions. These data may point to a potential role of the gut microbiome in sex-specific physiological effects of nicotine. It is well documented that nicotine exposure negatively affects body weight gain in humans and animals.4 Previous studies found that the acute thermogenic effect of nicotine was more pronounced in male smokers than females, and another study found smoking cessation caused persistence of body weight gain in females but not males, suggesting that the negative effect of nicotine on body weight gain was more distinct in males.47,48 In this study, we also found that nicotine

phenylalanine, tyrosine, glutamic acid, and GABA increased under nicotine exposure, whereas glycine, serine, and aspartic acid significantly decreased (Figure 5). In addition, we observed that some metabolites that play a role in regulating neuronal activity were also altered by nicotine exposure, as shown in Figure 6. For example, pyroglutamic acid was increased in nicotine-treated animals, with a stronger effect observed in male mice. Likewise, leucine, 2-ketoleucine, uric acid, and xanthurenic acid increased in nicotine-treated male mice only.

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DISCUSSION It is well documented that the normal community structures and functions of gut microbiome can be perturbed by a series of xenobiotics such as antibiotics, heavy metals, and pesticides.41 Xenobiotic-induced dysbiosis of the gut microbiome is generally associated with adverse health effects or diseases.41 For example, a previous study found that 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) exposure increased the level of Firmicutes and decreased the level of Bacteroidetes, which was correlated with liver and immune toxicity.42 Likewise, imazalil exposure caused severe gut microbiome shift and induced gut inflammation.43 Exposure to heavy metals also affected the gut microbiome and host health.41,44−46 In this study, we found that oral nicotine administration can change the normal composition, functional pathways, and fecal metabolite profiles of mouse gut microbiome in a sex-specific manner. With 2114


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Figure 4. Nicotine exposure perturbed the fecal metabolic profiles of female and male mice, as evidenced by a number of regulated metabolites in the cloud plots (A, female; C, male) and partial least-squares discriminant analysis (PLS-DA) plots (B, female; D, male).

Figure 5. Nicotine exposure significantly and differentially altered the levels of multiple neurotransmitters in fecal samples of female and male mice (F, female; M, male; ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, N.S. p > 0.05).

induced significantly lower body weight gain in male but not female mice, clearly indicating a sex-specific effect of nicotine on the metabolism of animals. Correspondingly, dramatically sex-specific effects of nicotine exposure on gut microbiome also

have been observed. As is known, gut bacteria play a critical role in host energy supplement and regulation that different perturbations of gut microbiome may differently alter the energy homoestasis in host body.49−51 Some potential links can 2115


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Figure 6. Nicotine exposure altered the abundances of metabolites that play a role in regulating the neuron activity (F, female; M, male; ∗∗p < 0.01, ∗p < 0.05, N.S. p > 0.05).

5). Unlike glutamate, GABA functions as the principal inhibitory neurotransmitter in the human brain and plays a key role in controlling the central reward system, which can be disrupted by the activation of GABA neurons.59 A previous study found that GABA level could be altered by nicotine exposure.60 Moreover, it is interesting to ask what causes the elevated GABA levels. An early study demonstrated that GABA could be synthesized by gut bacteria and was directly associated with the level of GABA in plasma.61 Some bacteria of the genus Pseudomonas and Arthrobacter can degrade nicotine into GABA.62 Considering the huge pool of genes in the gut bacteria, it is possible that some species in the gut can utilize nicotine to produce GABA. In this case, degradation by gut bacteria could reduce nicotine bioaccessibility to the host and the differential biotransformation capabilities of gut bacteria in the host may contribute to different sensitivity to nicotine exposure. As this study mainly focused on the changes of chemical signals in the gut, future study is warranted to examine whether and how neurotransmitters in the gut may affect their homeostasis in the brain. Other important neurotransmitters such as glycine, serine, and aspartic acid decreased in male animals only under nicotine exposure (Figure 5). Aspartic acid is structurally similar to glutamate and can function as a weak neurotransmitter to activate glutamate receptors.63 Glycine is the coagonist of Nmethyl-D-aspartate (NMDA) receptors.64 Previous studies found that the inhibitor of glycine transporter-1, which modulates glycine-binding to the NMDA receptor, can influence the activation of NMDA receptors in dopamine neurons.65 Serine has similar functions to glycine.66 Thus, specific decreases in glycine, serine, and aspartic acid in males might influence their levels in the host and weaken the nicotine-induced activation of glutamate receptors. Interestingly, a number of studies indicated that it is more difficult for

