biostudies-arrayexpressNicolaj BischoffHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15089This study provides critical insights into the adverse effects of titanium dioxide (E171) on human gastrointestinal health and its potential molecular mechanisms of action, marking a significant advancement in food toxicology. Through a human dietary intervention study involving 31 participants consuming 2 mg/kg body weight/day of E171 in yogurt, the research confirmed compliance by detecting significantly elevated titanium levels in feces during the intervention. Importantly, the study identified a statistically significant increase in superoxide radicals in whole blood, indicating that E171 induces oxidative stress. While systemic inflammation markers, including cytokine levels and hs-CRP, showed no relevant changes, gene expression analysis of colon biopsies revealed substantial transcriptomic alterations. Specifically, 73 enriched pathways were identified, including those related to metabolic reprogramming (consistent with the Warburg effect), colorectal cancer development, ribosome biogenesis, PPAR signaling, and chemical carcinogenesis involving reactive oxygen species. Additionally, the characterization of E171's physicochemical properties post-gastrointestinal digestion provided a clearer understanding of nanoparticle exposure in the human gut. This study is novel as it delivers human-specific data, bridging a critical gap in the knowledge of E171’s health risks. It also provides molecular evidence linking its consumption to hallmarks of colorectal cancer. These findings support the European Union’s decision to ban E171 as a food additive and highlight its potential risks, contributing significantly to food toxicology.biostudies-arrayexpressLibrary Construction - Samples containing purified RNA were prepared for sequencing using a ribosomal RNA (rRNA) depletion library preparation kit (Revvity, Groningen, The Netherlands) on a Zephyr G3 NGS workstation (CLS150362, Revvity, Groningen, The Netherlands).Nucleic Acid Extraction - The rectal biopsies were defrosted on ice before the remains of the RNAlater were carefully removed. Each biopsy was washed once in ice-cold RNAse-free water before transferring it into 1000 µL ice-cold QIAzol (Qiagen, The Netherlands). We used a bio-disrupter mixer on level 4 for 15 seconds to homogenize the biopsies in the QIAzol before 200 µL of chloroform was added to each sample. The samples were vigorously shaken for 15 seconds before being placed on the bench at room temperature for 3 minutes. The samples were centrifuged for 15 minutes at 12.000 x g at 4°C. The upper aqueous phase was transferred into a new collection tube, and 1.5 times the volume (~750 µl) of 100 % ethanol was added and mixed thoroughly by pipetting. Next, 700 µL of the sample was pipetted onto an RNease MinElute spin column in a 2 mL collection tube and centrifuged at 8000 x g for 15 seconds at room temperature. The flowthrough was discarded, and the previous step was repeated with the remaining sample. A DNase digestion step was conducted by adding 350 µL of Buffer RWT to the column before centrifugation at 8000 x g for 15 seconds. The flowthrough was discarded, and 80 µL of DNase I stock solution in Buffer RDD was added to the column, followed by 15 minutes of incubation at room temperature. Next, 350 µL of Buffer RWT were added to the column, and the RNease MinElute spin column was centrifuged at 8000 x g for 15 seconds. After discarding the flowthrough, we added 500 µL of Buffer RPE and centrifuged at 8000 x g for 15 seconds. Next, we discarded the flow through, added 500 µL of 80 % ethanol to the column, and centrifuged at 8000 x g for 15 seconds. The spin column was placed into a fresh collection tube and centrifuged at full speed for 5 minutes to dry the membrane and evaporate the ethanol residues. The spin column was then placed in a fresh 1.5 mL Eppendorf tube, and the RNA was eluded with 14 µL of RNase-free water through centrifugation at full speed for one minute.Sample Collection - Following both intervention periods, body weight and height were measured. We collected 20 mL of venous blood in EDTA tubes, which were shaken ten times and immediately placed on ice. The feces samples were weighed, the collection date was registered, and the samples were immediately put on ice. Fece samples were manually homogenized and transferred into 20 mL fecal collection