Abstract Intestinal microbiota play a significant role in nutrient metabolism, modulation of the immune system, obesity, and possibly in carcinogenesis, although the underlying mechanisms resulting in disease or impacts on longevity caused by different intestinal microbiota are mostly unknown. Herein we use isogenic Atm-deficient and wild type mice as models to interrogate changes in the metabolic profiles of urine and feces of these mice, which are differing in their intestinal microbiota. Using high resolution mass spectrometry approach we show that the composition of intestinal microbiota modulates specific metabolic perturbations resulting in a possible alleviation of a glycolytic phenotype. Metabolites including 3-methylbutyrolactone, kyneurenic acid and 3-methyladenine known to be onco-protective are elevated in Atm-deficient and wild type mice with restricted intestinal microbiota. Thus our approach has broad applicability to study the direct influence of gut microbiome on host metabolism and resultant phenotype. These results for the first time suggest a possible correlation of metabolic alterations and carcinogenesis, modulated by intestinal microbiota in A-T mice.

Citation: Cheema AK, Maier I, Dowdy T, Wang Y, Singh R, Ruegger PM, et al. (2016) Chemopreventive Metabolites Are Correlated with a Change in Intestinal Microbiota Measured in A-T Mice and Decreased Carcinogenesis. PLoS ONE 11(4): e0151190. https://doi.org/10.1371/journal.pone.0151190 Editor: Alexander V. Alekseyenko, Medical University of South Carolina, UNITED STATES Received: September 9, 2015; Accepted: February 24, 2016; Published: April 13, 2016 Copyright: © 2016 Cheema et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All files for this dataset have been uploaded in a universal format (netcdf) onto a public repository which can be accessed using the following link: http://datadryad.org/review?doi=doi:10.5061/dryad.s4617. Funding: This work was supported by RO1ES0951907 to RHS. Competing interests: RHS is the owner of Microbio Pharma, Holding, Inc. with no salary. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Abbreviations: A-T, Ataxia telangiectasia; RM, Restricted microbiota; CM, Conventional microbiota; KO, Knockout; UPLC, Ultra performance liquid chromatography; LC-MS, Liquid chromatography-mass spectrometry; TCA, Tricarboxylic acid

Introduction Ataxia telangiectasia (A-T) is a recessive genetic disorder of childhood that occurs in one out of 100,000 humans worldwide. A-T is a multi-system, progressive disorder associated with a high incidence of lymphoid malignancies [1]. Approximately 30–40% of A-T patients develop neoplasia during their life and more than 40% of all tumors in A-T patients are non-Hodgkin’s lymphomas, ~20% acute lymphocytic leukemias, and 5% Hodgkin’s lymphomas [2–6]. A-T patients suffer from compromised immune function, neurodegeneration, infections of the respiratory system, and increased mortality due to 1000-fold increase in malignancy [1, 7–9]. Although ground breaking studies have led to a better and deeper understanding of ataxia telangiectasia mutated (ATM) gene function [10, 11], no effective therapy is currently available to prevent cancer or progressive neurodegeneration. Several laboratories have generated different strains of Atm-deficient (Atm-/-) mice and reported that Atm-/- mice developed lymphomas and died between 2 and 5 months of age [12–16]. Later studies, including our own, showed that the onset of lymphoma can be drastically delayed till 7–12 months, which was not a result of genetic diversity [17–20]. Instead, it was found to be associated with changes in intestinal microbiota, including an increase in Lactobacillus johnsonii [21], and reduced lymphoma incidence was achieved by administering the antioxidant N-acetyl cysteine [19]. The inflammatory effect of normal intestinal microbiota compositions could shorten lifespan of mice compared to mice housed under sterile housing conditions. Subsequently, we generated Atm-/- mice harboring different but well characterized intestinal microbiota, termed conventional microbiota (CM), and restricted microbiota (RM). Restricted microbiota harbor relatively few bacterial species, dominated by unclassified Bacteroidetes, and are free of known pathogens [22]. RM mice were originally obtained by Caesarean section and re-derived into the mouse colony with restricted flora adoptive mothers [23]. Sterile housing and a restriction of intestinal microbiota (in RM mice) yielded a 4.5-fold lower level of genetic instability, differences in acute leukocyte genotoxicity, inflammation markers, and a 2.5-fold delayed onset of lymphoma resulting in longevity as compared to mice with CM [21]. We further demonstrated that increased levels of L. johnsonii present in RM mice, led to low basal levels of chromosomal genotoxicity and inflammation in Atm-/- mice compared to the CM mice [21, 24]. Given that energy metabolism has been implicated