be found between the sex-specific alterations of gut microbiota and body weight gain. For example, we found that Christensenellaceae was significantly decreased in treated females but moderately increased in males. Christensenellaceae was enriched in individuals with low body mass index.52 Moreover, butyrate has been considered as the major energy source of intestinal epithelial cells,53 and we found its synthesis genes were enriched in female mice (Figure 2B). However, acetate, the synthesis genes of which were enriched in male mice, can suppress appetite, and a previous study clearly demonstrated that increased acetate in the colon could improve brain acetate levels and reduce appetite.54 Therefore, differential perturbations of the gut microbiome and their functional genes by nicotine oral administration may partly contribute to nicotineinduced sex-specific regulation of host body weight gain. Producing neurotransmitters and their precursors is a major approach for gut microbiota to influence brain activity.55 In this study, we found that nicotine exposure changed the levels of multiple neurotransmitters in fecal samples, which may alter the pool of those neurotransmitters in hosts. Tyrosine and phenylalanine are dopamine precursors that can be converted to dopamine in the brain. Previous studies found that large doses of tyrosine and phenylalanine could improve dopamine synthesis, while dietary tyrosine and phenylalanine depletion reduce dopamine in the brain.56,57 Nicotine can induce dopamine release and increase extracellular dopamine to activate the reward system in the brain, which plays a key role in addiction.8,9 Likewise, glutamate increased significantly in nicotineexposed animals (Figure 5); glutamate is the main excitatory neurotransmitter in the brain and has been found to be deeply involved in the nicotine-induced brain reward system activation.58 Interestingly, we also detected highly increased GABA in the fecal samples of nicotine-treated animals (Figure 2116


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females to quit smoking, and self-administration studies found that females had higher motivation to consume more nicotine.9 The differential alteration of neurotransmitters between female and male mice might partially contribute to sex-specific dependence on nicotine. In addition to neurotransmitters, some metabolites that have important regulation functions in nervous systems are altered in the fecal samples of nicotine-treated mice (Figure 6). Pyroglutamic acid can cross the blood−brain barrier, and orally administered pyroglutamic acid can accumulate in high concentrations in the brain.67 Pyroglutamic acid can inhibit glutamate uptake by the synaptosome and induce neuron damage in brain.68 Besides, previous studies found that the transformation between branched-chain amino acids and 2-keto derivatives are very important to the homeostasis of glutamate metabolism in CNS.69,70 Nicotine-induced perturbation of leucine and 2-ketoleucine (Figure 6) might affect the balance of glutamate metabolism in males. In addition, we observed that xanthurenic acid and uric acid significantly increased in the fecal samples of nicotine-treated males, with no significant alteration in females (Figure 6). Xanthurenic acid is a product of the kynurenine pathway of tryptophan degradation; recent research found it has a neurotransmitter-like function and can activate group II metabotropic glutamate receptors.71,72 Uric acid can protect neurons from glutamate toxicity by reducing oxidative stress, which is beneficial for neurodegeneration diseases.73,74 The nicotine-induced differential alteration of neuroactive compounds, such as uric acid and xanthurenic acid, in female and male mice (Figure 6) may also contribute to sex-specific effects of nicotine.

Kun Lu: 0000-0002-8125-2394 Funding

This work was partially supported by the University of Georgia, University of North Carolina at Chapel Hill and the National Institute of Health/National Institute of Environmental Health Sciences (Grant No. R01ES024950). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS PCoA, principle coordinate analysis; GABA, gamma-aminobutyric acid; CNS, central nervous system; PLS-DA, partial least-squares-discriminant analysis; NMDA, N-methyl-D-aspartate

