Oxidized Polyunsaturated Fatty Acid Promotes Colitis and Colitis-Associated Tumorigenesis in Mice

Weicang Wang; Yuxin Wang; Katherine Z Sanidad; Yige Wang; Jianan Zhang; Wenqi Yang; Quancai Sun; Ipek Bayram; Renhua Song; Haixia Yang; David Johnson; Heather L Sherman; Daeyoung Kim; Lisa M Minter; Justin J-L Wong; Melody Y Zeng; Eric A Decker; Guodong Zhang

doi:10.1093/ecco-jcc/jjae148

. 2024 Sep 16;19(3):jjae148. doi: 10.1093/ecco-jcc/jjae148

Oxidized Polyunsaturated Fatty Acid Promotes Colitis and Colitis-Associated Tumorigenesis in Mice

Weicang Wang ^1,^2,^#,^✉, Yuxin Wang ^3,^4,^#, Katherine Z Sanidad ^5,^6,^7,^8,^#, Yige Wang ⁹, Jianan Zhang ¹⁰, Wenqi Yang ¹¹, Quancai Sun ¹², Ipek Bayram ¹³, Renhua Song ^14,¹⁵, Haixia Yang ¹⁶, David Johnson ¹⁷, Heather L Sherman ¹⁸, Daeyoung Kim ¹⁹, Lisa M Minter ^20,²¹, Justin J-L Wong ^22,²³, Melody Y Zeng ^24,²⁵, Eric A Decker ²⁶, Guodong Zhang ^27,^28,^✉

¹ Department of Food Science, University of Massachusetts, Amherst, MA, USA

² Department of Food Science, Purdue University, West Lafayette, IN, USA

³ Department of Food Science, University of Massachusetts, Amherst, MA, USA

⁴ Department of Food Science, Purdue University, West Lafayette, IN, USA

⁵ Department of Food Science, University of Massachusetts, Amherst, MA, USA

⁶ Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA, USA

⁷ Gale and Ira Drukier Institute for Children’s Health, Weill Cornell Medicine, New York, NY, USA

⁸ Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA

⁹ Department of Nutrition, University of California, Davis, Davis, CA, USA

¹⁰ Department of Food Science, University of Massachusetts, Amherst, MA, USA

¹¹ Department of Nutrition, University of California, Davis, Davis, CA, USA

¹² Department of Health, Nutrition, and Food Sciences, Florida State University, Tallahassee, FL, USA

¹³ Department of Food Science, University of Massachusetts, Amherst, MA, USA

¹⁴ Epigenetics and RNA Biology Program Centenary Institute, The University of Sydney, Camperdown, New South Wales, Australia

¹⁵ Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales, Australia

¹⁶ Department of Food Science, University of Massachusetts, Amherst, MA, USA

¹⁷ Department of Food Science, University of Massachusetts, Amherst, MA, USA

¹⁸ Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA

¹⁹ Department of Mathematics & Statistics, University of Massachusetts, Amherst, MA, USA

²⁰ Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA, USA

²¹ Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA

²² Epigenetics and RNA Biology Program Centenary Institute, The University of Sydney, Camperdown, New South Wales, Australia

²³ Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales, Australia

²⁴ Gale and Ira Drukier Institute for Children’s Health, Weill Cornell Medicine, New York, NY, USA

²⁵ Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA

²⁶ Department of Food Science, University of Massachusetts, Amherst, MA, USA

²⁷ Department of Food Science, University of Massachusetts, Amherst, MA, USA

²⁸ Department of Nutrition, University of California, Davis, Davis, CA, USA

Weicang Wang, Yuxin Wang and Katherine Z Sanidad contributed equally to this work.

^✉

Corresponding author: Weicang Wang, PhD, Department of Food Science, Purdue University, 745 Agriculture Mall Dr, West Lafayette, IN 47907, USA. Tel: 765-496-4298; Email: wang6205@purdue.edu

^✉

Corresponding author: Guodong Zhang, PhD, Department of Nutrition, University of California Davis, 3209 Meyer Hall, One Shields Avenue, Davis, CA 95616, USA. Tel: 530-752-4630; Email: gdzhang@ucdavis.edu

PMCID: PMC13032040 PMID: 39279209

Abstract

Background and Aims

Human studies suggest that a high intake of polyunsaturated fatty acid (PUFA) is associated with an increased risk of inflammatory bowel disease (IBD). PUFA is highly prone to oxidation. To date, it is unclear whether unoxidized or oxidized PUFA is involved in the development of IBD. Here, we aim to compare the effects of unoxidized PUFA vs oxidized PUFA on the development of IBD and associated colorectal cancer.

Methods

We evaluated the effects of unoxidized and oxidized PUFA on dextran sodium sulfate (DSS)-induced and IL-10 knockout-induced colitis, and azoxymethane/DSS-induced colon tumorigenesis in mice. Additionally, we studied the roles of gut microbiota and Toll-like receptor 4 (TLR4) signaling involved.

Results

Administration of a diet containing oxidized PUFA, at human consumption-relevant levels, increases the severity of colitis and exacerbates the development of colitis-associated colon tumorigenesis in mice. Conversely, a diet rich in unoxidized PUFA does not promote colitis. Furthermore, oxidized PUFA worsens colitis-associated intestinal barrier dysfunction and leads to increased bacterial translocation, and it fails to promote colitis in TLR4 knockout mice. Finally, oxidized PUFA alters the diversity and composition of gut microbiota, and it fails to promote colitis in mice lacking the microbiota.

Conclusions

These results support that oxidized PUFA promotes the development of colitis and associated tumorigenesis in mouse models via TLR4- and gut microbiota-dependent mechanisms. Our findings highlight the potential need to update regulation policies and industrial standards for oxidized PUFA levels in food.

Keywords: Polyunsaturated fatty acid, lipid oxidation, colitis, colitis-associated tumorigenesis

1. Introduction

The incidence and prevalence of inflammatory bowel disease (IBD), an inflammatory disease of the intestines that is a significant risk factor for developing colorectal cancer, have increased dramatically in recent decades.¹ Although the exact cause of IBD remains unknown, emerging research suggests that exposure to environmental or dietary risk factors contributes to the development of IBD and may be primarily responsible for the rapid increase in its incidence.^2–4 Identifying these risk factors is crucial since they are potentially preventable. However, the specific environmental or dietary risk factors remain poorly defined, hindering our efforts to reduce the risks of IBD.^2–4

Polyunsaturated fatty acids (PUFAs), such as linoleic acid (LA, 18:2 ω-6) and α-linolenic acid (ALA, 18:3), are essential components of the human diet and are widely recognized for their potential health benefits.⁵ However, recent human studies have suggested that high PUFA intake may be linked to an increased risk of developing IBD. A systematic analysis of 19 human studies, which included 2609 IBD patients and >4000 controls, revealed that high PUFA intake is associated with an increased risk of developing IBD.⁶ Furthermore, the European Prospective Investigation into Cancer and Nutrition (EPIC) study, a prospective cohort study comprising 203 193 human subjects, indicated that a high intake of LA is associated with more than double the risk of developing IBD.⁷ Other human studies also support the association between high PUFA intake and increased IBD risk.^8–14 Nevertheless, a limitation of the epidemiological studies is that they only establish a potential association between PUFA intake and IBD risk; whether PUFA is causally involved in IBD development remains largely unknown.⁴

PUFA in foods is commonly found in both unoxidized and oxidized forms.¹⁵ This is because PUFA is highly susceptible to oxidation during food production and storage; the addition of antioxidants to foods can slow the oxidation of PUFAs, but it cannot completely stop this process, as the antioxidants will eventually be consumed.¹⁵ Recent studies have shown that PUFA oxidation-derived compounds are frequently detected in PUFA-rich foods.^16,17 In previous human studies investigating PUFA intake and IBD, few studies have characterized the oxidative status of PUFA.^7–14 To date, it is unclear whether unoxidized or oxidized PUFA is involved in the development of IBD. Our recent study demonstrated that compared to a control diet rich in saturated fatty acids (SFAs), a diet high in column chromatography-purified corn oil, which is rich in unoxidized PUFA, did not impact the development of colitis in a well-established IBD model, the Il-10^−/− mice. This supports the notion that unoxidized PUFA has a limited role in IBD development.¹⁸ Therefore, we hypothesize that oxidized PUFA, rather than unoxidized PUFA, is a risk factor for IBD and associated diseases. In this study, we investigated the effects of unoxidized PUFA vs oxidized PUFA on the development of colitis and colitis-associated colorectal cancer in mice.

