Spatial Reconstruction of the Early Hepatic Transcriptomic Landscape After an Acetaminophen Overdose Using Single-Cell RNA-Sequencing

Abstract

An acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. A hallmark characteristic of APAP hepatotoxicity is centrilobular necrosis. General, innate mechanisms such as lower amounts of GSH and higher Cyp2e1 expression in pericentral (PC) hepatocytes are known to contribute to the differences in susceptibility to cell injury between periportal (PP) hepatocytes and PC hepatocytes. While a sequence of molecular events involving formation of the reactive metabolite N-acetyl-p-benzoquinone imine, GSH depletion, oxidative stress, and JNK activation define the early cell stress trajectory following APAP exposure, their activation in PC vs PP hepatocytes is not well characterized. By using single-cell RNA sequencing, we provide the first reconstruction of the early transcriptomic APAP liver lobule after validation of our methodology using human liver scRNA-seq data. Two hours after APAP treatment, we find that PP hepatocytes progress along the APAP stress axis to oxidative stress, before resolving injury due to innate and adaptive mechanisms. However, PC hepatocytes continue along this stress axis as indicated by activation of mitogen-activated protein kinase (MAPK) genes, which is absent in PP hepatocytes. We also identify a population of glutamine synthetase enriched PC hepatocytes in close proximity to the central vein, where a stepwise induction of a stress program culminated in cell death. Collectively, these findings elucidate a molecular sequence of events distinguishing the differential response to APAP exposure between PP and PC hepatocytes and identify a subset of uniquely susceptible PC hepatocytes.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls

“…g ., NAPQI) leading to the release of NRF2 by KEAP1 187 , whereby NRF2 can translocate to the nucleus and activate antioxidant defense protocols 188 . Importantly, induction of critical genes involved in hepatic GSH resynthesis ( Gclc and Gclm ) following APAP toxicity 36 , 189 can mitigate injury as GSH can directly sequester NAPQI and detoxify reactive oxygen and peroxynitrite. Other canonical genes regulated by NRF2 include NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione peroxidase, and the conjugation enzymes, UDP-glucuronosyltransferases (UGT) and glutathione- S -transferases (GST) 190 .…”

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole

“…APAP-induced liver injury is initiated by its metabolism by cytochrome P450, predominantly CYP2E1, to a reactive metabolite. This mainly occurs in the hepatocytes surrounding the central vein in the liver, where CYP2E1 is highly expressed . This zonated pattern of CYP2E1 expression correlates with the characteristic necrosis of hepatocytes surrounding the central vein seen after an APAP overdose.…”

“…This mainly occurs in the hepatocytes surrounding the central vein in the liver, where CYP2E1 is highly expressed. 12 This zonated pattern of CYP2E1 expression correlates with the characteristic necrosis of hepatocytes surrounding the central vein seen after an APAP overdose. This dependence of hepatotoxicity on regional metabolism within the liver lobule illustrates the importance of understanding local variations in APAP metabolism within the liver since regional concentrations of APAP-derived metabolites may be differentially toxic to hepatocytes around the central vein or the portal triad.…”

Advances in Single-Cell Toxicogenomics in Environmental Toxicology

“…As for the digestive system, Umbaugh et al reconstructed the early transcriptomic acetaminophen liver lobule using single-cell RNA sequencing and elucidated the molecular sequences of periportal and pericentral hepatocytes in different response to acetaminophen exposure. The authors found that periportal hepatocytes developed along the acetaminophen stress axis to oxidative stress, while pericentral hepatocytes continued to develop along the stress axis activated by mitogen-activated protein kinase genes . Zhang et al used single-cell RNA-seq to investigate the mechanism of hepatotoxicity of AFB 1 to S phase-arrested L 02 cells.…”