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(1) Schievelbein, H., Eberhardt, R., Löschenkohl, K., Rahlfs, V., and Bedall, F. K. (1973) Absorption of nicotine through the oral mucosa I. Measurement of nicotine concentration in the blood after application of nicotine and total particulate matter. Agents Actions 3, 254−258. (2) Chaturvedi, P., Mishra, A., Datta, S., Sinukumar, S., Joshi, P., and Garg, A. (2015) Harmful effects of nicotine. Indian J. Med. Paediatr. Oncol. 36, 24−31. (3) Jensen, K., Afroze, S., Munshi, M. K., Guerrier, M., and Glaser, S. S. (2012) Mechanisms for nicotine in the development and progression of gastrointestinal cancers. Transl. Gastrointest. Cancer 1, 81−87. (4) Yildiz, D. (2004) Nicotine, its metabolism and an overview of its biological effects. Toxicon 43, 619−632. (5) Grunberg, N. E., Bowen, D. J., and Winders, S. E. (1986) Effects of nicotine on body weight and food consumption in female rats. Psychopharmacology (Berl.) 90, 101−105. (6) Levin, E. D., McClernon, F. J., and Rezvani, A. H. (2006) Nicotinic effects on cognitive function: behavioral characterization, pharmacological specification, and anatomic localization. Psychopharmacology (Berl.) 184, 523−539. (7) Rezvani, A. H., and Levin, E. D. (2001) Cognitive effects of nicotine. Biol. Psychiatry 49, 258−267. (8) Di Chiara, G. (2000) Role of dopamine in the behavioural actions of nicotine related to addiction. Eur. J. Pharmacol. 393, 295−314. (9) Pogun, S., and Yararbas, G. (2009) Sex Differences in Nicotine Action, in Nicotine Psychopharmacology, Henningfield, J. E., London, E. D., and Pogun, S., Eds., pp 261−291, Springer, Berlin Heidelberg. (10) Yu, Z. J., and Wecker, L. (1994) Chronic nicotine administration differentially affects neurotransmitter release from rat striatal slices. J. Neurochem. 63, 186−194. (11) Toth, E., Sershen, H., Hashim, A., Vizi, E., and Lajtha, A. (1992) Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions: role of glutamic acid. Neurochem. Res. 17, 265−271. (12) Singer, S., Rossi, S., Verzosa, S., Hashim, A., Lonow, R., Cooper, T., Sershen, H., and Lajtha, A. (2004) Nicotine-induced changes in neurotransmitter levels in brain areas associated with cognitive function. Neurochem. Res. 29, 1779−1792. (13) Bäckhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A., and Gordon, J. I. (2005) Host-bacterial mutualism in the human intestine. Science 307, 1915−1920. (14) Guarner, F., and Malagelada, J.-R. (2003) Gut flora in health and disease. Lancet 361, 512−519. (15) Bravo, J. A., Forsythe, P., Chew, M. V., Escaravage, E., Savignac, H. M., Dinan, T. G., Bienenstock, J., and Cryan, J. F. (2011) Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc. Natl. Acad. Sci. U. S. A. 108, 16050−16055.

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CONCLUSIONS Taken together, we show that nicotine can induce differential alterations in gut bacteria including bacterial community compositions, functional genes, and the metabolome in female and male mice. These perturbations influence the chemical signaling of gut−brain interactions and may further mediate the effects of nicotine on the nervous system. Future studies are warranted to delineate more detailed molecular and signaling pathways involved in nicotine−microbiome−brain interactions. In addition, our study is limited by the focus to define the effect of nicotine on the gut microbiome and its metabolic functions. Future studies are needed to examine the disruption of key metabolites in the host, especially the brain, and also to demonstrate the causative role of the gut microbiome perturbation in mediating the chemical signaling of gut−brain interactions. Another limitation of this study comes from the exposure route difference between human inhalation exposure and oral nicotine administration used herein.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00162. Significantly altered carbohydrate metabolism pathways of nicotine-treated female and male mice (PDF)