2. Methods

2.1. Study design

Our study aims to investigate the impact of unoxidized PUFA and oxidized PUFA on the development of colitis and related diseases. To do so, we prepared unoxidized PUFA and oxidized PUFA samples.

Consumption of vegetable oils is a major source of PUFA intake in humans.^19,20 In this study, we utilized corn oil as a representative PUFA-rich vegetable oil (see fatty acid profiles of corn oil in Supplementary Table S1). To prepare the sample of unoxidized PUFA, we purified commercially available fresh corn oil using silicic acid-activated charcoal chromatography.²¹ To prepare the sample of oxidized PUFA, we subjected the purified oil to oxidation under conditions that mimic household or commercial storage or transport of vegetable oil. Finally, we administered the PUFA diet and oxidized PUFA diet to mice using multiple mouse models (see the scheme of the overall experiment in Supplementary Figure S1).

2.2. Preparation of column chromatography-purified corn oil (unoxidized PUFA)

Commercial samples of fresh corn oil (Mazola, Cordova, TN) were purchased. Since oxidized PUFA compounds are commonly found in commercial samples of vegetable oils,¹⁶ we further purified the oil using silicic acid-activated charcoal chromatography, as described previously.²¹ The oxidative status of the purified corn oil was analyzed using a peroxide value (PV) assay (see details below). The fatty acid profile was analyzed by gas chromatography-mass spectrometry (GC-MS) (Supplementary Table S1).

2.3. Preparation of oxidized corn oil (oxidized PUFA)

To prepare oxidized corn oil, the purified corn oil (as described above, without the addition of tocopherols) was stored in a sealed bottle at 37°C and shielded from light, until it reached a PV of approximately 10 mEq/kg, a process typically taking 10-20 days. This process mimics household or commercial storage and transport conditions. We chose this PV since the current industry standard states that the maximum PV of acceptable fresh vegetable oil is 10 mEq/kg.¹⁵ The prepared oxidized oil was fortified with 400 ppm tocopherols (Sigma-Aldrich, St. Louis, MO), flushed with N₂, aliquoted, and stored at −80°C until use. The fatty acid profile was analyzed by GC-MS (Supplementary Table S1).

2.4. PV assay

The oil samples (~20 µL) were weighed, dissolved in 2.8 mL of methanol/butanol solution (2:1, vol/vol), and incubated with 30 µL ammonium thiocyanate (3.94 M)/ferrous solution (0.072 M) (1:1, vol/vol) at room temperature for 20 minutes. After incubation, the absorbance was measured at 510 nm using a plate reader (Molecular Devices, Sunnyvale, CA). The concentration of fatty acid hydroperoxides was calculated from a cumene hydroperoxide standard curve.²²

2.5. Liquid chromatography-tandem mass spectrometry-based lipidomics analysis of lipid oxidation compounds in the oils

A commercial sample of corn oil was purified using silicic acid-activated charcoal chromatography.²¹ The purified oil (without the addition of external antioxidants) was stored in a sealed bottle at 37°C and shielded from light for 10-20 days. On Days 10, 15, and 20, the oxidized oil was collected and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) lipidomics analysis.

To extract free lipid oxidation compounds from the oil, 10 μL of the collected purified or oxidized corn oil was mixed with 10 μL isotope-labeled surrogate standard solution, 10 μL antioxidant solution (containing 0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), 200 μL methanol (containing 0.1% acetic acid and 0.1% butylated hydroxytoluene), and 1600 μL Millipore water. The resulting solution was vortexed for 10 minutes and then extracted using solid phase extraction (SPE) columns. Briefly, Waters Oasis HLB SPE columns (60 mg, 3cc cartridges; Waters, Milford, MA) were preconditioned by washing twice with 3 mL ethyl acetate, twice with 3 mL methanol, and twice with 3 mL SPE washing buffer (95:5 water/methanol with 0.1% acetic acid). The oil sample was then loaded onto the column and washed twice with 3 mL washing buffer. The SPE filter was dried under vacuum for 20 minutes. Lipid oxidation compounds were eluted with 0.5 mL methanol and 1.5 mL ethyl acetate into 2-mL collection tubes containing 10 μL trap solution (30% glycerol in methanol). Samples were dried by vacuum centrifugation and reconstituted in 50 μL methanol containing 200 nM 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) as a surrogate recovery standard. The reconstituted samples were filtered using Ultrafree-MC VV Centrifugal Filter (0.1 μm; EMD Millipore, Bedford, MA) for LC-MS/MS analysis.

To extract total lipid oxidation compounds (a combination of free and esterified oxidation compounds) from the oil, 10 μL of the collected purified or oxidized corn oil was mixed with 10 μL isotope-labeled surrogate standard solution, 10 μL antioxidant solution, 200 μL methanol (containing 0.1% acetic acid and 0.1% butylated hydroxytoluene), and 200 μL 0.25 M sodium carbonate solution (prepared by dissolving 1.13 g sodium carbonate in a mixture of 21.3 mL water and 21.3 mL methanol). The solution was vortexed, heated with constant shaking for 30 minutes at 60°C, and then cooled to room temperature. Subsequently, 25 μL acetic acid was added to adjust the pH below 7, followed by 1575 μL Millipore water. The sample was then extracted using SPE columns as described above.

The LC-MS/MS analysis was performed on an Agilent 1200SL HPLC system coupled to a 4000 QTRAP MS/MS, as described in our previous reports.^23,24 Peaks were identified based on retention time and specific multiple reaction monitoring (MRM) transitions of the lipid metabolite standards. The concentrations of the lipid metabolites were calculated using calibration curves with standards.

2.6. Animal experiments

The animal experiments were conducted by the protocols approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Amherst.

2.7. Experimental diets

We treated mice with completely defined isocaloric diets, which contain the same amount of total fat (10 wt/wt%) but have different fat contents. The PUFA diet has a fat content of 10 wt/wt% of the column chromatography-purified corn oil, and the oxidized PUFA diet has a fat content of 10 wt/wt% of oxidized corn oil (see diet composition information in Supplementary Table S2).

2.8. Animal Protocol 1: effects of PUFA diet and oxidized PUFA diet on basal inflammation in healthy mice

C57BL/6 mice (age = 6 weeks; Charles River, Wilmington, MA) were treated with a PUFA diet or oxidized PUFA diet for 4 or 15 weeks. The diets were freshly prepared and changed every other day to minimize PUFA oxidation. At the end of the experiment, the mice were sacrificed for analysis.

2.9. Animal Protocol 2: effects of PUFA diet and oxidized PUFA diet on dextran sodium sulfate-induced colitis in mice

C57BL/6 mice (age = 6 weeks) were treated with a PUFA diet or oxidized PUFA diet throughout the experiment. The diets were prepared and changed every other day. After 3 weeks of feeding, the mice were administered 2% wt/vol dextran sodium sulfate (DSS; 36-50 kDa; MP Biomedicals, Solon, OH) in drinking water to induce colitis. After 7 days, the mice were sacrificed for analysis.

2.10. Animal Protocol 3: effects of PUFA diet and oxidized PUFA diet on spontaneous colitis in Il-10^−/− mice

Il-10 ^−/− mice (B6.129P2-Il10^tm1Cgn/J, age = 5 weeks) were purchased from Jackson Laboratories (Bar Harbor, ME) and were fed with a PUFA diet or oxidized PUFA diet for 15 weeks. The diets were prepared and changed every other day. At the end of the experiment, the mice were sacrificed for analysis.

2.11. Animal Protocol 4: effects of PUFA diet and oxidized PUFA diet on azoxymethane/DSS-induced colitis-associated colon cancer in mice

C57BL/6 mice (age = 6 weeks) were treated with a PUFA diet or oxidized PUFA diet throughout the experiment. The diets were prepared and changed every other day. After 3 weeks of feeding, the mice were injected intraperitoneally (i.p.) with 10 mg/kg azoxymethane (AOM; Sigma-Aldrich); 1 week later, the mice were treated with 2% DSS in drinking water for a week. At Week 7 after the AOM injection, the mice were sacrificed for analysis.

