Mice
Male CD2F1 mice, six to seven weeks old and specific pathogen free were purchased from Envigo (Dublin, VA, USA) and housed in the Uniformed Services University animal facility, an Association for Assessment and Accreditation of Laboratory Animal Care-International accredited facility as described earlier [31, 32]. All animal procedures were performed according to a protocol approved by Uniformed Services University Institutional Animal Care and Use Committee (IACUC). Research was conducted according to the Guide for the Care and Use of Laboratory Animals [33]. Moribundity was used as a surrogate for mortality, and euthanasia was used in order to minimize pain and distress to animals.
Experimental design and drug administration
There were three groups of mice (16 mice per group) for this metagenomics and metabolomics study. There were four cages in each group with four mice in each cage. Group 1 received vehicle (0.5% Methocel A4M + 3% Kollidon 25) (termed ‘control’ throughout the text), and Groups 2 and 3 received either BIO 300 OS or BIO 300 OP (200 mg/kg/day) for six days prior to irradiation. BIO 300 OS is an aqueous liquid suspension of synthetic genistein nanoparticles with a median particle size of 200 nm. BIO 300 OP is an amorphous solid dispersion of genistein produced as a free-flowing dry powder with a median particle size of 160 µm. BIO 300 OP is prepared by hot-melt extrusion and milled to the final particle size. BIO 300 OP was slowly dispersed into vehicle and kept suspended with continuous stirring until dosing. Mice were administered their respective treatment twice per day po with 12 h between dosing. The drug/vehicle was administered using a 1 ml syringe and a 20-gauge feeding cannula, which had a ball tip. One sterile syringe and feeding needle was used for mice in each cage (four mice). Although the mice in each cage share the sipper tube in the water bottle and oral microorganisms as well, the feeding needle was wiped and disinfected between dosing on a gauze sponge moistened with 70% ethanol to reduce the microorganisms and saliva on the needle as an extra precaution. To avoid irritation to the mucosa, the feeding needle was wiped with a gauze sponge that was moistened with purified water. The volume of vehicle or drug administered was 200 µl/mouse.
Irradiation
Mice were exposed to high dose 60Co γ-radiation at the Armed Forces Radiobiology Research Institute’s facility, as described previously [31]. After 6 days of vehicle or drug administration, mice were placed in compartmentalized and ventilated Plexiglas boxes and exposed to bilateral γ-irradiation (9.2 Gy, 0.6 Gy/min, approximately LD70/30 dose). After irradiation, mice were returned to their home cage and monitored for 30 days. Radiation dosimetry was based primarily on the alanine/EPR (Electron Paramagnetic Resonance) system [34], one of the most accurate methods currently accepted, and used for comparison between National Metrology institutions.
Fecal pellet collection
As stated above, the drug or vehicle was administered for 6 days prior to irradiation. The day of irradiation was considered to be study day zero (SD0). Fecal pellets were collected from mice prior to drug or vehicle administration/irradiation in addition to post-treatment/post-irradiation, for a total of 5 time points (SD − 7, − 1, 3, 14, and 30 in relation to irradiation). For collecting feces samples, individual mice were placed in a sterile ventilated Plexiglas box. Using sterile forceps, two to three fecal pellets were collected in individually labeled sterile 1.5 ml Eppendorf tubes immediately after defecation. To prevent contamination between samples collected from different mice, the surface of the Plexiglas boxes and forceps were sterilized using 70% ethanol. The tubes containing the fecal pellets were immediately stored on dry ice before being transferred to − 80 °C until further analysis.
Isolation of DNA from fecal pellets
Total DNA was isolated from the fecal pellets using QIAamp PowerFecal Pro DNA Kit (Qiagen, Germantown, MD, USA), and the procedure was accomplished according to the manufacturer’s guidelines. In brief, a single pellet was loaded into a PowerBead Pro tube containing solution CD1. The fecal samples were homogenized on a vortex mixer for 10 min at maximum speed and then centrifuged at 15,000×g for 1 min. To remove inhibitors, the supernatant was mixed with solution CD2, vortexed and centrifuged at 15,000×g for 1 min. To bind DNA, the resulting supernatant was mixed with solution CD3 and passed over an MB spin column. The column was then washed with solutions EA and C5, and bound DNA was eluted using 50 μl of solution C6. Finally, the concentration of DNA in the eluate was estimated using a Qubit™ 4 Fluorometer (Life Technologies, Carlsbad, CA, USA). The isolated DNA was stored at − 80 °C until further use.
