# Single-cell transcriptomics of acetaminophen-induced responses in human 2D and 3D liver microtissues

## TL;DR

This study uses single-cell RNA sequencing to compare how human liver cells in 2D and 3D cultures respond to acetaminophen, revealing that 3D models show more complex and realistic responses.

## Contribution

The novel contribution is the use of single-cell transcriptomics to uncover hypoxia-driven differences in drug metabolism between 2D and 3D liver models.

## Key findings

- 3D liver spheroids showed greater transcriptional diversity and higher ribosomal gene expression compared to 2D cultures.
- 3D cultures exhibited hypoxia-associated signaling, especially in endothelial cells, even under baseline conditions.
- Acetaminophen exposure altered cytochrome P450 and conjugation enzyme expression in hypoxic hepatocytes, indicating compromised detoxification.

## Abstract

Drug-induced liver injury remains a major obstacle in pharmaceutical development and a leading cause of acute liver failure, underscoring the need for predictive and human-relevant in vitro models. 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 h to low (350 µM) and high (2687 µM) 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.

The online version contains supplementary material available at 10.1007/s00204-025-04296-6.