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REFERENCES

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Chemical Research in Toxicology (16) Sampson, T. R., and Mazmanian, S. K. (2015) Control of brain development, function, and behavior by the microbiome. Cell Host Microbe 17, 565−576. (17) De Vadder, F., Kovatcheva-Datchary, P., Goncalves, D., Vinera, J., Zitoun, C., Duchampt, A., Bäckhed, F., and Mithieux, G. (2014) Microbiota-generated metabolites promote metabolic benefits via gut− brain neural circuits. Cell 156, 84−96. (18) O’Mahony, S., Clarke, G., Borre, Y., Dinan, T., and Cryan, J. (2015) Serotonin, tryptophan metabolism and the brain-gut-microbiome axis. Behav. Brain Res. 277, 32−48. (19) Cryan, J. F., and Dinan, T. G. (2012) Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat. Rev. Neurosci. 13, 701−712. (20) Lu, K., Mahbub, R., Cable, P. H., Ru, H., Parry, N. M., Bodnar, W. M., Wishnok, J. S., Styblo, M., Swenberg, J. A., Fox, J. G., et al. (2014) Gut microbiome phenotypes driven by host genetics affect arsenic metabolism. Chem. Res. Toxicol. 27, 172−174. (21) Lu, K., Abo, R. P., Schlieper, K. A., Graffam, M. E., Levine, S., Wishnok, J. S., Swenberg, J. A., Tannenbaum, S. R., and Fox, J. G. (2014) Arsenic exposure perturbs the gut microbiome and its metabolic profile in mice: an integrated metagenomics and metabolomics analysis. Environ. Health Perspect. 122, 284−291. (22) Hopkins, M., Sharp, R., and Macfarlane, G. (2002) Variation in human intestinal microbiota with age. Dig. Liver Dis. 34, S12−S18. (23) Turnbaugh, P. J., Ridaura, V. K., Faith, J. J., Rey, F. E., Knight, R., and Gordon, J. I. (2009) The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci. Transl. Med. 1, 6ra14−16ra14. (24) Bercik, P., Denou, E., Collins, J., Jackson, W., Lu, J., Jury, J., Deng, Y., Blennerhassett, P., Macri, J., McCoy, K. D., et al. (2011) The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology 141, 599−609. (25) Cryan, J. F., and O’Mahony, S. (2011) The microbiome-gut− brain axis: from bowel to behavior. Neurogastroenterol. Motil. 23, 187− 192. (26) Perkins, K. A., Donny, E., and Caggiula, A. R. (1999) Sex differences in nicotine effects and self-administration: review of human and animal evidence. Nicotine Tob. Res. 1, 301−315. (27) Perkins, K. A. (1999) Nicotine discrimination in men and women. Pharmacol., Biochem. Behav. 64, 295−299. (28) Hatchell, P. C., and Collins, A. C. (1977) Influences of genotype and sex on behavioral tolerance to nicotine in mice. Pharmacol., Biochem. Behav. 6, 25−30. (29) Cronan, T., Bryson, R., and McNair, E. (1984) Effect of sex and castration on nicotine-induced activity responses. Pharmacol., Biochem. Behav. 21, 675−677. (30) Koylu, E., Demirgören, S., London, E. D., and Pöǧün, S. (1997) Sex difference in up-regulation of nicotinic acetylcholine receptors in rat brain. Life Sci. 61, PL185−PL190. (31) Yilmaz, Ö ., Kanit, L., Okur, B., and Pöğün, Ş. (1997) Effects of nicotine on active avoidance learning in rats: sex differences. Behav. Pharmacol. 8, 253−260. (32) Cong, X., Xu, W., Janton, S., Henderson, W. A., Matson, A., McGrath, J. M., Maas, K., and Graf, J. (2016) Gut Microbiome Developmental Patterns in Early Life of Preterm Infants: Impacts of Feeding and Gender. PLoS One 11, e0152751. (33) Chi, L., Bian, X., Gao, B., Ru, H., Tu, P., and Lu, K. (2016) Sexspecific Effects of Arsenic Exposure on the Trajectory and Function of the Gut Microbiome. Chem. Res. Toxicol. 29, 949−951. (34) Gao, B., Bian, X., Mahbub, R., and Lu, K. (2017) Sex-specific effects of organophosphate diazinon on the gut microbiome and its metabolic functions. Environ. Health Perspect. 125, 198−206. (35) Rowell, P. P., Hurst, H. E., Marlowe, C., and Bennett, B. D. (1983) Oral administration of nicotine: its uptake and distribution after chronic administration to mice. J. Pharmacol. Methods 9, 249− 261. (36) Meyer, F., Paarmann, D., D’Souza, M., Olson, R., Glass, E. M., Kubal, M., Paczian, T., Rodriguez, A., Stevens, R., Wilke, A., Edwards, R., et al. (2008) The metagenomics RAST server−a public resource