in oncogenesis, we hypothesized that the microbiome is likely to influence host metabolism resulting in an altered phenotype. In this study, we used a comparative fecal and urine metabolomics profiling approach to discern how either of the microbiota compositions, CM or RM, would impact the metabolic phenotype of Atm-/- mice. Gut microbiome are known to have a profound influence of a myriad of diseases including dementia, obesity and cancer, although the molecular implications of dysbiosis are yet to be elucidated [25]. For example, microbiota have been shown to influence energy metabolism, beta-oxidation of lipids, bile acid, glutamine and tryptophan metabolism, as well as oxidative stress and immune response metabolites [26, 27]. Most of recently reported studies focus on the regulatory and signaling pathways that are directly affected by microbiome in different patho-physiological conditions; however, there is a lack in studies that directly examine the metabolic consequences of alterations in gut microbiome. Metabolomics is a qualitative and quantitative evaluation of alterations in endogenous metabolism in response to a specific perturbation in a biological system [28]. It represents the endpoint of genetic regulation and its impact on altered enzymatic activities and endogenous biochemical reactions in a cell [29]; thus characterizing precise changes in metabolism is likely to enable better understanding of how cells adapt to oxidative stress and minimize DNA damage and genomic instability. Moreover, correlation of metabolic profiles with gut microbiota offers to provide valuable insights into host-microbe interactions as well as valuable steps for achieving a breakthrough in personalized therapy paradigm [30–33]. Since ATM function influences cell signaling and metabolism [34], a metabolomics approach using isogenic mouse models differing only in microbiome composition should allow for a direct assessment of accumulated bacterial metabolites and the effects of the microbiome on metabolism [35].

Methods Reagents and standards Acetonitrile (ACN), methanol, and water (all LC-MS grade) were purchased from Fisher Scientific (NJ, USA). Debrisoquine, 4-nitrobenzoic acid (4-NBA), and heparin were purchased from Sigma (USA). All standard compounds used for tandem mass spectrometry based validation of metabolite identification were bought from Sigma. Lipid standards were ordered from Avanti Polar Inc. Other reagents for preparation and analyses of samples are described in the respective sections. Animal housing and husbandry The study was performed under the guidelines of the UCLA Animal Research Committee and specifically approved procedures for animal breeding, housing and sample collection in this study. Mice were housed under two types of specific pathogen free (SPF) conditions at UCLA Department of Laboratory and Animal Research, where either sterile (for RM), or non-sterile (for CM) food, water, and bedding were employed. Atm-/- mice harboring RM and CM microbiota were created by rederivation as described in Fujiwara et al. [23], and by antibiotic treatment [36] followed by orogastric gavage of CM feces, respectively. The same procedures were applied in wild type mice. Mice of the RM colony were maintained under strictly aseptic and sterile conditions at the animal facility of the Radiation Oncology Department for several years. Atm-/- mice were obtained by intercrossing Atm+/- mice and identified by genotyping as previously described [21]. CM and RM mice for the metabolomics study were born in the respective colonies and were not treated with antibiotics. Urine and fecal samples from male Atm-/- and Atm+/+ (referred to as wild type, or WT) mice aged 6–8 months were investigated by metabolomics-based molecular phenotyping. The different mice strains used in this study were defined as follows: Atm-KO-RM (Atm knock out mice carrying restricted microbiota); Atm-KO-CM (Atm knock out mice carrying conventional microbiota); WT-RM (wild type mice carrying restricted microbiota); and WT-CM (wild type mice carrying conventional microbiota). All urines and feces were separately collected in Eppendorf tubes from individual mice. The urine samples were centrifuged and the supernatant was aliquoted and frozen at -80°C until further use. All experiments were performed using at least 5 mice per genotype for metabolomics and at least 3–4 mice for 16S RNA sequencing experiments. Intestinal bacterial analysis For this study, Illumina-based high-throughput sequencing of bacterial 16S rRNA genes from intestinal mucosa samples [37] was performed as described [38]. DNA was extracted from these samples using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), and a 30-second bead-beating step using a Mini-Beadbeater-16 (BioSpec Products, Bartlesville, OK, USA). Metabolite extraction and metabolomics profiling Urine samples were processed as described previously [39] using 66% acetonitrile in water to