2.12. Animal Protocol 5: effects of PUFA diet and oxidized PUFA diet on colitis-associated gut barrier dysfunction

C57BL/6 mice (age = 6 weeks) were treated with a PUFA diet or oxidized PUFA diet throughout the experiment. The diets were prepared and changed every other day. After 3 weeks of feeding, the mice were administered 2% DSS in drinking water for 7 days to induce colitis. On Day 7 of the DSS treatment, the mice were treated with 600 mg/kg fluorescein isothiocyanate (FITC)-dextran (70 kDa, Sigma-Aldrich) by oral gavage; after 4 hours, blood samples were obtained by cardiac puncture. Plasma was isolated from the blood by centrifuging at 1500 g for 10 minutes at 4°C, and the fluorescence intensity of FITC-dextran in the plasma was quantified at 485 nm/528 nm and was calculated according to a standard curve.

2.13. Animal Protocol 6: effects of PUFA diet and oxidized PUFA diet on DSS-induced colitis in wild-type and Tlr4^−/− mice

Tlr4 ^−/− mice (B6.B10ScN-Tlr4^lps-del/JthJ, age = 6-7 weeks) and wild-type (WT) mice (C57BL/6J, https://www.jax.org/strain/000664, age = 6 weeks) were purchased from the Jackson Laboratories and were fed with PUFA diet or oxidized PUFA diet throughout the experiment. The diets were prepared and changed every other day. After 3 weeks of feeding, Tlr4^−/− and WT mice were administered with DSS (2% wt/vol) in drinking water to induce colitis.

2.14. Animal Protocol 7: effects of antibiotic cocktail-mediated suppression of gut microbiota on effects of PUFA vs oxidized PUFA on DSS-induced colitis

We used a well-established broad-spectrum antibiotic cocktail (1.0 g/L ampicillin and 0.5 g/L neomycin) from previous studies,^25,26 having shown that the antibiotic cocktail can effectively suppress the microbiota in mice.^27–30 C57BL/6 mice (age = 6 weeks) were treated with drinking water with the antibiotic cocktail during the whole experiment. Five days after antibiotic treatment, the mice were fed with a PUFA diet or oxidized PUFA diet for the next 4 weeks. The diets were prepared and changed every other day. After 3 weeks of diet treatment, the mice were stimulated with 2% DSS in drinking water for 7 days to induce colitis.

2.15. Animal Protocol 8: effects of diets rich in PUFA, oxidized PUFA, and SFA on DSS-induced colitis

C57BL/6 mice (age = 6 weeks) were randomly assigned to 4 groups and were treated with a completely defined isocaloric diet which contains the same amount of total fat (10 wt/wt%) but has a different fat content. The fat content of the diet is 10% column chromatography-purified corn oil (PUFA diet), 10% oxidized corn oil (oxidized PUFA diet), 9% lard + 1% column chromatography-purified corn oil (SFA Diet-1), or 9% lard + 1% oxidized corn oil (SFA Diet-2). The detailed diet composition is given in Supplementary Table S3. During animal feeding, the diets were prepared and changed every other day. After 3 weeks of feeding, the mice were administered with 2% DSS in drinking water to induce colitis.

2.16. Flow cytometry analysis of immune cell infiltration in colon tissues

Distal colon tissues were dissected, washed with cold phosphate-buffered saline (PBS), and digested with Hank’s Balanced Salt Solution (HBSS, Lonza, Basel, Switzerland) supplemented with 1 mM dithiothreitol (DTT) and 5 mM ethylenediaminetetraacetic acid (EDTA) for 2 hours at 4°C. The released cells were stained with FITC-conjugated anti-mouse CD45 antibody, PerCP/Cy5.5-conjugated anti-mouse F4/80 antibody, and isotype control antibody (BioLegend, San Diego, CA). The stained cells were analyzed using BD LSRFortessa cell analyzer, and data were analyzed using FlowJo software.

2.17. 16S rRNA sequencing of fecal microbiota

C57BL/6 male mice were fed with a PUFA diet or oxidized PUFA diet for 3 weeks. The fecal samples were collected, and total fecal DNA was extracted using QIAamp DNA Stool Mini Kit (Qiagen) following the manufacturer’s instructions. The quantity of the extracted DNA was measured using a NanoDrop Spectrophotometer. PCR was performed to amplify the bacteria community with primers (see Supplementary Table S4) that bound the V3-4 regions of the 16S rRNA gene. PCRs were performed in a 96-well format on a Veriti thermal cycler (Life Technology) with 2 × KAPA HiFi Hotstart ReadyMix (KAPA Biosystem). After quantification and qualification, samples were pooled in equimolar amounts, and pair-end 2 × 300 bp sequencing was performed on the Illumina MiSeq platform and MiSeq Reagent Kit V3 (Illumina).

2.18. Bioinformatic analysis

The 16S rRNA raw sequences were imported to Quantitative Insights Into Microbial Ecology version 2 (QIIME2) software for demultiplexing and quality filtering.³¹ We used DADA2 to denoise sequences and construct a feature table.³² A Custom Naïve Bayesian classifier was constructed for taxonomic classification against the SILVA database release 132 based on the amplified 16S region.³³ The difference in α diversity was tested using the Kruskal-Wallis test. The difference in β diversity was tested using the Permutational Multivariate Analysis of Variance (PERMANOVA), followed by Benjamini-Hochber (BH) correction. Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) was used for the differential abundance analysis.³⁴

2.19. Histology

For hematoxylin and eosin (H&E) staining, formalin-fixed tissue was embedded in paraffin, sliced into 5-µm sections, dewaxed in serial xylene, rehydrated through graded ethanol solutions, stained with H&E (Sigma-Aldrich), and examined with light microscopy. The pathological scores were evaluated by a blinded observer according to the parameters including crypt architecture disruption, degree of inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess. The histological damage score is the sum of each score.³⁵

2.20. Immunohistochemical staining

For immunohistochemistry analysis, antigen retrieval was performed by heating the sections in 0.01 M citrate buffer (pH 6.0) to 95°C for 10 minutes. Samples were incubated with anti-proliferating cell nuclear antigen (PCNA) antibody (Dako, Carpinteria, CA; 1:1000 dilution) and anti-β-catenin antibody (BD Biosciences, Franklin Lakes, NJ; 1:1000 dilution) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies were then applied to the sections, followed by the chromogen 4-diaminobenzidine staining (Abcam, Cambridge, MA).

2.21. ELISA analysis

For plasma cytokines analysis, blood samples were harvested via cardiac puncture of the mice. The concentrations of cytokines in plasma were determined using a CBA Mouse Inflammation Kit (BD Biosciences) according to the manufacturer’s instructions. For colonic cytokines analysis, the distal section of the colon was excised and cut into 1-cm² sections. Tissues were washed in PBS containing penicillin and streptomycin, and the weight of each section was recorded. The colon section was placed in complete RPMI medium 1640 with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin and cultured at 37°C for 24 hours. The supernatants were harvested, and cytokines were determined using the CBA Mouse Inflammation Kit.

2.22. qRT-PCR analysis of gene expression in colon tissues

Colon tissues from the same locations were grounded after being frozen in liquid nitrogen. Total RNA was isolated from the colon tissues using TRIzol reagent (Ambion, Austin, TX). The quality of the extracted RNA was measured using a NanoDrop Spectrophotometer, and the extracted RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). qRT-PCR was performed in a DNA Engine Opticon system (Bio-Rad Laboratories, Hercules, CA) with Maxima SYBR-Green Master Mix (Thermo Fisher Scientific). The sequences of mouse-specific primers (Thermo Fisher Scientific) are listed in Supplementary Table S4. The results of target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh).

2.23. qRT-PCR analysis of 16S rRNA in blood

Whole blood samples were collected, and the total DNA was extracted using QIAamp DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) following the manufacturer’s instruction with the addition of a bead-beating step. The quality of the extracted DNA was measured using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA), and qRT-PCR was performed using the same amount of DNA (5 ng/μL) in a DNA Engine Opticon system with Maxima SYBR-Green Master Mix. The sequences of 16S rRNA primers are listed in Supplementary Table S4.

