Hazardous effects of 6PPD on human small intestinal organoids
Data files
Oct 27, 2025 version files 304.19 MB
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original_images__data_and_description.zip
304.19 MB
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README.md
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Abstract
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.
Dataset DOI: 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*104(μm2)", 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*104(um2)" 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.