Dietary inclusion of nitrite-containing frankfurter exacerbates colorectal cancer pathology and alters metabolism in APCmin mice

animal model

40 Female Apc^min/+ Mice were purchased from Charles River and shipped to Axis Bioservices (Coleraine, Northern Ireland) where the study was performed. All mice were 4 weeks old at birth. All mice were fed an ad libitum maintenance diet of AIN76 during a 2-week acclimation period (Fig. 1a). Mice were caged in groups of 3 or 4 on a 12 h light, 12 h dark cycle. Mice had unrestricted access to their respective chow and water for the duration of the 12-week study, except for the 12-hour period prior to blood collection when they were fasted. Body weight was measured every 3–4 days. An attempt was made to measure food intake in a similar fashion, but (for welfare reasons) the meals had to be plated in the home cage, so this led to highly erroneous data and was subsequently abandoned. Mice were randomly assigned to one of the four treatment groups described below. Studies in mice underwent ethical review and were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.

diet

Each treatment group received one of four diets, including the diet group, for the duration of the study. Controls consisting of 100% AIN76 (Altromin, Germany). Sausage meat (15%)/AIN76 (85%), Frankfurt (15%)/AIN76 (85%), raw pork (15%)/AIN76 (85%). Sausage meat, as well as raw pork, were manufactured and supplied by Finnebrogue Artisan (Downpatrick, UK). Frankfurters containing nitrites were manufactured by Herta (Illkich, France) and purchased from a local supermarket. All meat is freeze-dried and powdered by European Freeze-Dry Company (UK). Altromin incorporated this into pellets composed of 15% meat meal and 85% AIN76. The nutritional properties of each diet were analyzed after pelleting (Supplementary Table 2).

biological sample

Tail blood samples were taken from all animals at three time points: 0, 3, 6 weeks, and a terminal cardiac puncture blood was taken at the end of the study (8 weeks). Urine was collected from individual animals at baseline and week 8. Fecal samples were collected from each cage at the same time points.

Gastrointestinal Tumor Scoring

Sacrifice an animal using CO₂ Suffocation and organ extraction.as explained before²⁹, the intestinal tract was removed, washed with phosphate-buffered saline, and cut into duodenum, jejunum, ileum, and colon. Sections were opened longitudinally, fixed in 10% formalin, colon stained with 0.05% methylene blue solution (Sigma) for 5 minutes, and small intestine stained with 0.05% methylene blue for 48 hours. All tumors present were counted blindly by one investigator using 25× magnification. The minimum size for accepting and counting tumors was 0.5 mm in diameter.

Identification of abnormal crypt foci and mucin-depleting foci

Aberrant crypt foci (ACF) were measured in the colon by a well-described method.³⁰Colons were washed with saline and stained with 0.2% methylene blue for 5 minutes. They were placed on glass slides and visualized with a light microscope using 32x magnification. Mucin depletion foci (MDF) were also measured in the colon and sections were washed with saline and stained with a 1% high iron diamine alcian blue procedure for 30 min. All scoring was performed by one of his investigators and verified by a second investigator in a blinded fashion.

lipid peroxidation

Serum samples from all time points were analyzed for malondialdehyde (MDA) using an ELISA method (Abcam, Cambridge UK). Briefly, thiobarbiturates, sera, and standards were added to 96-well microtiter plates, incubated at 95 °C for 60 min, and chilled on ice. An aliquot from each well was added to a colorimetric plate and measured at OD532 nm using a plate reader. Baseline and week 8 urine samples were analyzed for 1,4-dihydroxynonane mercapturic acid (DHN-MA) using the Bertin Bioreagent enzyme immunoassay (Wolverhampton UK). Briefly, buffer, standards, and urine were added to each well, detection antibody was added, and the plate was incubated overnight at 4 °C. Ellman’s reagent was added and absorbance was measured at 414 nm.

