Dietary titanium dioxide (E171) increases systemic ROS levels and alters the colon transcriptome – Evidence from a human dietary intervention study
Dietary titanium dioxide (E171) increases systemic ROS levels and alters the colon transcriptome – Evidence from a human dietary intervention study
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.   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. 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).