precipitate any proteins or cellular debris. The samples were centrifuged, dried under vacuum, and stored at -80°C until analysis. Fecal samples were processed by initially sonicating in extraction solvent containing water and methanol (50% v/v) and internal standards, followed by addition of chloroform (1:1 v/v). The samples were centrifuged and the two biphasic layers were separated carefully. To each of the solvent phase two volumes of acetonitrile was added and mixed thoroughly by vortexing. The samples were incubated at -20°C for 4 hours to allow protein precipitation followed by centrifugation. The supernatant was combined and dried under vacuum, and resuspended in water containing 5% methanol for MS analysis. Metabolites extracted from urine and fecal samples were analyzed in the same batch with two technical replicates for each sample to assess chromatographic reproducibility. The sample queue was randomized to avoid bias. Each sample (5 μl) was injected onto a reverse-phase 50 × 2.1 mm Acquity 1.7μm BEH C18 column (Waters Corp, Milford, MA), using an Acquity UPLC (Waters Corporation, USA) system online with an electrospray quadrupole time-of-flight tandem mass spectrometer (ESI-Q-TOF) (QTOF Premiere, Waters Corporation, USA). Positive and negative ion mode were operated; the details of tune page parameters have been described before [39, 40]. Together, we performed a broad range metabolite extraction and used reversed phase chromatography that would support the detection of a broad class of metabolites for urine and fecal samples. These data were normalized to the ion intensity of the internal standards (debrisoquine and 4, nitrobenzoic acid) and weight of the fecal pellet (for fecal samples), or creatinine (for urine samples). A 500 pg/μL solution of sulfadimethoxine in 50% acetonitrile ([M + H] +, m/z 311.0814) was infused at 0.08 μL/min flow rate as the reference mass (lock mass) for accurate mass measurements. The quality control (QC) samples for each matrix comprised an aliquot of all samples in the study set, thus representing a universal set of metabolites. The QC sample was used to initially condition the column and thereafter, it was injected after every ten injections to account for reproducibility of the LC-MS data [41]. The coefficient of variance was examined for internal standards and creatinine in the QC samples and was found to be less than 15%. As explained above, five biological replicates per group were used for comparative profiling, while pooled QC samples were used for assessing technical reproducibility throughout each batch acquisition. Statistical analysis of LC-MS data and Pathway Analysis The UPLC-QTOF raw data files were converted into NetCDF format (Network Common Data Form) using the MassLynx software (Waters Corp, Milford, MA). Subsequently, the LC-MS data were preprocessed using XCMS software as described [42]. The normalized data were processed using Metaboanalyst software (2.0) [43] and univariate analysis methods. Principal components analysis (PCA) was performed as an exploratory analysis to check for inter-group variability based on overall metabolite profiles. The candidate markers were selected by examining each volcano plot thereby considering fold change threshold of 2 and statistical p-value less than 0.05. A Benjamini-Hochberg correction was applied, which estimates conservative q-values [44], to ensure control of the false discovery rate (FDR) at a significance level of 0.05. Relative quantitation was achieved for molecular ions using the UPLC-QTOF system by taking a ratio of normalized intensity of the respective for the ions of interest in the comparative groups. This approach has been used by several laboratories including ours in previously reported studies [45–48]. The significant peaks from the FDR-adjusted analysis were putatively identified based on their m/z against Metabosearch, which performs accurate mass-based metabolite search through four main online databases; the Human Metabolome Database (HMDB), Lipid Maps, Madison Metabolomics Consortium Database (MMCD), and Metline [49]. The mass tolerance was kept at 5 parts per million to minimize false positive identification. The identity of metabolites was subsequently confirmed by comparisons of fragmentation spectra and retention time with commercially available standard compounds using tandem mass spectrometry (MS-MS). Signal intensities of the differentially abundant metabolites were visualized as a heat map, wherein the log transformed data were hierarchically clustered by Pearson correlation and average linkage clustering. The mean signal intensity is colored black, red indicates above-mean intensity, green denotes below-mean intensity and the degree of color saturation reflects the magnitude of intensity relative to the mean. Both, fecal and urine metabolomics data sets were analyzed using the Ingenuity Pathway Analysis (IPA) tool to score for correlative biochemical pathways.