Metabolite profiling

Amino acids, acylcarnitines, bile acids, biogenic amines, carboxylic acids, ceramides, cholesterol esters, diacylglycerols, fatty acids, glycerophospholipids, glycosylceramides, Sphingolipids, triacylglycerols in serum samples from baseline and week 8. Samples were dispensed into 96-well plates and dried at room temperature for 30 minutes before adding 50 µL of phenylisothiocyanate (PITC). After an additional 20 minutes incubation at room temperature, the samples were dried under nitrogen for 1 hour. Ammonium acetate (3 mM) was added and the plates were shaken at room temperature for 30 minutes and centrifuged at 500× for 2 minutes.g150 μL from each well was then transferred to a 96-deep well plate and an equal volume of dH was added.₂O. Shake a 96-well plate containing 490 µL of FIA solvent with 10 µL of extract for 5 min at room temperature. 5 µL of sample sample from each well was injected onto the MxP Quant 500 kit column system and analyzed for amino acids and biogenic amines. Other metabolites were semi-quantified using the same mass spectrometer without column separation by flow injection analysis (FIA). Metabolite concentrations were calculated using Analyst/MetIDQ software and expressed as µM in serum.

microbial profiling

DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Manchester, UK). All reagents were provided by Qiagen unless otherwise noted. A fecal sample was weighed (220 mg), added to a 2 ml eppendorf tube with 1.4 ml of ASL buffer, vortexed and heated at 95°C for 5 min. The contents were added to a 2 ml screw cap tube containing 0.5 g of both 0.1 mm and 0.5 mm zirconium beads. The tubes were then added to a FastPrep 24 g5 bead beater (MP biomedicals, Eschwege, Germany) in 5 × 20 s bursts. The suspension was then spun at 14,000×.g 3 minutes. Supernatants (1.2 ml) were collected, transferred to QIAamp silica gel membrane tubes, spun for 3 minutes and DNA extracted according to the manufacturer’s instructions. Amplicon sequencing was performed on the Illumina MiSeq. Variable regions V4-V5 of the bacterial 16S ribosomal RNA gene were amplified from extracted DNA using PCR conditions and custom primers as described in the Microbiome Helper protocol.³¹Forward (515FB = GTGYCAGCMGCCGCGGTAA) and reverse (926 R = CCGYCAATTYMTTTRAGTTT) primers used Nextera Illumina index tags and sequence adapters fused to 16 S sequences. Each sample was amplified with a different combination of index barcodes to allow sample identification after multiplex sequencing. After amplification, paired-end 300 + 300 bp v3 sequencing was performed on all samples on the Illumina MiSeq.Raw sequence reads were trimmed as previously described³².

Analysis of 16S sequence data

Analysis of the 16 S sequence data was performed on the Hardiman Laboratory GRANDE server. Primer sequences were stripped from sequencing reads using cutadapt (v 1.14).³³and primer-trimmed files imported into QIIME2 (v. 2019.10.0)³⁴Forward and reverse paired-end reads were combined using VSEARCH (v 2.9.0).³⁵ Input to Deblur³⁶ Correct the reads to obtain the amplicon sequence variant (ASV).Taxonomy was assigned to ASV using the SILVA rRNA gene database³⁷ QIIME2, sklearn’s “feature-classifier” option. Rare sequences have been removed from the feature table. Sequences below any cutoff frequency of 100 were also removed. Estimates of alpha diversity (observed ASV, Shannon Diversity), beta diversity (weighted UniFrac) and relative abundance of ASV were obtained using QIIME2 (v 2019.10.0).Abundance difference tests were performed using the Analysis of Microbiome Composition (ANCOM) plugin³⁸.

statistical analysis

All data were tested for normality using Kologorov-Smirnoff and appropriate data were transformed. Group comparisons of tumor number, ACF and MDF were performed by one-way ANOVA with LSD post hoc test (two-tailed; SPSS version 21). Lipid hyperbinding markers DHN-MA and MDA at week 8 were compared between groups while controlling for baseline concentrations by ANCOVA. Metabolomics data were cube root transformed, mean centered scaled and compared using univariate (ANOVA), multivariate and hierarchical clustering tools within MetaboAnalyst 4.0 (http://metaboanalyst.ca).

Report overview

For more information on the study design, see the Nature Research Reporting Summary linked to this article.