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Acetaminophen, a widely used analgesic with well-characterized dose-dependent hepatotoxicity, serves as a benchmark compound for evaluating liver toxicity mechanisms. Here, we applied single cell RNA sequencing to characterize cellular responses to acetaminophen exposure in two distinct liver cell culture formats: two-dimensional monolayers (2D) and three-dimensional (3D) spheroids composed of primary human hepatocytes, Kupffer cells, and liver endothelial cells. Cultures were exposed for 24\u00a0h to low (350\u00a0\u00b5M) and high (2687\u00a0\u00b5M) acetaminophen concentrations, with 2D cultures receiving only the low dose. Compared to 2D monolayers, 3D spheroids exhibited greater transcriptional diversity and elevated expression of ribosomal genes, indicative of enhanced metabolic and biosynthetic activity. Reactome pathway analysis revealed pronounced hypoxia-associated signaling in 3D cultures even under baseline conditions, likely due to restricted oxygen diffusion within spheroids. These hypoxia signatures were most prominent in endothelial cells. Upon acetaminophen exposure, hypoxic hepatocytes displayed elevated expression of cytochrome P450 enzymes, while conjugation enzymes involved in detoxification declined with increasing dose, suggesting compromised phase II metabolism under oxygen-limited conditions. Normoxic hepatocytes showed minimal transcriptional response. These results suggest a dynamic interplay between oxygen availability and acetaminophen metabolism, where oxygen tension influences metabolic activity, and drug metabolism in turn alters the hypoxic landscape. Our findings underscore the physiological relevance of 3D liver models and highlight the importance of spatial microenvironmental context for improving mechanistic insights into hepatotoxicity.<\/jats:p>","DOI":"10.1007\/s00204-025-04296-6","type":"journal-article","created":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T20:15:24Z","timestamp":1768421724000},"page":"1411-1424","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Single-cell transcriptomics of acetaminophen-induced responses in human 2D and 3D liver microtissues"],"prefix":"10.1007","volume":"100","author":[{"given":"Brian","family":"Bwanya","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcha C. 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J.","family":"Jennen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Twan","family":"van den Beucken","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3325-0466","authenticated-orcid":false,"given":"Florian","family":"Caiment","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,1,14]]},"reference":[{"key":"4296_CR1","doi-asserted-by":"publisher","first-page":"22","DOI":"10.1186\/s13036-019-0148-5","volume":"13","author":"J Ahn","year":"2019","unstructured":"Ahn J, Ahn JH, Yoon S, Nam YS, Son MY, Oh JH (2019) Human three-dimensional in vitro model of hepatic zonation to predict zonal hepatotoxicity. 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