16S rRNA sequencing library preparation
The amplicon library for the V3-V4 region of the 16S rRNA gene was prepared using QIAseq 16S/ITS Region Panel library construction kit (Qiagen, Germantown, MD, USA) as per the manufacturer’s directions. Briefly, the V3-V4 region of the 16S rRNA gene was PCR amplified in a 10 μl reaction volume containing UCP master mix, V3-V4 primers, and 1 ng of DNA. The following cycling conditions were used for PCR amplification: denature at 95 °C for 2 min, (95 °C 30 s, 50 °C 30 s, 72 °C 2 min) × 12 cycles, and final extension at 72 °C for 7 min. PCR reactions were set up in triplicate. After PCR was completed, the reactions were pooled and the amplified DNA was purified using QIAseq beads. Sample indices and sequencing adapters were added to the purified PCR product from the above step using dual indexed “phased primers” from Qiagen. The indexing PCR reaction contained purified PCR product from the previous step and UCP master mix in a total reaction volume of 50 μl. Cycling conditions for indexing PCR were: denature at 95 °C for 2 min, (95 °C 30 s, 60 °C 30 s, 72 °C 2 min) × 19 cycles, and a final extension at 72 °C 7 min. The amplified library from the above process was purified using QIAseq beads and eluted in 25 μl volume of nuclease free water. Size and quality of the indexed library was checked using Agilent DNA 1000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), and the library concentration was determined using the KAPA Library Quantification Kit for Illumina (KAPA Biosystems, Inc., Wilmington, MA, USA).
Sequencing
The metagenomic library was prepared for sequencing on a MiSeq sequencing platform, as described in the MiSeq Reagent kit v3 Reagent Preparation Guide (Illumina, San Diego, CA, USA). Briefly, libraries were normalized to 4 nM and pooled volumetrically. The pooled sample library was denatured by mixing 5 μl of library and 5 μl of freshly prepared 0.2 N sodium hydroxide, vortexed and centrifuged at 250×g for 1 min. The tube was incubated for 5 min at room temperature and the reaction was stopped by adding 5 μl of Tris-Cl pH 7.0, mixed by vortexing, and centrifuged at 250×g for 1 min. Following this step, 985 μl of buffer HT1 was immediately added to the tube containing denatured library, and was then mixed and kept on ice. This process yielded a 20 pM library. The final concentration of the denatured library to be used for sequencing was determined based on the targeted cluster density. A MiSeq v3, 600 Cycle Reagent Cartridge (Illumina, San Diego, CA, USA) was used for sequencing. It was a paired end, dual indexed (151 Read I and 151 Read II, 8 Index Read 1, 8 Index Read 2) sequencing run.
16S rRNA sequence analysis
Raw 16S rRNA amplicons were converted to Amplicon Sequence Variants (ASVs) using the default DADA2 recommendations (version 1.18.0) [35]. In brief, the forward and reverse reads were trimmed to a length of 250 and 210, respectively. Additionally, reads were truncated to the first instance of a quality score less than or equal to 2. The maximum number of expected errors was set to 2 for the forward reads and 7 for the reverse reads. After assessing error rates, the reads were merged, chimera checked, and assigned taxonomic classification using the Silva Project’s 16S rRNA non-redundant reference database (version 132).
Reads were not rarefied [https://doi.org/10.1371/journal.pcbi.1003531], but were filtered out if they were associated with ASVs that were present in less than 5% of the samples. Furthermore, all reads classified as “uncharacterized” at the phylum level were removed from the study. Changes in microbial composition upon irradiation and/or drug treatment were inferred by performing comparative group analysis. Relative abundance data for each ASV was compared across samples via Bray–Curtis Dissimilarity matrix construction. Significant differences in Principal Component Analysis (PCoA) ordination clusters were computed using the analysis of variance of distance matrices test (Adonis) from within vegan R package. 999 permutations were performed for each Adonis test.
Liquid chromatography–Mass Spectrometry (LC–MS) methods
Each fecal pellet sample was mixed with 600 µL of an extraction solution comprising of 10% (v/v) water, 30% (v/v) isopropanol, 20% (v/v) methanol, and 40% (v/v) chloroform. The extraction solution contained 0.1% debrisoquine (1 mg/ml in water) and 0.5% 4-Nitrobenzoic acid (1 mg/ml in methanol) as internal standards. The samples were homogenized on ice then kept at 4 °C for 15 min before being centrifuged at 15,493×g for 20 min at 4 °C. The supernatants were transferred to new tubes and a volume of 600 µl of chilled acetonitrile was added. Extracts were vortexed, and then kept at − 20 °C for 18 h. The samples were centrifuged as before at 15,493×g for 20 min at 4 °C and the supernatants were transferred to new tubes. Finally, the samples were vacuum dried, resuspended in 200 µl of 50/50 water/methanol, and then transferred to MS vials for LC–MS analysis.