tubes before storing them at -20 °C until further analysis. Whole blood was immediately treated with 1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) to a final concentration of 0.5 mM CMH and snap-frozen in liquid nitrogen for Electrospin (ESR) resonance spectrometry measurements for reactive oxygen species (ROS). To obtain plasma, we centrifuged the tubes at 2100 rpm for 10 minutes and transferred the plasma into 1.5 mL Eppendorf tubes, which were frozen and stored at – 80 °C until analysis. Participants were accompanied to the endoscopy ward of Zuyderland Medical Centre, where trained endoscopy nurses harvested rectal biopsies (Radial JawTM 4, 2.8 mm, Boston Scientific BV, Kerkrade, The Netherlands). Biopsy samples were snap-frozen in a Corning cooling rack on dry ice or treated with RNAse later at room temperature before being transferred to a 4 °C fridge overnight. Snap-frozen biopsies were immediately transferred into a -80 °C freezer, ensuring the integrity of the samples until further analysis. RNAse later treated biopsies were also moved to the -80 °C freezer after 24 hours of incubation at 4 °C.Sequencing - After library preparation, the samples were sequenced on an S2 flow cell (Illumina, San Diego, USA) on the NovaSeq 6000 system (Illumina, San Diego, USA) using Rapid Directional RNA-sq Kit 2.0 (Revvity, Groningen, The Netherlands).OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - Raw RNA sequencing data were obtained as BCL files and converted to fastq files using BCL2FastQ (v2.20.0.422). The fastq files were processed using the R-ODAF pipeline (30). In short, reads were trimmed with fastp (v0.20.0) with a head crop of 12 bases. MultiQC (v1.7) quality control after trimming indicated good sequencing quality (all bases > Q35) with a sequencing depth between 41.7 M and 66.2 M per sample. These reads were mapped to the genome (GRCh38_112) with STAR (v2.7.9a) and quantified using RSEM (v1.3.1).UnknownTranscriptomicsGenomicsProteomicsmiRNeasy Micro kitZephyr G3 NGS workstationRadial JawTMIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensDietary titanium dioxide (E171) increases systemic ROS levels and alters the colon transcriptome – Evidence from a human dietary intervention studyNicolaj BischoffTheo de KokNicolaj S. Bischoff, Anna K. Undas, Greet van Bemmel, Marcel van Herwijnen, Marcha Verheijen, Simone G. Van Breda, Jacco J. Briedé, Lana Drenth-Brink, Marcella Peelen-Buijs, Ad A. van Bodegraven, Dick T.H.M. Sijm, Theo M. de KokfalseDietary titanium dioxide (E171) increases systemic ROS levels and alters the colon transcriptome – Evidence from a human dietary intervention studyThis study provides critical insights into the adverse effects of titanium dioxide (E171) on human gastrointestinal health and its potential molecular mechanisms of action, marking a significant advancement in food toxicology. Through a human dietary intervention study involving 31 participants consuming 2 mg/kg body weight/day of E171 in yogurt, the research confirmed compliance by detecting significantly elevated titanium levels in feces during the intervention. Importantly, the study identified a statistically significant increase in superoxide radicals in whole blood, indicating that E171 induces oxidative stress. While systemic inflammation markers, including cytokine levels and hs-CRP, showed no relevant changes, gene expression analysis of colon biopsies revealed substantial transcriptomic alterations. Specifically, 73 enriched pathways were identified, including those related to metabolic reprogramming (consistent with the Warburg effect), colorectal cancer development, ribosome biogenesis, PPAR signaling, and chemical carcinogenesis involving reactive oxygen species. Additionally, the characterization of E171's physicochemical properties post-gastrointestinal digestion provided a clearer understanding of nanoparticle exposure in the human gut. This study is novel as it delivers human-specific data, bridging a critical gap in the knowledge of E171’s health risks. It also provides molecular evidence linking its consumption to hallmarks of colorectal cancer. These findings support the European Union’s decision to ban E171 as a food additive and highlight its potential risks, contributing significantly to food toxicology.2025-09-30T00:00:00Z2025-09-30T01:04:50.842Z2025-04-24T16:48:39.357ZE-MTAB-15089ERP171953EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184