Single‐Cell Proteomic Profiling of Hepatocytes Reveals Heterogeneous Responses to Hepatotoxic Drugs Nevirapine, Efavirenz, and Acetaminophen

Drug‐induced hepatotoxicity remains a leading cause of the withdrawal of drugs from the global market. Changes in the proteomic profile of hepatocytes following exposure to known hepatotoxic drugs could provide insight into the mechanisms underlying drug‐induced hepatotoxicity. Thus, we aimed to employ mass spectrometry (MS)‐based proteomics to investigate the impact of a subset of known hepatotoxic drugs, namely, efavirenz (EFV), nevirapine (NVP), and acetaminophen (APAP) on the human hepatocyte proteome in vitro . In addition, to add further granularity to the analysis and to understand potential cell‐to‐cell variability in the proteomic response to the above‐mentioned drugs, single‐cell proteomics was employed. Cultured cryoplateable hepatocytes were incubated with each drug individually at their IC 50 or IC 90 values to induce hepatotoxicity. For single‐cell proteomic analysis, after 40‐hour treatment individual hepatocytes were sorted using fluorescence‐activated cell sorting (FACS) and protein lysates were digested with trypsin, labeled with unique tandem mass tags (TMT), and pooled together for multiplex analysis using a timsTOF Flex mass analyzer. Preliminary single‐cell proteomic results were obtained from 385 cells analyzed at a depth of approximately 350 proteins per cell, and an approximate 1,700 proteins per treatment could be readily quantified using reporter ion‐based quantification with SequestHT with Percolator validation. A principal component analysis (PCA) separated the hepatotoxic drug‐treated cells and DMSO control‐treated cells into two mostly discrete clusters thereby confirming the hepatocytes’ proteome change upon hepatotoxic drug treatment. Further, these data revealed a heterogeneous response to hepatotoxic across single hepatocytes. From proteomic analysis on bulk hepatocytes, we found that the abundance of 36 proteins was significantly increased following exposure of cultured hepatocytes to IC 50 and IC 90 concentrations of NVP, EFV, and APAP ( p‐value < 0.05, Log2 fold change >1 ). These proteins included calnexin, an endoplasmic reticulum (ER) stress‐induced protein. Interestingly, single‐cell proteomic analysis also showed that the abundance of calnexin was significantly increased following treatment of NVP, EFV, and APAP at IC 90 ; however, the levels of calnexin were divergent across the single cells with certain cells exhibiting no increase in calnexin following drug treatment. These data demonstrate hepatocyte‐to‐hepatocyte heterogeneity in the response to hepatotoxic drugs and suggest that proteomics performed on bulk/total hepatocytes may not fully shed light on hepatoxicity mechanisms.