Discussion Despite increasing efforts in the field of A-T research, the mechanisms by which enteric microbiota lead to changes in Atm-mediated lymphomagenesis remain elusive. ATM regulates the cellular response to oxidative stress and it senses double-stranded DNA breaks, thus inhibiting cell cycle progression [11, 54]. It has been demonstrated that intestinal microbiota can induce cancer development [35], for example Helicobacter pylori, inducing gastric cancer and lymphoma in WT mice and humans, as well as potentially influencing the response of cancer to therapies [55, 56]. We have previously shown reduction of cancer susceptibility and an impact on longevity by specific alterations in gut microbiota composition in Atm-/- mice. Using an untargeted, high resolution mass spectrometry approach, we then asked how this translates into metabolic changes in mice that are genetically identical but differ only in their gut microbiome composition. Surprisingly, we did not find differently abundant metabolites in blood profiles (data not shown). Moreover, our results from the analysis of urine and fecal samples suggest that distinct intestinal microbiota cause a metabolic shift towards the upregulation of metabolites including kyneurenic acid, methyladenine and 3-methybutyrolactone that may attenuate cancer-promoting signaling pathways, reported to be associated with an onco-protective phenotype independent of the genotype of the mice. We found that intestinal microbiota restriction in Atm-deficient mice led to a 2.5-fold extension of lymphoma latency and 4 fold increased longevity, and significant differences in chromosomal genotoxicity, oxidative DNA damage and inflammation; our research was the first to show a relationship between intestinal microbiota and lymphoma onset [21]. Lactobacillus johnsonii, one of the indicator phylotypes that we identified to be more abundant in RM than CM mice, is known to increase the number of Paneth cells, which are a host cell type that produces antimicrobial compounds, and are located together with stem cells in the intestinal crypt [57]. The extended lifespans of our RM mouse models appear to be caused by an intestinal microbiota comprised largely of unclassified members of the Bacteroidetes (Table 1), which in turn result in specific metabotypes. Potentially chemopreventive metabolites were found to be differentially abundant in a microbiota-dependent manner in Atm-KO or WT mice; for example, the urinary levels of 3-methylbutyrolactone were elevated in both genotypes harboring RM microbiota. This metabolite is known to be produced by the intestinal bacteria and exhibits strong anti-aromatase properties [58]. Further, this metabolite inhibits fatty acid synthase (FAS), which activates carnitine palmitoyltransferase 1beta (CPT-1beta) and ultimately results in beta-oxidation of long chain fatty acids and prevention of lipid accumulation [59]. In addition, alpha-methylbutyrolactone acts as a selective alkylating agent for thiol rich enzymes like phosphofructokinase, DNA polymerase and glycogen synthase; thus regulating cell proliferation [60]. Additionally, mice with RM microbiota showed elevation of methyladenine, which is a potent inhibitor of phosphoinositide 3-kinases (PI3K) and has also been shown to exhibit tumor suppressor activity [61–64]. There was an elevation in the levels of kynurenic acid (a tryptophan metabolite) in feces of RM mice. Walczak et al. have reported the potential chemopreventative role of this metabolite in colon cancer [53]. Elevation of kynurenic acid has been shown to positively correlate with immune tolerance [65–67]. An upregulation of metabolites participating in oxidative phosphorylation was found in RM as compared to CM in Atm- KO (Fig 5, Panel A) mice, as well as an upregulation of TP53 regulated molecular networks in RM mice (Fig 5, Panel B). Interestingly, TP53 is known to regulate the expression of TIGAR (TP53-induced Glycolysis and Apoptosis Regulator) that has been shown to downregulate glycolysis by lowering the endogenous levels of fructose 1,6 biphosphate [68]. This is an interesting finding, generating new hypothesis for investigating how alleviation of glycolytic phenotype in RM mice is induced. Future investigations in our laboratory will specifically follow the molecular links and associations proposed herein. Our results demonstrate that microbiome composition leads to specific changes in overall host metabolism, which may have direct implications on phenotype. This proof of principle investigation opens up several relevant and challenging questions; particularly, how lipids measured in feces of these mice indirectly regulate apoptosis, or if the altered metabolic profile accounts at least in part for extended life and decreased incidence of carcinogenesis in eukaryotes and will be a part of future studies. Further investigation is also needed to specify the regulatory role of certain microbiota compositions in permitting genotoxicity by reducing local or systemic autophagy. In conclusion, our pilot study demonstrates metabolite profiles likely modulated by specific phylotypes in RM mice that correlate with our previously reported observations of decreased tumor incidence and general leukocyte genotoxicity in RM mice as compared to Atm-KO-CM mice [21]. These alterations in the mean abundance of the interrogated metabolites, including 3-methylbutyrolactone and 3-methyadenine (Fig 2) and kynurenic acid (Fig 4) were more pronounced in Atm-KO mice as compared to the WT RM mice, which may help explain genotype based susceptibility. Together, these findings lend credence to the notion that manipulating microbial composition could be used as an effective strategy to prevent or alleviate cancer susceptibility. The onco-protective effect of RM microbiota is true for WT as well as Atm-KO mice—the only difference lies in cancer susceptibility for both genotypes. Remarkably, our findings suggest that composition of the gut microbiota influences and alters central carbon metabolism in a genotype independent manner. In future, it is our hope that the use of probiotics-containing RM would be a potential chemopreventive for normal humans, while the same type of microbiota would decrease tumor incidence in cancer susceptible populations.

Acknowledgments The authors would like to acknowledge the Proteomics and Metabolomics shared resource partially supported by NIH/NCI grant P30-CA051008.

Author Contributions Conceived and designed the experiments: RHS AKC IM AF. Performed the experiments: IM AKC RS PMR JB. Analyzed the data: IM AKC YW TD. Contributed reagents/materials/analysis tools: RHS AKC AF IM JB. Wrote the paper: AKC IM RHS AF JB.