A volume of 2 µl from each sample was injected onto a Waters Acquity BEH C18 1.7 μm, 2.1 × 50 mm column, which was kept at 40 °C for the metabolomics acquisition, and a Waters Acquity CSH C18 1.7 μm, 2.1 × 100 mm column, which was kept at 65 °C for the lipidomic acquisition utilizing an Acquity UPLC system coupled to a Xevo G2-S quadrupole-time-of-flight mass spectrometer with an electrospray ionization source (UPLC-ESI-QToF-MS—Ultra-performance liquid chromatography-lectrospray ionization-Quadrupole-time-of-flight) (Waters Corporation, Milford, MA). For the metabolomics profiling, the mobile phases consisted of 100% water with 0.1% formic acid (solvent A), acetonitrile containing 0.1% formic acid (solvent B), and 90:10 isopropanol/acetonitrile with 0.1% formic acid (solvent C). All solvents used were of LC–MS grade and were purchased from Fisher Scientific (Waltham, MA). The solvent flow rate was set to 0.4 ml/min with the column set at 40 °C. The LC gradient was as follows: Initial—95% A, 5% B; 0.5 min—95% A, 5% B; 8.0 min—2% A, 98% B; 9.0 min—11.8% B, 88.2% C; 10.5 min—11.2% B, 88.2% C; 11.5 min—50% A, 50% B; 12.5 min—95% A, 5% B; 13.0 min—95% A, 5%. The lipidomic solvents consisted of 50% water + 50% acetonitrile (ACN) + 0.1% formic acid + 10 mm ammonium formate (solvent A) and 90% isopropanol + 10% acetonitrile + 0.1% formic acid + 10 mm ammonium formate (solvent D). The flow rate was set to 0.45 ml/min and the LC gradient was as follows: Initial—60% A, 40% B; 0.5 min—60% A, 40% B; 8.0 min—0% A, 100% B; 8.5 min—0% A, 100.0% B; 9.0 min—60% A, 40% B; 11.0 min—60% A, 40% B.
The column eluent was introduced into the Xevo G2 mass spectrometer by electrospray operating in either negative or positive electrospray ionization mode. Positive mode had a capillary voltage of 3.00 kV and a sampling cone voltage of 30 V. Negative mode had a capillary voltage of 2.80 kV and had a sampling cone voltage of 30 V. The desolvation gas flow was set to 1000 L/hour and the desolvation temperature was set to 500 °C. The cone gas flow was 25 L/hour and the source temperature was set to 120 °C. The data was acquired in the sensitivity MS mode with a scan time of 0.300 s and an interscan time of 0.014 s. Accurate mass was maintained by infusing leucine enkephalin (556.2771 [M + H]+/554.2615 [M-H]−) in 50% aqueous acetonitrile (1.0 ng/ml) at a rate of 10 µl/min via the Lockspray interface every 10 s. The data was acquired in centroid mode from a mass range of 50 to 1200 m/z TOF–MS (Time-of-flight mass spectrometry) scanning. An aliquot of each sample was pooled and run as a quality control (QC) sample, which represented all metabolites present. The QC sample was run at the beginning of each acquisition to condition the column and then injected every 10 samples to ensure consistent retention times and intensities.
Statistical analysis
The untargeted data was converted to NetCDF format using the Databridge tool in MassLynx (Waters Corporation, Milford, MA), and the XCMS [36] R package (version 3.12.0 URL: https://www.bioconductor.org/packages/release/bioc/html/xcms.html) was used for peak detection with ordered bijective interpolated warping algorithm utilized for retention time correction and parameters optimized using the Isotopologue Parameter Optimization (IPO) [37] R package (version 1.16.0 URL: https://bioconductor.org/packages/release/bioc/html/IPO.html). The mass to charge ratio and retention time (mz/rt) features were normalized to the internal standards debrisoquine and 4-nitrobenzoic acid present in the extraction solution in positive and negative modes respectively, then log transformed and pareto scaled. Unpaired t-tests were used to calculate up or down-regulation of each feature statistically, and significant metabolites were selected for MS/MS data acquisition and validation by the National Institute of Standards and Technology (NIST) 2017 database [38] fragmentation pattern matching. Kaplan Meier curves were constructed to view overall survival function throughout the study. Chi-square tests were performed to compare 30-day survival between the drug groups and the vehicle group. p-values of less than 0.05 were considered statistically significant and have been noted with an asterisk (*). The Wilcoxon-Mann–Whitney test was used to test the difference of Firmicutes/Bacteroidetes ratios between each group.