2.24. Assessment of TLR4 ligand levels in serum

We used a Toll-like receptor 4 (TLR4) reporter assay to measure the levels of TLR4 ligands in mouse serum. HEK-Blue mTLR4 cells (InvivoGen, San Diego, CA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% FBS and HEK-Blue Selection at 37°C under an atmosphere with 5% CO₂. A suspension of 2 × 10⁴ HEK-Blue mTLR4 cells was prepared in 200 µL of HEK-Blue detection medium per well in a 96-well plate, and then 4 µL of mouse serum (2% vol/vol) was added to each well. After 24-hour incubation at 37°C, the production of secreted embryonic alkaline phosphatase (SEAP) was assessed by reading the absorbance at 620 nm with a plate reader (Molecular Devices).

2.25. Effects of corn oil and oxidized corn oil on TLR4 activation in vitro

HEK-Blue mTLR4 cells were treated with different compounds for 16 hours to analyze their direct effects on the activation of TLR4. PBS buffer was used as a negative control, and the PBS extract of mouse feces was used as a positive control. To prepare the extract, 0.23 g of mouse feces was dissolved in 1 mL of PBS buffer. Unoxidized corn oil was used at a concentration of 10 µg/mL, while oxidized corn oil was used at concentrations ranging from 0.1 to 10 µg/mL.

2.26. Lipopolysaccharide determination in plasma

Plasma lipopolysaccharide (LPS) levels were quantified using the LPS ELISA kit (MBS261904, MyBiosource, San Diego, CA) following the manufacturer’s instructions.

2.27. Data analysis

All data are expressed as the mean ± SEM. For the comparison between diet treatment groups, the Shapiro-Wilk test was used to verify the normality of data. When data were normally distributed, statistical significance was determined using a 2-sided t test; otherwise, significance was determined by the Mann-Whitney U test. Analysis of inflammation in Tlr4^−/− mouse experiments according to mouse type and treatment was performed by 2-way ANOVA, followed by Tukey-Kramer’s method, and H&E histology data in these experiments was analyzed by 2-way ANOVA Poisson Generalized Linear Model, followed by the Tukey-Kramer’s multiple comparison method. The statistical analyses were performed using SAS statistical software, and p-values <0.05 were considered statistically significant.

3. Results

3.1. Preparation of unoxidized and oxidized PUFA

Our study aims to compare the effects of unoxidized PUFA vs oxidized PUFA on the development of colitis and associated colorectal cancer. To do so, we prepared unoxidized and oxidized PUFA samples. A major source of PUFA intake comes from vegetable oil and its products,^19,20 and here we used corn oil which contains high levels of PUFA. To prepare unoxidized PUFA, we used column chromatography to purify the commercial sample of corn oil,²¹ since oxidized compounds are commonly found in commercial oil samples.¹⁶ The peroxide assay showed that the PV (a maker of PUFA oxidation) of the purified corn oil was as low as 0.12 ± 0.02 mEq/kg, validating its low oxidative status. To prepare an oxidized corn oil sample, the purified corn oil, which is highly susceptible to oxidation due to the removal of antioxidants by chromatography purification,²¹ was stored in a sealed bottle without light until it reached a PV of ~10 mEq/kg. We chose this PV since this is the maximum PV of acceptable fresh vegetable oil according to the current industry standard.^15,36

PUFA oxidation generates a large array of oxidation compounds.^15,36 We used an LC-MS/MS-based lipidomics approach to analyze the profiles of PUFA oxidation products in oxidized corn oil. This lipidomics approach can analyze over 80 compounds that are produced from the enzymatic or nonenzymatic oxidation of several PUFAs, including LA, arachidonic acid (ARA, 20:4 ω-6), ALA, eicosapentaenoic acid (EPA, 20:5 ω-3), and docosahexaenoic acid (DHA, 22:6 ω-3) (see the list of compounds included in our LC-MS/MS method in Supplementary Table S5).³⁷ We analyzed both free and esterified oxidation compounds in the oils (see the experimental details in the “Methods” section). Compared to the purified corn oil, the concentrations of a series of LA- and ALA-derived oxidation products are increased in the oxidized corn oil, with the LA oxidation products being more abundant than ALA products. The increased compounds include (±)-9-hydroxyoctadecadienoic acid (9-HODE), (±)-13-hydroxyoctadecadienoic acid (13-HODE), 9-oxo-octadecadienoic acid (9-OxoODE), 13-oxo-octadecadienoic acid (13-OxoODE), 9-oxo-11-(3-pentyl-2-oxiranyl)-undecenoic acid (EKODE), 9,10,13-trihydroxy-octadecenoic acid (9,10,13-TriHOME), and 9,12,13-trihydroxy-octadecenoic acid (9,12,13-TriHOME) derived from the oxidation of LA (Supplementary Figure S2A), as well as 9-hydroxyoctadecatrienoic acid (9-HOTrE), 13-hydroxyoctadecatrienoic acid (13-HOTrE), and 9,10-epoxyoctadecadienoic acid (9,10-EpODE) derived from ALA oxidation (Supplementary Figure S2B). The detected oxidation compounds are predominately present as esterified forms since the concentrations of esterified oxidation compounds are higher than those of free oxidation compounds (Supplementary Figure S2). Besides the oxidation products from LA and ALA, the oxidation products from other PUFAs were not detected. Overall, these results support that during corn oil oxidation, LA and ALA, which are the 2 PUFAs in corn oil, are oxidized.

3.2. Oxidized PUFA does not induce basal colonic inflammation in healthy mice

We treated mice with a completely defined isocaloric diet which contains the same amount of total fat (10 wt/wt%) but has a different fat content. The PUFA diet has a fat content of 10 wt/wt% of the column chromatography-purified corn oil, and the oxidized PUFA diet has a fat content of 10 wt/wt% of oxidized corn oil (see diet composition in Supplementary Table S2). After 4 or 15 weeks of treatment, compared with the PUFA diet, treatment with the oxidized PUFA diet did not cause any significant effect on basal colonic inflammation in mice, as assessed by colon length, spleen weight, concentrations of proinflammatory cytokines in plasma, expression of proinflammatory genes in the colon, and histology of colon tissue (Supplementary Figures S3 and S4). These results suggest that oxidized PUFA does not induce basal colonic inflammation in mice.

3.3. Oxidized PUFA increases the severity of chemically induced colitis in mice

We treated mice with PUFA diet or oxidized PUFA diet and then stimulated the mice with DSS to induce colitis (see the scheme of experiment in Figure 1A). Compared with PUFA diet, treatment with oxidized PUFA diet reduced colon length (p < 0.01, Figure 1B), enlarged spleen tissue (p < 0.05, Figure 1C), exaggerated crypt damage in the colon (p < 0.001, Figure 1D), increased infiltration of leukocytes (CD45⁺) and macrophages (CD45⁺ F4/80⁺) into the colon (p < 0.05, Figure 1E and F, see representative FACS images in Supplementary Figure S5), enhanced expression of proinflammatory genes Il-1β and Tnf-α in the colon (p < 0.05, Figure 1G), and increased concentrations of TNF-α in both plasma and colonic explant (p < 0.05, Figure 1H and I). Together, these results suggest that oxidized PUFA increased the severity of DSS-induced colitis in mice.

Oxidized PUFA increases the severity of DSS-induced colitis in mice. A, C57BL/6 mice were treated with PUFA diet or oxidized PUFA diet and then stimulated with DSS to induce colitis. B, Colon length. C, Spleen weight. D, H&E staining of the colon (magnification ×300). E, FACS quantification of CD45⁺ cells in the colon. F, FACS quantification of CD45⁺ F4/80⁺ cells in the colon. G, Gene expressions in the colon (n = 5-8 mice per group). H, Concentrations of TNF-α in plasma. I, Concentrations of TNF-α in the colonic explant. The data are mean ± SEM, n = 7-10 mice per group. Abbreviations: DSS, dextran sodium sulfate; H&E, hematoxylin and eosin; Oxi-PUFA, oxidized polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid.

