DOI: 10.5061/dryad.jwstqjqpd
title: Hazardous effects of 6PPD on human small intestinal organoids

description: N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is the most
 ubiquitously utilized antioxidant in the manufacture of rubber products.
 While it has been associated with the development of human colorectal
 cancer (CRC), its intestinal hazardousness in humans remains
 experimentally unconfirmed. In this study, human small intestinal
 organoids were used to assess the intestinal toxicity of 6PPD. The
 findings of this study demonstrated that exposure to 6PPD at
 concentrations ranging from 300 to 900 μg·L⁻¹ resulted in impaired
 organoid growth in a concentration-dependent manner, as evidenced by
 reduced organoid count and size. Although even the highest concentration
 tested (900 μg·L⁻¹) did not induce overt cell death, viability was
 significantly inhibited across this concentration range, as evidenced by
 decreased Calcein fluorescence and CCK-8 metabolic activity.
 Immunofluorescence analysis further revealed suppressed proliferation and
 downregulation of epithelial and goblet cell markers, whereas markers of
 Paneth, villus, and enteroendocrine cells remained unchanged. Notably, at
 higher concentrations (≥1000 μg·L^−1^), 6PPD rapidly induced organoid
 disintegration and significant increases in cell death. Together, these
 results demonstrate that 6PPD disrupts human intestinal epithelial
 development and survival in a concentration-dependent manner, providing
 direct experimental evidence of its cytotoxic potential in human guts and
 highlighting the need for further mechanistic and regulatory
 investigations.
description: # Hazardous effects of 6PPD on human small intestinal organoids Dataset
 DOI: [10.5061/dryad.jwstqjqpd](https://doi.org/10.5061/dryad.jwstqjqpd) ##
 Description of the data and file structure ### File:
 original_images__data_and_description.zip The data for the four figures in
 the manuscript (Figures 1-4) are organized into separate folders labeled
 F1 through F4, respectively. Each folder contains the original images and
 processed data used to create its corresponding figure. Please note that
 the folder prefixes 'F' and 'D' have distinct
 meanings: F1-F4 refer to the figure number, while D1-D4 refer to the
 experimental day (e.g., Day 1). For each folder, the image counts and
 relevant statistical data are recorded in a CSV file within that same
 folder. Folder F1 contains data related to the culture and 6PPD treatment
 of human small intestinal organoids. Folder F1A includes images of human
 small intestine organoids thawed from cryopreservation and cultured under
 standard conditions. Images were taken using bright-field (BF) from the
 day of thawing (day 0, D0) to day 3. Scale bars = 500 μm. 6PPD was first
 dissolved in DMSO at a stock concentration of 1000 mg·L^-1^, and then
 diluted with culture medium to final concentrations of 0, 300, 600, and
 900 μg·L^-1^ for organoid treatment. Morphological changes of organoids
 were monitored over 3 days. The images for the groups with concentrations
 of 0, 300, 600, and 900 are in folder 0, folder 300, folder 600, and
 folder 900, respectively, which are all in folder F1B. In the F1.SCV file,
 the table in the top left shows the "Organoid count/image", and
 the bottom left provides the related statistical analysis (Tukey's
 multiple comparisons test, mean difference, (Mean Diff.), 95% confidence
 interval of the difference (95.00% CI of diff.), Below threshold, Summary,
 Adjusted P Value). The table in the top right shows the "Organoid
 total BF area*10^4^(μm^2^)", and the bottom right provides the
 related statistical analysis. Folder F2 contains raw data and images on
 the effects of 6PPD on the viability of human small intestinal
 organoids. Representative images of live/dead cell staining after
 treatment with 0, 300, 600, and 900 μg·L^-1^ 6PPD for 3 days are shown in
 folder 0, folder 300, folder 600, and folder 900, respectively. The live
 cells were stained green with Calcein-AM, while the dead cells were
 stained red with propidium iodide (PI). Scale bars = 500 μm. The Excel
 spreadsheet F2BC contains raw data and graphs for quantification of
 Calcein fluorescence intensity and measurement of cell viability with the
 CCK8 assay. Within the F2 folder, the merged images represent the combined
 results of Calcein and Pi signals. For example: 1-merge is the merged
 result of 1-Calcein and 1-Pi signals, while 2-merge is the merged result
 of 2-Calcein and 2-Pi signals. In the F2BC.SCV file, the table in the
 top left shows the "relative Calcein level", and the
 bottom left provides the related statistical analysis (Dunnett's T3
 multiple comparisons test, Mean Diff., 95.00% CI of diff., Below
 threshold, Summary, Adjusted P Value). The table in the top right shows
 the "Cell viability (%)", and the bottom right provides the
 related statistical analysis. The data and images in folder F3 are
 about the impact of 6PPD on the differentiation of human small intestinal
 organoids. Raw images for assessment of cell proliferation (Ki67) are in
 the folder Ki67. Raw images for assessing epithelial integrity
 (E-cadherin) of MUC2 (goblet cells) in folder E-cad-muc2. Raw images
 of the assessment of Villin (villus enterocytes), and LYZ (Paneth cells)
 are in Folder VILLIN-Lyz, and raw images for the evaluation of CHGA
 (enteroendocrine cells) are in Folder CHGA. Folder 0, folder 600, and
 folder 900 in the above folders refer to the 6PPD concentrations used
 being 0, 600, and 900 μg·L^-1^, respectively. Within the F3 folder, the
 “merge” images represent the combined results of CHGA and DAPI staining
 methods. For example, each merged image in the CHGA folder is the result
 of merging corresponding CHGA and DAPI staining methods. In the
 F3.SCV file, for the tables in the top, shows the "Relative
 Ki67/E-cad/MUC2 level" from left to right, and below the table is the
 corresponding data analysis (Unpaired t with Welch's correction, Mean
 Diff., 95.00% CI of diff., Below threshold, Summary, Adjusted P
 Value). For the tables at the bottom, show the "Relative
 Villin/Lyz/CHGA level" from left to right, and below the table is the
 corresponding data analysis. Raw data and images in folder F4 demonstrate
 that high concentrations of 6PPD induce cell death in human small
 intestinal organ tissues. Images in folder F4A are representative images
 after 1 day of treatment with 1000 and 2000 μg·L^-1^ concentrations of
 6PPD and the corresponding quantification of organoid count per image and
 total area of organoid bright-field (BF) for each image. Scale bar = 500
 μm. Representative images and quantification of GreenNuc/PI cell staining
 after treatment with 0, 1000, and 2000 μg·L^-1^ 6PPD for 1 day are in
 folder F4A and Excel sheet F4B, respectively. Scale bars = 500 μm. Inside
 the F4B folder, the “merge” images represent the combined results of the
 green and pi staining methods. In the F4.SCV file, for the A section,
 table in the left shows the "Organoid count/image", table in the
 right shows the "Organoid total BF area*10^4(um^2)" and below
 the tables are the corresponding data analysis (Uncorrected Fisher's
 LSD, Mean Diff., 95.00% CI of diff., Below threshold, Summary, Individual
 P Value). For the B section, table on the left shows the "Relative
 GreenNuc level(%)", table in the right shows the "PI positive
 cells(%)", and below the tables are the corresponding data analysis.