for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinf. 9, 386. (37) Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., Lesniewski, R. A., Oakley, B. B., Parks, D. H., Robinson, C. J., et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537−7541. (38) White, J. R., Nagarajan, N., and Pop, M. (2009) Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS Comput. Biol. 5, e1000352. (39) Bowler, C., Montagu, M. v., and Inze, D. (1992) Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 83−116. (40) Penninckx, M. (2000) A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses. Enzyme Microb. Technol. 26, 737−742. (41) Jin, Y., Wu, S., Zeng, Z., and Fu, Z. (2017) Effects of environmental pollutants on gut microbiota. Environ. Pollut. 222, 1−9. (42) Lefever, D. E., Xu, J., Chen, Y., Huang, G., Tamas, N., and Guo, T. L. (2016) TCDD modulation of gut microbiome correlated with liver and immune toxicity in streptozotocin (STZ)-induced hyperglycemic mice. Toxicol. Appl. Pharmacol. 304, 48−58. (43) Jin, C., Zeng, Z., Fu, Z., and Jin, Y. (2016) Oral imazalil exposure induces gut microbiota dysbiosis and colonic inflammation in mice. Chemosphere 160, 349−358. (44) Wu, J., Wen, X. W., Faulk, C., Boehnke, K., Zhang, H., Dolinoy, D. C., and Xi, C. (2016) Perinatal lead exposure alters gut microbiota composition and results in sex-specific bodyweight increases in adult mice. Toxicol. Sci. 151, 324−333. (45) Gao, B., Chi, L., Mahbub, R., Bian, X., Tu, P., Ru, H., and Lu, K. (2017) Multi-Omics Reveals that Lead Exposure Disturbs Gut Microbiome Development, Key Metabolites, and Metabolic Pathways. Chem. Res. Toxicol. 30, 996−1005. (46) Zhang, S., Jin, Y., Zeng, Z., Liu, Z., and Fu, Z. (2015) Subchronic exposure of mice to cadmium perturbs their hepatic energy metabolism and gut microbiome. Chem. Res. Toxicol. 28, 2000−2009. (47) Perkins, K. A., Epstein, L. H., Sexton, J. E., Stiller, R. L., and Jacob, R. G. (1991) Effects of dose, gender, and level of physical activity on acute metabolic response to nicotine. Pharmacol., Biochem. Behav. 40, 203−208. (48) Hill, A. L., Roe, D. J., Taren, D. L., Muramoto, M. M., and Leischow, S. J. (2000) Efficacy of transdermal nicotine in reducing post-cessation weight gain in a Hispanic sample. Nicotine Tob. Res. 2, 247−253. (49) Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., and Gordon, J. I. (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027−1131. (50) Turnbaugh, P. J., and Gordon, J. I. (2009) The core gut microbiome, energy balance and obesity. J. Physiol. 587, 4153−4158. (51) Musso, G., Gambino, R., and Cassader, M. (2010) Gut microbiota as a regulator of energy homeostasis and ectopic fat deposition: mechanisms and implications for metabolic disorders. Curr. Opin. Lipidol. 21, 76−83. (52) Fischbach, M. A., and Segre, J. A. (2016) Signaling in hostassociated microbial communities. Cell 164, 1288−1300. (53) Hague, A., Singh, B., and Paraskeva, C. (1997) Butyrate acts as a survival factor for colonic epithelial cells: further fuel for the in vivo versus in vitro debate. Gastroenterology 112, 1036−1040. (54) Frost, G., Sleeth, M. L., Sahuri-Arisoylu, M., Lizarbe, B., Cerdan, S., Brody, L., Anastasovska, J., Ghourab, S., Hankir, M., Zhang, S., Carling, D., et al. (2014) The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. Nat. Commun. 5, 3611. (55) Carabotti, M., Scirocco, A., Maselli, M. A., and Severi, C. (2015) The gut−brain axis: interactions between enteric microbiota, central and enteric nervous systems. Ann. Gastroenterol. 28, 203−209. (56) Montgomery, A. J., McTavish, S. F., Cowen, P. J., and Grasby, P. M. (2003) Reduction of brain dopamine concentration with dietary 2118