3.4. Oxidized PUFA, but not unoxidized PUFA, increases the severity of chemically induced colitis in mice

To better understand the effects of oxidized PUFA on colitis, we treated mice with diets rich in unoxidized PUFA (column chromatography-purified corn oil), oxidized PUFA (oxidized corn oil), or SFAs (lard) and then stimulated the mice with DSS to induce colitis (see the scheme of experiment in Figure 2A and diet composition in Supplementary Table S3). Compared with the diet rich in SFAs, treatment with the diet rich in unoxidized PUFA does not affect DSS-induced colitis, akin to our previous study in Il-10^−/− mice¹⁸; in contrast, treatment with oxidized PUFA diet significantly increased the severity of DSS-induced colitis in mice, with increased infiltration of macrophages and enhanced crypt damage in the colon (Figure 2B and C). These findings support the conclusion that oxidized PUFA, rather than PUFA itself (unoxidized PUFA), exacerbates the development of colitis.

Oxidized PUFA, but not unoxidized PUFA, exacerbates the development of DSS-induced colitis in mice. A, C57BL/6 mice were treated with diets that contain the same amount of total fat (10 wt/wt%) but have different fat content and then stimulated with DSS to induce colitis. The fat content of the diet is 10% column chromatography-purified corn oil (PUFA diet), 10% oxidized corn oil (Oxi-PUFA diet), 9% lard + 1% purified corn oil (SFA Diet-1), or 9% lard + 1% oxidized corn oil (SFA Diet-2). B, FACS quantification of immune cells in the colon. C, H&E staining of the colon (magnification ×300, n = 6-7 mice per group). The data are mean ± SEM, n = 8-9 mice per group. Abbreviations: DSS, dextran sodium sulfate; H&E, hematoxylin and eosin; Oxi-PUFA, oxidized polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid.

3.5. Oxidized PUFA increases the development of spontaneous colitis in Il-10^−/− mice

We further studied the effect of oxidized PUFA on colitis in another colitis model, the Il-10^−/− mice. We treated Il-10^−/− mice with PUFA diet or oxidized PUFA diet for 15 weeks (see the scheme of experiment in Figure 3A). We found that treatment with oxidized PUFA diet reduced colon length (p < 0.05, Figure 3B), increased infiltration of CD45⁺ cells into the colon and small intestine (Figure 3C, see representative FACS images in Supplementary Figure S6), enhanced expression of proinflammatory cytokines (Tnf-α, Tlr-4, Mcp-1, and Ifn-γ) in the colon (p < 0.05, Figure 3D), increased plasma concentrations of TNF-α and IFN-γ (p < 0.05, Figure 3E), and exaggerated crypt damage in the colon (p < 0.05, Figure 3F). Together, these results demonstrate that oxidized PUFA exacerbated spontaneous colitis in the Il-10^−/− mice, further supporting the colitis-enhancing effects of oxidized PUFA.

Oxidized PUFA increases the development of spontaneous colitis in *Il-10*^−/− mice. A, *Il-10*^−/− mice were treated with PUFA diet or oxidized PUFA diet for 15 wk. B, Colon length. C, FACS quantification of CD45⁺ cells in the colon and small intestine. D, qRT-PCR analysis of gene expressions in the colon. E, Concentrations of TNF-α and IFN-γ in plasma. F, H&E staining of the colon (magnification ×300, n = 5-6 mice per group). The data are mean ± SEM, n = 8-10 mice per group. Abbreviations: Oxi-PUFA, oxidized polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid.

3.6. Oxidized PUFA increases the development of colitis-associated colorectal cancer in mice

We treated mice with PUFA diet or oxidized PUFA diet and then stimulated the mice with AOM and DSS to induce colorectal tumorigenesis (see the scheme of experiment in Figure 4A). Compared with the AOM/DSS mice treated with PUFA diet, the AOM/DSS mice treated with oxidized PUFA diet had increased tumor size and total tumor burden (p < 0.05, Figure 4B), higher expression of β-catenin and PCNA in the colon (p ≤ 0.01, Figure 4C), increased colonic infiltration of CD45⁺ and CD45⁺ F4/80⁺ immune cells (p < 0.05, Figure 4D, see representative FACS images in Supplementary Figure S7), enhanced colonic expressions of proinflammatory genes (Il-1β, Tlr-4, Mcp-1, and Tnf-α) and reduced expressions of genes that are critical in maintaining gut barrier functions (Occludin and Tff3) (p < 0.05, Figure 4E), and increased concentrations of proinflammatory cytokines (MCP-1 and TNF-α) in colonic explants (p < 0.05, Figure 4F). Together, these results demonstrate that oxidized PUFA increased the development of colitis-associated colorectal tumorigenesis in mice.

Oxidized PUFA increases AOM/DSS-induced colon tumorigenesis in mice. A, C57BL/6 mice were treated with PUFA diet or oxidized PUFA diet, and stimulated with AOM and DSS to induce colon tumorigenesis. B, Quantification of colon tumorigenesis in mice (n = 11-12 mice per group). C, Representative images of immunohistochemical staining of PCNA, β-catenin, and H&E (magnification ×300,), and quantification of immunohistochemical images (n = 6 mice per group). D, FACS quantification of immune cells in the colon (n = 6-9 mice per group). E, Gene expressions in the colon (n = 4-5 mice per group). F, Concentrations of MCP-1 and TNF-α in the colonic explant (n = 7-9 mice per group). The data are mean ± SEM. Abbreviations: AOM, azoxymethane; DSS, dextran sodium sulfate; H&E, hematoxylin and eosin; Oxi-PUFA, oxidized polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid.

3.7. Oxidized PUFA increases colitis through TLR4-dependent mechanisms

TLR4 is an important microbial receptor involved in the recognition of bacteria and bacterial products such as LPS and has been linked to the development of colitis and colorectal cancer.³⁸ To investigate the role of TLR4 in the colitis-enhancing effects of oxidized PUFA, we analyzed the effects of oxidized PUFA treatment on the abundance of TLR4 ligands in circulation (see the scheme of the experiment in Figure 5A). Our results showed that the plasma from oxidized PUFA diet-treated DSS mice had a higher abundance of TLR4 ligands compared to mice treated with PUFA diet, as assessed using a TLR4 reporter cell line-based assay (p < 0.05, Figure 5B). We also measured the circulating concentrations of TLR4 ligands, such as LPS and bacteria.³⁸ Our results showed that treatment with oxidized PUFA diet increased the concentration of LPS (p < 0.05, Figure 5C) and upregulated the expression of 16S rRNA gene (p < 0.05, Figure 5D), a marker of bacterial abundance, in the circulation. We also tested whether oxidized PUFA has any direct effects on TLR4 activation but found no significant effects using the TLR4 reporter cell line assay (Supplementary Figure S8). Overall, our data suggest that the oxidized PUFA diet induces the translocation of bacteria or LPS, many of which are TLR4 ligands, from the gut into the systemic circulation.

Oxidized PUFA exacerbates colitis-associated gut barrier dysfunction, leading to increased bacterial translocation. A, C57BL/6 mice were treated with PUFA diet or oxidized PUFA diet and then stimulated with DSS to induce colitis. B, The abundance of TLR4 ligands in mouse plasma assessed using a TLR4 reporter assay (n = 7-8 mice per group). C, Concentrations of LPS in the plasma (n = 5-6 mice per group). D, Expression of *16S rRNA* gene in the blood (n = 8 mice per group). E, Plasma concentrations of FITC-dextran measured 4 h after oral gavage of FITC-dextran (n = 8 mice per group). F, Gene expressions in the colon (n = 8-9 mice per group). The results are mean ± SEM. Abbreviations: DSS, dextran sodium sulfate; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; Oxi-PUFA, oxidized polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid; TLR, Toll-like receptor.

Next, we determined the mechanisms by which oxidized PUFA induced bacterial translocation. We hypothesize that oxidized PUFA exacerbates colitis-associated gut barrier dysfunction, leading to increased translocation of bacteria and/or bacterial products from the gut into systemic circulation. Using a FITC-dextran-based permeability assay, we found that compared with the DSS mice treated with the PUFA diet, the DSS mice treated with oxidized PUFA diet had enhanced leakage of FITC-dextran from the gut to the circulation (p < 0.001, Figure 5E), suggesting that oxidized PUFA impaired intestinal barrier function. In agreement with the FITC-dextran permeability assay, we found that oxidized PUFA diet reduced colonic expressions of Muc3, Tff3, and Occludin, which are important mediators of intestinal barrier function (p ≤ 0.01, Figure 5F).³⁹ Together, these results support that treatment with oxidized PUFA diet impaired intestinal barrier function, leading to enhanced bacterial translocation and resulting in increased levels of TLR4 ligands in the circulation.

Finally, we examined the functional roles of TLR4 in the colitis-enhancing effects of oxidized PUFA. We treated WT mice or Tlr4^−/− mice with PUFA diet or oxidized PUFA diet and then stimulated the mice with DSS to induce colitis (see the scheme of experiment in Figure 6A). In the WT mice, treatment with oxidized PUFA diet exacerbated the development of DSS-induced colitis, with enhanced colonic infiltration of CD45⁺ and CD45⁺ F4/80⁺ immune cells (p < 0.05, see representative FACS images in Supplementary Figure S9), and exaggerated crypt damage in the colon tissues (p < 0.0001); while all these effects were abolished in the Tlr4^−/− mice (Figure 6B–D). Two-way ANOVA showed that there was a significant interaction between mouse type (Tlr4^−/− mice vs WT mice) and diet (PUFA diet vs oxidized PUFA diet) on the development of DSS-induced colitis (p < 0.05, Figure 6B–D). These results demonstrate that TLR4 is required for the colitis-enhancing effect of oxidized PUFA.

TLR4 is required for the colitis-enhancing effects of oxidized PUFA. A, WT and *Tlr4*^−/− mice were treated with PUFA diet or oxidized PUFA diet and then stimulated with DSS to induce colitis. B, C, FACS quantification of immune cells in the colon. D, H&E staining of the colon. The results are mean ± SEM, n = 6-8 mice per group. Statistical significance (p-value) of the interaction effect between mouse type (*Tlr4*^−/− mice vs WT mice) and treatment (PUFA diet vs oxidized PUFA diet) on colitis was determined by 2-way ANOVA analysis. Abbreviations: ANOVA, analysis of variance; DSS, dextran sodium sulfate; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; Oxi-PUFA, oxidized polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid; TLR, Toll-like receptor; WT, wild type.

3.8. Oxidized PUFA increases colitis through gut microbiota-dependent mechanisms

Having demonstrated that the microbial receptor TLR4 is critical for the colitis-enhancing effects of oxidized PUFA, we studied the role of the gut microbiota. First, we analyzed the effects of oxidized PUFA on the diversity and composition of gut microbiota. We treated mice with PUFA diet or oxidized PUFA diet for 3 weeks and then performed 16S rRNA sequencing to analyze the microbiota (Figure 7A). We found that treatment with oxidized PUFA diet showed a trend to reduce α diversity, as assessed using Shannon (p = 0.048), Simpson (p = 0.0504), and Observed (p = 0.0697) indices (Figure 7B). Oxidized PUFA diet also modulated β diversity of the microbiota (p = 0.011, Figure 7C). In addition, oxidized PUFA diet changed the composition of the microbiota at both phylum and genus levels (Figure 7D and E and Supplementary Tables S6 and S7). The oxidized PUFA diet increased the relative abundance of the genus Streptococcus (0.06 ± 0.02% in the PUFA group vs 0.22 ± 0.05% in the oxidized PUFA group, p = 0.005) in the microbiota (Supplementary Table S7). Previous studies have shown that some bacteria from this genus are linked with colitis.⁴⁰ Overall, these results support that dietary intake of oxidized PUFA alters the microbiota.

Oxidized PUFA alters the gut microbiota in mice. A, C57BL/6 mice were treated with PUFA diet or oxidized PUFA diet for 21 d, then the fecal samples were collected for sequencing. B, Alpha diversity of the microbiota. C, Beta diversity of the microbiota. D, Composition of the microbiota at phylum levels. E, Composition of the microbiota at genus levels. The data are mean ± SEM, n = 13 mice per group. Abbreviation: PUFA, polyunsaturated fatty acid.

Next, we determined the roles of gut microbiota in the biological effects of oxidized PUFA. We have shown that when the mice were maintained on normal drinking water, the mice treated with oxidized PUFA demonstrated increased severity of DSS-induced colitis compared with those treated with PUFA (Figures 1 and 2). Here, we tested the extent to which antibiotic-mediated suppression of gut microbiota modulates the effects of PUFA vs oxidized PUFA on DSS-induced colitis (see the scheme of experiment in Figure 8A). We used a well-established broad-spectrum antibiotic cocktail from previous studies,^25,26 and our previous studies have shown that the cocktail effectively suppressed the microbiota in mice.^27–30 Here, we found that treatment with the antibiotic cocktail caused a dramatic reduction of total fecal bacteria in mice (Supplementary Figure S10), further validating its suppressing effects on the microbiota. When mice were treated with antibiotics, those given PUFA or oxidized PUFA showed similar development of DSS-induced colitis, as assessed by colon length, colonic infiltration of immune cells, and colon histology (Figure 8B–E). This result supports that gut microbiota plays a critical role in the colitis-enhancing effect of oxidized PUFA.

Oxidized PUFA increases colitis through gut microbiota-dependent mechanisms. A, We used antibiotic-mediated suppression of gut microbiota to determine the roles of the microbiota in the colitis-enhancing effects of oxidized PUFA. B, Colon length. C, FACS quantification of CD45⁺ immune cells in the colon. D, FACS quantification of CD45⁺F4/80⁺ macrophages in the colon. E, H&E staining of the colon. The data are mean ± SEM, n = 7-9 mice per group. Abbreviations: H&E, hematoxylin and eosin; ns, not significant; PUFA, polyunsaturated fatty acid.

4. Discussion

Oxidized PUFA is a common component of the human diet.^15–17 To date, there are no governmentally established limits to define the levels of oxidized PUFA in foods. The food industry has presented its limits, with a maximum PV of 10 mEq/kg for acceptable fresh vegetable oil, but the industrial regulation is not strictly enforced.^15,36 The weak regulation is, at least in part, due to a lack of data regarding the health impact of oxidized PUFA, since previous studies generally support that the oral toxicity of oxidized PUFA is low.^41–43 Herein, our central finding is that dietary intake of oxidized PUFA-rich vegetable oil, even at low oxidative status (PV ≤ 10 mEq/kg, which is within the current industry standard of fresh vegetable oil), increased the severity of colitis and exacerbated the development of colitis-associated colorectal cancer in mouse models. These findings demonstrate that dietary intake of oxidized PUFA, at human consumption levels, could negatively affect gut health in mouse models. A better understanding of the effects of oxidized PUFA on gut health could help establish regulatory policies or industrial standards about the levels of oxidized PUFA in foods.

Human epidemiological studies support that a high intake of PUFA is associated with increased risks of developing IBD.^4,6–14 However, whether PUFA is causally involved in the development of IBD remains largely unknown.⁴ Here, we showed that oxidized PUFA, rather than unoxidized PUFA, is a dietary risk factor for IBD and associated diseases in mouse models. We showed that compared with a diet rich in SFAs, administration of a diet rich in unoxidized PUFA did not impact DSS-induced colitis, akin to our previous study in Il-10^−/− mice¹⁸; in contrast, treatment with a diet rich in oxidized PUFA increased the severity of DSS-induced colitis in mice. Additionally, treatment with the oxidized PUFA diet also increased spontaneous colitis in Il-10^−/− mice and exacerbated the development of AOM/DSS-induced colorectal tumorigenesis in mice. Our results suggest that the effects of oxidized PUFA on colitis are dose-dependent. When mice were treated with a low level of oxidized PUFA in the context of an SFA-rich diet, the oxidized PUFA treatment did not promote the development of colitis. Together, our findings support that oxidized PUFA, rather than PUFA itself, promotes the development of IBD and associated colorectal cancer in mouse models. These results could, at least in part, explain the previously observed IBD-enhancing effects of dietary PUFA in human epidemiological studies. Based on our findings, individuals with or prone to IBD could be susceptible to the adverse effects of oxidized PUFA, and these high-risk individuals may need to reduce the consumption of food products containing high levels of oxidized PUFA, though further studies are needed before dietary recommendations could be established.