Article

Chemical Research in Toxicology tyrosine plus phenylalanine depletion: an [11C] raclopride PET study. Am. J. Psychiatry 160, 1887−1889. (57) Lou, H., Lykkelund, C., Gerdes, A., Udesen, H., and Bruhn, P. (1987) Increased vigilance and dopamine synthesis by large doses of tyrosine or phenylalanine restriction in phenylketonuria. Acta Paediatr. 76, 560−565. (58) Mansvelder, H. D., and McGehee, D. S. (2000) Long-term potentiation of excitatory inputs to brain reward areas by nicotine. Neuron 27, 349−357. (59) Van Zessen, R., Phillips, J. L., Budygin, E. A., and Stuber, G. D. (2012) Activation of VTA GABA neurons disrupts reward consumption. Neuron 73, 1184−1194. (60) Al-Wadei, H. A., Plummer, H. K., and Schuller, H. M. (2009) Nicotine stimulates pancreatic cancer xenografts by systemic increase in stress neurotransmitters and suppression of the inhibitory neurotransmitter γ-aminobutyric acid. Carcinogenesis 30, 506−511. (61) Schafer, D., and Jones, E. A. (1982) Hepatic encephalopathy and the γ-aminobutyric-acid neurotransmitter system. Lancet 319, 18− 20. (62) Brandsch, R. (2006) Microbiology and biochemistry of nicotine degradation. Appl. Microbiol. Biotechnol. 69, 493−498. (63) Chen, P. E., Geballe, M. T., Stansfeld, P. J., Johnston, A. R., Yuan, H., Jacob, A. L., Snyder, J. P., Traynelis, S. F., and Wyllie, D. J. (2005) Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by sitedirected mutagenesis and molecular modeling. Mol. Pharmacol. 67, 1470−1484. (64) Kleckner, N. W., and Dingledine, R. (1988) Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241, 835. (65) Schmitz, Y., Castagna, C., Mrejeru, A., Lizardi-Ortiz, J. E., Klein, Z., Lindsley, C. W., and Sulzer, D. (2013) Glycine transporter-1 inhibition promotes striatal axon sprouting via NMDA receptors in dopamine neurons. J. Neurosci. 33, 16778−16789. (66) Mothet, J.-P., Parent, A. T., Wolosker, H., Brady, R. O., Linden, D. J., Ferris, C. D., Rogawski, M. A., and Snyder, S. H. (2000) D-serine is an endogenous ligand for the glycine site of the N-methyl-Daspartate receptor. Proc. Natl. Acad. Sci. U. S. A. 97, 4926−4931. (67) Caccia, S., Garattini, S., Ghezzi, P., and Zanini, M. (1982) Plasma and brain levels of glutamate and pyroglutamate after oral monosodium glutamate to rats. Toxicol. Lett. 10, 169−175. (68) Dusticier, N., Errami, M., Nieoullon, A., et al. (1985) Effects of pyroglutamic acid on corticostriatal glutamatergic transmission. Neuropharmacology 24, 903−908. (69) Hutson, S. M., Lieth, E., and LaNoue, K. F. (2001) Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J. Nutr. 131, 846S−850S. (70) Yudkoff, M., Daikhin, Y., Nissim, I., Horyn, O., Luhovyy, B., Lazarow, A., and Nissim, I. (2005) Brain amino acid requirements and toxicity: the example of leucine. J. Nutr. 135, 1531S−1538S. (71) Gobaille, S., Kemmel, V., Brumaru, D., Dugave, C., Aunis, D., and Maitre, M. (2008) Xanthurenic acid distribution, transport, accumulation and release in the rat brain. J. Neurochem. 105, 982−993. (72) Taleb, O., Maammar, M., Brumaru, D., Bourguignon, J.-J., Schmitt, M., Klein, C., Kemmel, V., Maitre, M., and Mensah-Nyagan, A. G. (2012) Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20. PLoS One 7, e48553. (73) Du, Y., Chen, C. P., Tseng, C. Y., Eisenberg, Y., and Firestein, B. L. (2007) Astroglia-mediated effects of uric acid to protect spinal cord neurons from glutamate toxicity. Glia 55, 463−472. (74) Stover, J. F., Lowitzsch, K., and Kempski, O. S. (1997) Cerebrospinal fluid hypoxanthine, xanthine and uric acid levels may reflect glutamate-mediated excitotoxicity in different neurological diseases. Neurosci. Lett. 238, 25−28.

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