Our findings are largely in agreement with recent studies that showed that oxidized PUFA or PUFA oxidation-derived compounds promote intestinal inflammation. Notably, recent studies showed that a PUFA-rich diet induced small intestinal inflammation in Gpx4^+/−IEC mice, which had reduced expression of glutathione peroxidase 4 (GPX4), an antioxidant enzyme that protects against PUFA oxidation; while the PUFA-rich diet had no such effects in WT mice.^12,44 In addition, enhancing PUFA oxidation (through knockdown of Gpx4 or addition of iron) increases PUFA’s effects on cytokine production in vitro, while inhibiting PUFA oxidation (through the addition of iron chelators or other antioxidants) attenuates these effects.⁴⁴ These results are largely consistent with our conclusion that oxidized PUFA, rather than unoxidized PUFA, increases the risks of IBD.

PUFA oxidation produces a wide range of oxidation compounds.^15,36 In this study, we employed an LC-MS/MS-based lipidomics method to analyze the profiles of oxidation compounds in oxidized corn oil. We observed increased levels of LA and ALA oxidation products in oxidized corn oil compared to purified oil, with the oxidation compounds being predominantly found in esterified forms. LC-MS/MS analysis revealed that concentrations of the detected LA oxidation products ranged from nanomolar to low micromolar (Supplementary Figure S2). In contrast, the LA concentration in corn oil is calculated to be 1-2 M. This suggests that only a small fraction of LA undergoes oxidation during the storage-induced oxidation process, consistent with our GC-MS analysis showing similar LA levels between the purified and oxidized corn oils (Supplementary Table S1). To date, the specific lipid oxidation compounds responsible for the pro-colitis effects of oxidized PUFA are unknown. Here, we found that the concentration of EKODE is increased in the oxidized corn oil compared to the purified oil (Supplementary Figure S2). Our recent study demonstrated that systemic treatment with EKODE, as well as other LA oxidation products, exacerbated DSS-induced colitis or AOM/DSS-induced colorectal tumorigenesis in mouse models.^24,45,46 We would like to point out that in our studies,^24,45,46 we treated mice with the oxidation compounds, such as EKODE, via i.p. injection, but not via oral administration, because these compounds are chemically or metabolically unstable.^24,45,46 Thus, it is likely that other PUFA oxidation-derived compounds, or a combination thereof, contribute to the observed oral toxicity of oxidized PUFA. Identifying the specific oxidation compounds responsible for these health effects will enhance our understanding of the underlying molecular mechanisms and aid in developing potential biomarkers to improve food quality.

Our results support that the gut microbial factors play critical roles in mediating the colitis-enhancing effects of oxidized PUFA. Previous studies showed that after consumption of dietary fatty acids, most of the fatty acids are absorbed in the small intestine, while the remaining unabsorbed fatty acids could enter the colon.^47,48 In addition, the PUFA oxidation-derived compounds could enter the gastrointestinal tract and have substantial interactions with commensal microbes that reside in the intestines. We found that oxidized PUFA intake altered the diversity and composition of gut microbiota. We would like to point out that a limitation of our study is that we did not analyze the basal gut microbiota (t = Week 0 in the animal experiment of Figure 7A). Using antibiotic-mediated suppression of gut microbiota, we showed that microbiota plays a critical role in the colitis-enhancing effects of oxidized PUFA. In addition, our results support that the microbial receptor TLR4 also contributes to the colitis-promoting effects of oxidized PUFA. In colitis mice, the oxidized PUFA diet exacerbated colitis-associated gut barrier dysfunction, leading to increased translocation of bacteria or bacterial products, many of which are TLR4 ligands, from the gut into the systemic circulation. The oxidized PUFA diet failed to promote colitis in Tlr4^−/− mice, supporting that TLR4 is required for the effects of oxidized PUFA. In our study, we did not use the approach of germ-free mice, because it usually requires irradiation to create a sterile feed for germ-free mice⁴⁹; but during the irradiation process, the oils in the diets, notably the column chromatography-purified corn oil (rich in unoxidized PUFA), could be degraded and/or oxidized, making it difficult to compare the effects of unoxidized PUFA vs oxidized PUFA. Overall, these results support that gut microbial factors play critical roles in mediating the colitis-enhancing effects of oxidized PUFA.

In summary, our findings showed that oxidized PUFA, rather than PUFA itself (unoxidized PUFA), exacerbated colitis and associated disease and could therefore contribute to the previously observed IBD-enhancing effects of dietary PUFA in human studies. Based on our findings, individuals with or prone to IBD might need to reduce their consumption of oxidized PUFA. In our study, we used corn oil which is rich in ω-6 PUFA. Besides ω-6 PUFA, previous studies have shown that other dietary PUFA, notably ω-3 PUFA, is also highly prone to oxidation,^16,17 and the oxidized ω-3 PUFA could also have adverse effects on gut health. Further studies are needed to better understand the impact of unoxidized vs oxidized PUFA on human health, which could help to establish novel regulation policies, industrial standards, and/or dietary recommendations.

Supplementary Material

jjae148_suppl_Supplementary_Materials

jjae148_suppl_supplementary_materials.pdf^{(976KB, pdf)}

Contributor Information

Weicang Wang, Department of Food Science, University of Massachusetts, Amherst, MA, USA; Department of Food Science, Purdue University, West Lafayette, IN, USA.

Yuxin Wang, Department of Food Science, University of Massachusetts, Amherst, MA, USA; Department of Food Science, Purdue University, West Lafayette, IN, USA.

Katherine Z Sanidad, Department of Food Science, University of Massachusetts, Amherst, MA, USA; Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA, USA; Gale and Ira Drukier Institute for Children’s Health, Weill Cornell Medicine, New York, NY, USA; Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA.

Yige Wang, Department of Nutrition, University of California, Davis, Davis, CA, USA.

Jianan Zhang, Department of Food Science, University of Massachusetts, Amherst, MA, USA.

Wenqi Yang, Department of Nutrition, University of California, Davis, Davis, CA, USA.

Quancai Sun, Department of Health, Nutrition, and Food Sciences, Florida State University, Tallahassee, FL, USA.

Ipek Bayram, Department of Food Science, University of Massachusetts, Amherst, MA, USA.

Renhua Song, Epigenetics and RNA Biology Program Centenary Institute, The University of Sydney, Camperdown, New South Wales, Australia; Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales, Australia.

Haixia Yang, Department of Food Science, University of Massachusetts, Amherst, MA, USA.

David Johnson, Department of Food Science, University of Massachusetts, Amherst, MA, USA.

Heather L Sherman, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA.

Daeyoung Kim, Department of Mathematics & Statistics, University of Massachusetts, Amherst, MA, USA.

Lisa M Minter, Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA, USA; Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA.

Justin J-L Wong, Epigenetics and RNA Biology Program Centenary Institute, The University of Sydney, Camperdown, New South Wales, Australia; Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales, Australia.

Melody Y Zeng, Gale and Ira Drukier Institute for Children’s Health, Weill Cornell Medicine, New York, NY, USA; Department of Pediatrics, Weill Cornell Medicine, New York, NY, USA.

Eric A Decker, Department of Food Science, University of Massachusetts, Amherst, MA, USA.

Guodong Zhang, Department of Food Science, University of Massachusetts, Amherst, MA, USA; Department of Nutrition, University of California, Davis, Davis, CA, USA.

Funding

This research was supported by the USDA NIFA 2016-67017-24423, USDA NIFA 2019-67017-29248, and USDA/Hatch MAS00492 (to G.Z.); USDA NIFA 2022-67017-36483 (to E.A.D.); and NIH K01DK114376, R21CA270998, and R01HD110118 (to M.Y.Z.).

Conflict of Interest

None declared.

Author Contributions

W.W., Yuxin Wang, K.Z.S., Yige Wang, J.Z., W.Y., Q.S., I.B., H.Y., D.J., and H.L.S. performed the experiments and analyzed the data; R.S., D.K., L.M.M., J.J.-L.W., M.Y.Z., E.A.D., and G.Z. analyzed the data; W.W., Y.W., K.Z.S., E.A.D., and G.Z. designed the experiments; and W.W., Yuxin Wang, and G.Z. wrote the manuscript.

Data Availability

All data are reported in the manuscript and Supplementary Material. The 16S rRNA sequencing data have been deposited in the Sequence Read Archive under accession number GSE209580.

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28. Wang Y, Yang J, Wang W, et al. Soluble epoxide hydrolase is an endogenous regulator of obesity-induced intestinal barrier dysfunction and bacterial translocation. Proc Natl Acad Sci USA 2020;117:8431–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Wang G, Zhang H, Zhang J, et al. Metabolic fate of environmental chemical triclocarban in colon tissues: roles of gut microbiota involved. Sci Total Environ 2021;787:147677. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Zhang J, Walker ME, Sanidad KZ, et al. Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract. Nat Commun 2022;13:136. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Hall M, Beiko RG. 16S rRNA Gene Analysis with QIIME2. Methods Mol Biol 2018;1849:113–29. [DOI] [PubMed] [Google Scholar]
32. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 2016;13:581–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 2013;41:D590–596. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Lin H, Peddada SD. Analysis of compositions of microbiomes with bias correction. Nat Commun 2020;11:3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Erben U, Loddenkemper C, Doerfel K, et al. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol 2014;7:4557–76. [PMC free article] [PubMed] [Google Scholar]
36. Wang W, Yang H, Johnson D, Gensler C, Decker E, Zhang G. Chemistry and biology of omega-3 PUFA peroxidation-derived compounds. Prostaglandins Other Lipid Mediat 2017;132:84–91. [DOI] [PubMed] [Google Scholar]
37. Yang J, Schmelzer K, Georgi K, Hammock BD. Quantitative profiling method for oxylipin metabolome by liquid chromatography electrospray ionization tandem mass spectrometry. Anal Chem 2009;81:8085–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function. Nat Rev Immunol 2010;10:131–44. [DOI] [PubMed] [Google Scholar]
39. Bischoff SC, Barbara G, Buurman W, et al. Intestinal permeability—a new target for disease prevention and therapy. BMC Gastroenterol 2014;14:189. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Kojima A, Nakano K, Wada K, et al. Infection of specific strains of Streptococcus mutans, oral bacteria, confers a risk of ulcerative colitis. Sci Rep 2012;2:332. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Jackson V, Penumetcha M. Dietary oxidised lipids, health consequences and novel food technologies that thwart food lipid oxidation: an update. Int J Food Sci Technol 2018;54:1981–8. [Google Scholar]
42. Billek GH. Health aspects of thermoxidized oils and fats. Eur J Lipid Sci Technol 2000;102:587–93. [Google Scholar]
43. Esterbauer H. Cytotoxicity and genotoxicity of lipid-oxidation products. Am J Clin Nutr 1993;57:779S–785S; discussion 785S. [DOI] [PubMed] [Google Scholar]
44. Mayr L, Grabherr F, Schwärzler J, et al. Dietary lipids fuel GPX4-restricted enteritis resembling Crohn’s disease. Nat Commun 2020;11:1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Wang Y, Dattmore DA, Wang W, et al. Trans, trans-2,4-Decadienal, a lipid peroxidation product, induces inflammatory responses via Hsp90- or 14-3-3ζ-dependent mechanisms. J Nutr Biochem 2020;76:108286. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Wang Y, Wang W, Yang H, Shao D, Zhao X, Zhang G. Intraperitoneal injection of 4-hydroxynonenal (4-HNE), a lipid peroxidation product, exacerbates colonic inflammation through activation of Toll-like receptor 4 signaling. Free Radic Biol Med 2019;131:237–42. [DOI] [PubMed] [Google Scholar]
47. Gabert L, Vors C, Louche-Pélissier C, et al. 13C tracer recovery in human stools after digestion of a fat-rich meal labelled with [1,1,1-13C3]tripalmitin and [1,1,1-13C3]triolein. Rapid Commun Mass Spectrom 2011;25:2697–703. [DOI] [PubMed] [Google Scholar]
48. Vors C, Pineau G, Gabert L, et al. Modulating absorption and postprandial handling of dietary fatty acids by structuring fat in the meal: a randomized crossover clinical trial. Am J Clin Nutr 2013;97:23–36. [DOI] [PubMed] [Google Scholar]
49. Qv L, Yang Z, Yao M, et al. Methods for establishment and maintenance of germ-free rat models. Front Microbiol 2020;11:1148. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jjae148_suppl_Supplementary_Materials

jjae148_suppl_supplementary_materials.pdf^{(976KB, pdf)}

Data Availability Statement

All data are reported in the manuscript and Supplementary Material. The 16S rRNA sequencing data have been deposited in the Sequence Read Archive under accession number GSE209580.

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PERMALINK

Oxidized Polyunsaturated Fatty Acid Promotes Colitis and Colitis-Associated Tumorigenesis in Mice

Weicang Wang

Yuxin Wang

Katherine Z Sanidad

Yige Wang

Jianan Zhang

Wenqi Yang

Quancai Sun

Ipek Bayram

Renhua Song

Haixia Yang

David Johnson

Heather L Sherman

Daeyoung Kim

Lisa M Minter

Justin J-L Wong

Melody Y Zeng

Eric A Decker

Guodong Zhang

Abstract

Background and Aims

Methods

Results

Conclusions

1. Introduction

2. Methods

2.1. Study design

2.2. Preparation of column chromatography-purified corn oil (unoxidized PUFA)

2.3. Preparation of oxidized corn oil (oxidized PUFA)

2.4. PV assay

2.5. Liquid chromatography-tandem mass spectrometry-based lipidomics analysis of lipid oxidation compounds in the oils

2.6. Animal experiments

2.7. Experimental diets

2.8. Animal Protocol 1: effects of PUFA diet and oxidized PUFA diet on basal inflammation in healthy mice

2.9. Animal Protocol 2: effects of PUFA diet and oxidized PUFA diet on dextran sodium sulfate-induced colitis in mice

2.10. Animal Protocol 3: effects of PUFA diet and oxidized PUFA diet on spontaneous colitis in Il-10−/− mice

2.11. Animal Protocol 4: effects of PUFA diet and oxidized PUFA diet on azoxymethane/DSS-induced colitis-associated colon cancer in mice

2.12. Animal Protocol 5: effects of PUFA diet and oxidized PUFA diet on colitis-associated gut barrier dysfunction

2.13. Animal Protocol 6: effects of PUFA diet and oxidized PUFA diet on DSS-induced colitis in wild-type and Tlr4−/− mice

2.14. Animal Protocol 7: effects of antibiotic cocktail-mediated suppression of gut microbiota on effects of PUFA vs oxidized PUFA on DSS-induced colitis

2.15. Animal Protocol 8: effects of diets rich in PUFA, oxidized PUFA, and SFA on DSS-induced colitis

2.16. Flow cytometry analysis of immune cell infiltration in colon tissues

2.17. 16S rRNA sequencing of fecal microbiota

2.18. Bioinformatic analysis

2.19. Histology

2.20. Immunohistochemical staining

2.21. ELISA analysis

2.22. qRT-PCR analysis of gene expression in colon tissues

2.23. qRT-PCR analysis of 16S rRNA in blood

2.24. Assessment of TLR4 ligand levels in serum

2.25. Effects of corn oil and oxidized corn oil on TLR4 activation in vitro

2.26. Lipopolysaccharide determination in plasma

2.27. Data analysis

3. Results

3.1. Preparation of unoxidized and oxidized PUFA

3.2. Oxidized PUFA does not induce basal colonic inflammation in healthy mice

3.3. Oxidized PUFA increases the severity of chemically induced colitis in mice

Figure 1.

3.4. Oxidized PUFA, but not unoxidized PUFA, increases the severity of chemically induced colitis in mice

Figure 2.

3.5. Oxidized PUFA increases the development of spontaneous colitis in Il-10−/− mice

Figure 3.

3.6. Oxidized PUFA increases the development of colitis-associated colorectal cancer in mice

Figure 4.

3.7. Oxidized PUFA increases colitis through TLR4-dependent mechanisms

Figure 5.

Figure 6.

3.8. Oxidized PUFA increases colitis through gut microbiota-dependent mechanisms

Figure 7.

Figure 8.

4. Discussion

Supplementary Material

Contributor Information

Funding

Conflict of Interest

Author Contributions

Data Availability

References

Associated Data

2.10. Animal Protocol 3: effects of PUFA diet and oxidized PUFA diet on spontaneous colitis in Il-10^−/− mice

2.13. Animal Protocol 6: effects of PUFA diet and oxidized PUFA diet on DSS-induced colitis in wild-type and Tlr4^−/− mice

3.5. Oxidized PUFA increases the development of spontaneous colitis in Il-10^−/− mice